# PRIMER3 RELEASE 2.6.1 MANUAL

## CONTENTS

1. COPYRIGHT AND LICENSE
2. INTRODUCTION
3. CITING PRIMER3
4. FAIR USE OF PRIMER3
5. DIFFERENCE BETWEEN PRIMER3 AND PRIMER3PLUS
6. CHANGES FROM VERSION 2.2.3
7. INSTALLATION INSTRUCTIONS - UNIX/LINUX
8. INSTALLATION INSTRUCTIONS - Mac OSX
9. INSTALLATION INSTRUCTIONS - WINDOWS
10. SYSTEM REQUIREMENTS
11. INVOKING primer3_core
12. COMMAND LINE ARGUMENTS
13. INPUT AND OUTPUT CONVENTIONS
14. AN EXAMPLE
15. HOW TO MIGRATE TAGS TO IO VERSION 4
16. "SEQUENCE" INPUT TAGS
17. "GLOBAL" INPUT TAGS
18. "PROGRAM" INPUT TAGS
19. HOW PRIMER3 CALCULATES THE PENALTY VALUE
20. PRIMER3 SETTINGS FILE FORMAT
21. OUTPUT TAGS
22. EXAMPLE OUTPUT
23. ADVICE FOR PICKING PRIMERS
24. GENERAL THOUGHTS ON PRIMER BINDING
25. CAUTIONS
26. WHAT TO DO IF PRIMER3 CANNOT FIND ANY PRIMERS?
27. DIFFERENCES FROM EARLIER VERSIONS
28. EXIT STATUS CODES
29. PRIMER3 WWW INTERFACES
30. ACKNOWLEDGMENTS

## 1. COPYRIGHT AND LICENSE

    Copyright (c) 1996 - 2022
    Whitehead Institute for Biomedical Research, Steve Rozen
    (http://purl.com/STEVEROZEN/), Helen Skaletsky, Triinu Koressaar,
    Maido Remm and Andreas Untergasser. All rights reserved.
        This file is part of the Primer3 suite and libraries.
        The Primer3 suite and libraries are free software;
        you can redistribute them and/or modify them under the terms
        of the GNU General Public License as published by the Free
        Software Foundation; either version 2 of the License, or (at
        your option) any later version.
        This software is distributed in the hope that it will be useful,
        but WITHOUT ANY WARRANTY; without even the implied warranty of
        MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.  See the
        GNU General Public License for more details.
        You should have received a copy of the GNU General Public License
        along with this software (file gpl-2.0.txt in the source
        distribution); if not, write to the Free Software
        Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA
    THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS
    "AS IS" AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT
    LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR
    A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL THE COPYRIGHT
    OWNERS OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL,
    SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT
    LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE,
    DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY
    THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT
    (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE
    OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.

## 2. INTRODUCTION

Primer3 picks primers for PCR reactions, considering as criteria:
o oligonucleotide melting temperature, size, GC content, and primer-dimer possibilities,
o PCR product size,
o positional constraints within the source (template) sequence, and
o possibilities for ectopic priming (amplifying the wrong sequence)
o many other constraints.
All of these criteria are user-specifiable as constraints, and some are specifiable as terms in an objective function that characterizes an optimal primer pair.

## 3. CITING PRIMER3

We request but do not require that use of this software be cited in publications as
* Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M and Rozen SG.
Primer3--new capabilities and interfaces.
Nucleic Acids Res. 2012 Aug 1;40(15):e115.
The paper is available at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3424584/
and
* Koressaar T and Remm M.
Enhancements and modifications of primer design program Primer3.
Bioinformatics 2007;23(10):1289-1291.
The paper is available at https://www.ncbi.nlm.nih.gov/pubmed/17379693
If you use masker function, please cite:
* Koressaar T, Lepamets M, Kaplinski L, Raime K, Andreson R and Remm M.
Primer3_masker: integrating masking of template sequence with primer design software.
Bioinformatics 2018;34(11):1937-1938.
The paper is available at https://www.ncbi.nlm.nih.gov/pubmed/29360956
Source code available at https://github.com/primer3-org/primer3.

## 4. FAIR USE OF PRIMER3

The development of Primer3 is promoted by a small group of enthusiastic scientists mainly in their free time.They do not gain any financial profit with Primer3.
There are two groups of Primer3 users: end users, who run Primer3 to pick their primers and programmers, who use Primer3 in their scripts or software packages. We encourage both to use Primer3.
If you are an end user, we request but do not require that use of this software be cited in publications as listed above under CITING PRIMER3.
If you are a programmer, you will see that Primer3 is now distributed under the GNU General Public License, version 2 or (at your option) any later version of the License (GPL2). As we understand it, if you include parts of the Primer3 source code in your source code or link to Primer3 binary libraries in your executable, you have to release your software also under GPL2. If you only call Primer3 from your software and interpret its output, you can use any license you want for your software. If you modify Primer3 and then release your modified software, you have to release your modifications in source code under GPL2 as well.
We chose GPL2 because we wanted Primer3 to evolve and for the improvements to find their way back into the main distribution. If you are programming a new web interface which runs Primer3, please include in the about page of the tool the sentence "<your software name> uses Primer3 version ...". Please consider releasing your software under GPL2 as well, especially if you do not want to maintain it in the future.
There is no need to ask us for permission to include Primer3 in your tools.

## 5. DIFFERENCE BETWEEN PRIMER3 AND PRIMER3PLUS

Primer3Plus is a web interface to Primer3, so if you pick primers with Primer3Plus, it will collect and reformat your input, run the command line tool Primer3, collet and reformat it's output and display it to you.
In principle, both tools would give you the same output. In practice, the default settings ob both tools differ. While Primer3 default settings are usually kept for backward compatibility, the Primer3Plus default settings are adapted for regular wetlab use.

#### Primer3 Values:

      PRIMER_PRODUCT_SIZE_RANGE=100-300
      PRIMER_SECONDARY_STRUCTURE_ALIGNMENT=0
      PRIMER_NUM_RETURN=5
      PRIMER_MAX_HAIRPIN_TH=47.0
      PRIMER_INTERNAL_MAX_HAIRPIN_TH=47.0
      PRIMER_MAX_END_STABILITY=100.0
      PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE=-1
      PRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE=-1
      PRIMER_EXPLAIN_FLAG=0
      PRIMER_LIBERAL_BASE=0
      PRIMER_FIRST_BASE_INDEX=0
      PRIMER_MAX_TEMPLATE_MISPRIMING=-1.00
      PRIMER_MAX_TEMPLATE_MISPRIMING_TH=-1.00
      PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING=-1.00
      PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH=-1.00

#### Primer3Plus Values:

      PRIMER_PRODUCT_SIZE_RANGE=501-600 601-700 401-500 701-850 851-1000 1001-1500
                                1501-3000 3001-5000 401-500 301-400 201-300
                                101-200 5001-7000 7001-10000 10001-20000
      PRIMER_SECONDARY_STRUCTURE_ALIGNMENT=1
      PRIMER_NUM_RETURN=10
      PRIMER_MAX_HAIRPIN_TH=47.00
      PRIMER_INTERNAL_MAX_HAIRPIN_TH=47.00
      PRIMER_MAX_END_STABILITY=9.0
      PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE=3
      PRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE=3
      PRIMER_EXPLAIN_FLAG=1
      PRIMER_LIBERAL_BASE=1
      PRIMER_FIRST_BASE_INDEX=1
      PRIMER_MAX_TEMPLATE_MISPRIMING=12.00
      PRIMER_MAX_TEMPLATE_MISPRIMING_TH=47.00
      PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING=24.00
      PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH=47.00

## 6. CHANGES FROM VERSION 2.2.3

1. The tag PRIMER_THERMODYNAMIC_ALIGNMENT was replaced by two new tags:
- PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT which governs hairpin and oligo-oligo interactions. Therefore, whenever it is set to 1 (the default) thermodynamic alignments will be used for oligo-oligo interactions and hairpins.
- PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT which governs the oligo-template interactions and, when set to 1 (default is 0), will cause Primer3 to use the thermodynamic alignment against templates.
The reason for this change is to provide the option of using the old alignment for oligo-template interactions when the thermodynamic alignment is too slow or the template is too long (there is a hard limit THAL_MAX_SEQ=10k on the length of sequences involved in thermodynamic alignments).
2. We changed the following default values:
2.1. Changing default oligo temperature calculations
PRIMER_TM_FORMULA=1 (was 0)
PRIMER_SALT_CORRECTIONS=1 (was 0)
2.2. Making thermodynamic secondary structure calculations for oligos the default:
PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=1 (was 0)
2.3. The following need reasonable values to support the changes in 2.1 and 2.2 above:
PRIMER_SALT_DIVALENT=1.5 (was 0.0)
PRIMER_DNTP_CONC=0.6 (was 0.0)
2.4. To make old defaults easily accessible, we added a command line argument, --default_version=1, which directs primer3_core to use the old defaults. --default_version=2 directs primer3_core to use the new defaults. The default is --default_version=2.
2.5. IMPORTANT: because PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=1, PRIMER_THERMODYNAMIC_PARAMETERS_PATH must point to the right location. This tag specifies the path to the directory that contains all the parameter files used by the thermodynamic approach. In Linux, there are two *default* locations that are tested if this tag is not defined: ./primer3_config/ and /etc/primer3_config/. For Windows, there is only one default location: .\primer3_config\. If the the parameter files are not in one these locations, be sure to set PRIMER_THERMODYNAMIC_PARAMETERS_PATH.
2.6. Changed default for
PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS=0 (was 1)
(0 is almost always the the behavior one wants.)
2.7. To get the behavior of -default_version=1 when -default_version=2 set the following:
PRIMER_TM_FORMULA=0
PRIMER_SALT_CORRECTIONS=0
PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=0
PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT=0
PRIMER_SALT_DIVALENT=0.0
PRIMER_DNTP_CONC=0.0
PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS=1
2.8. To get the behavior of -default_version=2 when -default_version=1 set the following:
PRIMER_TM_FORMULA=1
PRIMER_SALT_CORRECTIONS=1
PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=1
PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT=0
PRIMER_SALT_DIVALENT=1.5
PRIMER_DNTP_CONC=0.6
PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS=0
3. We changed the NULL value for SEQUENCE_FORCE_{LEFT,RIGHT}_START_END to -1000000, and made it an error to select PRIMER_FIRST_BASE_INDEX <= this value. This is to correct an error when SEQUENCE_FORCE_{LEFT,RIGHT}_START_END was -1 (value previously used to indicate a NULL) but PRIMER_FIRST_BASE_INDEX was < 0, which caused the intended NULL value (-1) to be treated as a constraint on primer location (a constraint that was not possible to satisfy).
4. We changed the PRIMER_TASK called 'pick_detection_primers' to 'generic' while retaining 'pick_detection_primers' as an alias for backward compatibility.
5. The code now uses 'end' alignments when assessing template mispriming using thermodynamic alignments. This is consistent with the approach taken with the previous alignment algorithm and with checking for mispriming against repeat libraries.
6. We removed PRIMER_PAIR_MAX_HAIRPIN_TH (which was ignored previously).
7. Primer3 now requires the user to set SEQUENCE_TARGET, not SEQUENCE_INCLUDED_REGION when PRIMER_TASK=pick_discriminative_primers
8. When PRIMER_TASK=pick_discriminative_primers or PRIMER_TASK=pick_cloning_primers the value of SEQUENCE_INCLUDED_REGION is no longer changed to the entire input sequence.
9. The handling of divalent cations when PRIMER_SALT_CORRECTIONS=2 (not the default and not the recommended value) has been updated. The rationale is that, when divalent cations are present, the formula by Owczarzy et al., 2004 (used previously by Primer3) can be improved upon as described in (Ahsen et al., 2010; Owczarzy et al., 2008). Therefore we have updated the melting temperature calculation to follow the scheme in Figure 9 of (Owczarzy et al., 2008). Please find references to these papers below in the manual.
10. We modified the error handling of non-memory related errors that can occur during the thermodynamic alignment. They are no longer fatal errors.
11. We removed io_version 3, which was only kept for backward compatibility with very old versions of Primer3.
12. In addition, several error corrections:
12.1. Corrected a short, fixed-size buffer for the file/path names specified by the command line arguments -output, -error and for the settings file.
12.2. Two other corrected errors were in p3_set_gs_primer_self_end and p3_set_gs_primer_internal_oligo_self_end, which erroneously multiplied their 'val' arguments by 100.
12.3. Primer3 now detects and handles the situation in which user-supplied primers (SEQUENCE_PRIMER and SEQUENCE_PRIMER_REVCOMP) have the left primer to the right of the right primer. Also issue a warning if user-supplied primers occur in more than one location in the template.

## 7. INSTALLATION INSTRUCTIONS - UNIX/LINUX

Unzip and untar the distribution. DO NOT do this on a PC if you are going to compile and/or test on a different operation system. Primer3_core will not compile and various tests will fail if pc newlines get inserted into the code and test files. Instead, move the distribution (primer3-<release>.tar.gz) to Unix/Linux, and then
$ unzip primer3-<release>.tar.gz
$ tar xvf primer3-<release>.tar
$ cd primer3-<release>/src
If you do not use gcc, then you will probably have to modify the makefile to use your (ANSI) C compiler with the compile and link flags it understands.
$ make all
# You should have created executables primer3_core, ntdpal,
# olgotm, and long_seq_tm_test
$ make test
# You should not see 'FAILED' during the tests.
If your perl command is not called perl (for example, if it is called perl5) you will have to modify the Makefile in the test/ directory.
ntdpal (NucleoTide Dynamic Programming ALignment) is a stand-alone program that provides Primer3's alignment functionality (local, a.k.a. Smith-Waterman, global, a.k.a. Needleman-Wunsch, plus "half global"). It is provided strictly as is; for further documentation please see the code.

## 8. INSTALLATION INSTRUCTIONS - Mac OSX

How to install this software
============================
1. Double click on the .tar.gz file to extract the archive.
2. The binary files are located in the 'bin' [for 'binary'] folder
3. (Optional) To run the tests, cd to the new directory and then the test folder
4. (Optional) Within this folder run: a. 'perl -w p3test.pl'
5. (Optional) You should not see 'FAILED' during the tests.
6. (Optional) *NOTE*: If your perl command is not called perl (for example, if it is called perl5) you will have to modify the internals of the test scripts).
7. Copy the following files to a location of your choice:
a. bin/long_seq_tm_test
b. bin/ntdpal
c. bin/oligotm
d. bin/primer3_core
8. (Optional) Make sure this location is within your $PATH (see below)
Where to put the binary files
=============================
A good place to put these is within ~/bin/ (this means in your home folder, within a folder named `bin` [for 'binary']).
You can also just drag the 'bin' folder to a location within your home directory.
You can certainly also copy the files within 'bin' to /usr/local/bin (if you are an administrator) or another similar location.
You may need to adjust the permissions on the binaries if you get fancy.
Add the location to your $PATH
==============================
This is an optional step, but it will allow you to run Primer3 in any directory on your machine as your user just by typing its name (primer3_core).
*** You should be very careful when altering your $PATH as things can go very wrong. See below for an alternate method. ***
If you added the binaries to /usr/local/bin, then you do not need to do this.
If you added the binaries to a local directory (let's say ~/bin/), do the following:
1. Edit your ~/.bash_profile. You can edit this on the command line (Terminal) with:
nano ~/.bash_profile
2. Add the following line if it is not present (replacing '~/bin' if you used another directory):
PATH=$PATH:~/bin/
3. If a PATH line *is* present, ensure you add a colon to the end of what is there and then the directory, so if you have something like:
a) PATH=$PATH:/usr/local/genome/bin:/sw/bin
make it look like:
b) PATH=$PATH:/usr/local/genome/bin:/sw/bin:~/bin
4. Quit and restart terminal for the changes to take effect.
If you don't add the location to your $PATH
===========================================
Assuming you don't want to modify your $PATH, you can still run the binaries. Let's assume you put the files in '~/bin/. You may run primer3_core by doing either of the following:
1. ~/bin/primer3_core < yourInputFile
2. /Users/<your username>/bin/primer3_core < yourInputFile
The first option is just a shortcut to the second.

## 9. INSTALLATION INSTRUCTIONS - WINDOWS

Functionality warning
=====================
The windows version does not support the masker functionality.

How to install this software
============================
Get your copy of the source code
--------------------------------
1. Install Git for Windows from
https://git-scm.com/download/win
2. Open Git for Windows and run
git clone https://github.com/primer3-org/primer3.git
cd primer3
3. Use git commands to obtain tagged version
git tag
git checkout tags/v2.3.7
or 1. Download and unpack latest from
https://github.com/primer3-org/primer3/releases
Compile Primer3
---------------
1. Download and install TDM-GCC MinGW Compiler from
https://sourceforge.net/projects/tdm-gcc/ 2. Open the windows command line and change to the src folder in Primer3
3. Run: mingw32-make TESTOPTS=--windows
Running the tests
=================
You must install a perl distribution to run the windows tests.
We *strongly* recommend you install ActiveState perl (http://www.activestate.com/products/activeperl/) as this was used to test our Primer3 builds, and it is known to work.
1. Click on 'Start > Run...'
2. Type 'cmd' into the space provided
3. Hit enter (or select 'OK')
4. Navigate to the location of the tests:
A. if you put it in
C:/Documents and Settings/YourName/primer3-<release>/test/,
you would type
'cd c:/Documents and Settings/YourName/primer3-<release>/test/'
B. you can also type 'cd ' (don't forget the space after cd) and drag the primer3-<release> folder onto the command-line window from windows explorer, this will fill in the location for you
5. On the command line, run 'mingw32-make TESTOPTS=--windows' in this directory. You can also run the tests individually, for example 'perl p3test.pl --windows'.
6. You should see [OK] for the majority of the tests. The masker functionality is not supported on Windows and coresponding tests will fail.
Running the software
====================
To run the program, you must do so from the MS-DOS command-line. The intricacies of the DOS command line are beyond the scope of this document. Google for more information, if needed. Here is a quick summary:
1. Click on 'Start > Run...'
2. Type 'cmd' into the space provided
3. Hit enter (or select 'OK')
4. Navigate to the location of the binary:
A. if you put it in
C:/Documents and Settings/YourName/Temp,
you would type
'cd c:/Documents and Settings/YourName/Temp'
B. you can also type 'cd ' (don't forget the space after cd) and drag the Primer3 folder onto the command-line window from windows explorer, this will fill in the location for you
5. Run the example file by typing:
primer3_core.exe < example
Other files may be run in a similar fashion. If your input filename is 'MyData.txt' you can run Primer3 using this file (in the correct format; see README) with:
primer3_core.exe < MyData.txt
If your file is not in the folder containing primer3_core.exe, you could run the program from the primer3_core folder using:
primer3_core.exe < c:/someOtherFolder/someOtherFolder/MyData.txt
Finally, if you want to run the program without going to its folder, assuming primer3_core.exe is in c:/Temp, you could run:
c:/Temp/primer3_core.exe < c:/someOtherFolder/someOtherFolder/MyData.txt

## 10. SYSTEM REQUIREMENTS

Please see https://github.com/primer3-org/primer3 for up-to-date information. Primer3 should compile on any system using with an ANSI C compiler like Linux/Unix, MacOS 10 or MS Windows. The Makefile will probably need to be modified for compilation with C compilers other than gcc.

## 11. INVOKING primer3_core

By default, the executable program produced by the Makefile is called primer3_core. This is the C program that does the heavy lifting of primer picking. There are also two, more user-friendly, web interfaces (distributed separately).
The command line for Primer3 is:
primer3_core [ --format_output ] [ --default_version=1|--default_version=2 ] [ --io_version=4 ] [ --p3_settings_file=<file_path> ] [ --echo_settings_file ] [ --strict_tags ] [ --output=<file_path> ] [ --error=<file_path> ] [ input_file ]
A complete list of valid command line arguments can be found in COMMAND LINE ARGUMENTS below.
If no input file is specified, primer3_core will read its input from stdin.

## 12. COMMAND LINE ARGUMENTS

This section presents the list of arguments that may given in the command line. Any unique abbreviation of the arguments is allowed (e.g. -ab instead of -about) and each argument can be preceded by either one or two dashes. In the case of arguments that receive values, the '=' can be replaced by a space.

### --about

With this argument Primer3 generates one line of output indicating the release number and then exits. This allows scripts to query Primer3 for its version.

### --default_version=n

n=2 is the default, and directs primer3_core to use the latest default values (the ones documented here). n=1 directs primer3_core to use defaults from version 2.2.3 and before.

### --format_output

This argument indicates that primer3_core should generate user-oriented (rather than program-oriented) output.

### --strict_tags

This argument indicates that primer3_core should generate a fatal error if there is any tag in the input that it does not recognize. This tag also applies to the settings file (see documentation for the --p3_settings_file argument), if any, but with limitations: lines that do not begin with PRIMER_ or P3_FILE_ID are always ignored in the settings file.

### --p3_settings_file=file_path

This argument specifies a settings file, file_path. Primer3 reads the global ("PRIMER_...") parameters from this file first. Tags appearing in the settings file override default Primer3 settings. The values set in the settings files can be also overridden by tags in the input file. See Primer3 file documentation for details on the file format. WARNING: the --strict_tags flag applies only to tags beginning with the string "PRIMER_"; lines that do not begin with the string "PRIMER_" are ignored.

### --echo_settings_file

This argument indicates that primer3_core should print the input tags specified in the given settings file. If no settings file is provided or if the --format_output option is given, this argument will have no effect.

### --io_version=n

This argument is provided for backward compatibility. --io_version=4 is the only legal value and the default .

### --output=file_path

This argument specifies the file where the output should be written. If omitted, stdout is used.

### --error=file_path

This argument specifies the file where the error messages should be written. If omitted, stderr is used.

## 13. INPUT AND OUTPUT CONVENTIONS

By default, Primer3 accepts input in Boulder-IO format, a pre-XML, pre-RDF, text-based input/output format for program-to-program data interchange. By default, Primer3 also produces output in the same format.
When run with the --format_output command-line flag, Primer3 prints a more user-oriented report for each sequence.
Primer3 exits with 0 status if it operates correctly. See EXIT STATUS CODES below for additional information.
The syntax of the version of Boulder-IO recognized by Primer3 is as follows:

      o Input consists of a sequence of RECORDs.
      o A RECORD consists of a sequence of (TAG,VALUE) pairs, each terminated
        by a newline character (\n). A RECORD is terminated by  '='
        appearing by itself on a line.
      o A (TAG,VALUE) pair has the following requirements:
        o the TAG must be immediately (without spaces)
              followed by '='.

An example of a legal (TAG,VALUE) pair is
SEQUENCE_ID=my_marker
and an example of a Boulder-IO record is
SEQUENCE_ID=test1
SEQUENCE_TEMPLATE=GACTGATCGATGCTAGCTACGATCGATCGATGCATGCTAGCTAGCTAGCTGCTAGC
=
Many records can be sent, one after another. Below is an example of three different records which might be passed through a Boulder-IO stream:
SEQUENCE_ID=test1
SEQUENCE_TEMPLATE=GACTGATCGATGCTAGCTACGATCGATCGATGCATGCTAGCTAGCTAGCTGCTAGC
=
SEQUENCE_ID=test2
SEQUENCE_TEMPLATE=CATCATCATCATCGATGCTAGCATCNNACGTACGANCANATGCATCGATCGT
=
SEQUENCE_ID=test3
SEQUENCE_TEMPLATE=NACGTAGCTAGCATGCACNACTCGACNACGATGCACNACAGCTGCATCGATGC
=
Primer3 reads Boulder-IO on stdin and echos its input and returns results in Boulder-IO format on stdout. Primer3 indicates many user-correctable errors by a value in the output tag PRIMER_ERROR (see below). Primer3 indicates other errors, including system configuration errors, resource errors (such out-of-memory errors), and detected programming errors by a message on stderr and a non-zero exit status.
This document lists the input tags that Primer3 recognizes. Primer3 echos and ignores any tags it does not recognize, unless the --strict_tags flag is set on the command line, in which case Primer3 prints an error in the PRIMER_ERROR output tag (see below), and prints additional information on stdout; this option can be useful for debugging systems that incorporate Primer3.
Except for tags with the type "interval list" each tag should only appear ONCE in any given input record. This restriction is not systematically checked, and if a tag appears more than once, the new value silently over-writes the previous value.

## 14. AN EXAMPLE

One might be interested in performing PCR on an STS with a CA repeat in the middle of it. Primers need to be chosen based on the criteria of the experiment.
We need to create a Boulder-IO record to send to Primer3 via stdin. There are lots of ways to accomplish this. We could save the record into a text file called 'example', and then type the Unix command 'primer3_core < example'.
Let's look at the input record itself:
SEQUENCE_ID=example
SEQUENCE_TEMPLATE=GTAGTCAGTAGACNATGACNACTGACGATGCAGACNACACACACACACACAGCACACAGGTATTAGTGGGCCATTCGATCCCGACCCAAATCGATAGCTACGATGACG
SEQUENCE_TARGET=37,21
PRIMER_TASK=generic
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_INTERNAL_OLIGO=1
PRIMER_PICK_RIGHT_PRIMER=1
PRIMER_OPT_SIZE=18
PRIMER_MIN_SIZE=15
PRIMER_MAX_SIZE=21
PRIMER_MAX_NS_ACCEPTED=1
PRIMER_PRODUCT_SIZE_RANGE=75-100
P3_FILE_FLAG=1
SEQUENCE_INTERNAL_EXCLUDED_REGION=37,21
PRIMER_EXPLAIN_FLAG=1
=
A breakdown of the reasoning behind each of the TAG=VALUE pairs is below:
SEQUENCE_ID=example
The main intent of this tag is to provide an identifier for the sequence that is meaningful to the user, for example when Primer3 processes multiple records, and by default this tag is optional. However, this tag is _required_ when P3_FILE_FLAG is non-0 Because it provides the names of the files that contain lists of oligos that Primer3 considered.
SEQUENCE_TEMPLATE=GTAGTCAGTAGACNATGACNACTGACGATGCAGACNACACACACACACACAGCACACAGGTATTAGTGGGCCATTCGATCCCGACCCAAATCGATAGCTACGATGACG
The SEQUENCE_TEMPLATE provides the sequence from which Primer3 will design primers. Note that there is no newline until the sequence terminates completely.
SEQUENCE_TARGET=37,21
There is a simple sequence repeat in this sequence. The repeat starts at base 37, and has a length of 21 bases. We instruct Primer3 to choose primers that flank the repeat site (because we want to use the PCR product for determining the length of the repeat, which is likely to be polymorphic).
PRIMER_TASK=generic
The PRIMER_TASK tells Primer3 which type of primers to pick. You can select typical primers for PCR detection, primers for cloning or for sequencing.
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_INTERNAL_OLIGO=1
PRIMER_PICK_RIGHT_PRIMER=1
We would like to pick a left primer, an internal oligo and a right primer, so we set these flags to 1 (true). In combination with the PRIMER_TASK this tags control which primers are picked.
PRIMER_OPT_SIZE=18
Since our sequence length is rather small (only 108 bases long), we lower the PRIMER_OPT_SIZE from 20 to 18. It is more likely that Primer3 will succeed if it aims for smaller primers.
PRIMER_MIN_SIZE=15
PRIMER_MAX_SIZE=21
With the lowering of optimal primer size, it's good to lower the minimum and maximum sizes as well.
PRIMER_MAX_NS_ACCEPTED=1
Since the sequence is short with a non-negligible amount of unknown bases (N's) in it, we make Primer3's job easier by allowing it to pick primers that have at most 1 unknown base.
PRIMER_PRODUCT_SIZE_RANGE=75-100
We reduce the product size range from the default of 100-300 because our source sequence is only 108 base pairs long. If we insisted on a product size of 100 base pairs Primer3 would have few possibilities to choose from.
P3_FILE_FLAG=1
Because we have a short sequence with many Ns and with a simple sequence repeat that we must amplify, Primer3 might fail to pick and primers. Therefore, we want to get the list of primers it considered, so that we could manually pick primers ourselves if Primer3 fails to do so. Setting this flag to 1 asks Primer3 to output the primers it considered to a forwar primers and reverse primers to output files.
SEQUENCE_INTERNAL_EXCLUDED_REGION=37,21
Normally CA-repeats make poor hybridization probes (because they not specific enough). Therefore, we exclude the CA repeat (which is the TARGET) from consideration for the internal oligo.
PRIMER_EXPLAIN_FLAG=1
We want to see statistics about the oligos and oligo triples (left primer, internal oligo, right primer) that Primer3 examined.
=
The '=' character terminates the record.
There were many Boulder-IO input tags that were not specified, which is legal. For the tags with default values, those defaults will be used in the analysis. For the tags with NO default values (for example SEQUENCE_TARGET), the functionality requested by the those tags will be absent. Also, please note that it is not the case that we need to surround a simple sequence repeat every time we want to pick primers!

## 15. HOW TO MIGRATE TAGS TO IO VERSION 4

With Primer3 release 2.0, many Boulder-IO tags were modified and new tags were introduced. The new Primer3 tags are designed with the idea in mind that computer scripts and other programs use primer3_core. The modifications make it easier for programs to read and write Primer3 input and output.
Furthermore the Primer3 default values and the use of PRIMER_WT_TEMPLATE_MISPRIMING and PRIMER_PAIR_WT_TEMPLATE_MISPRIMING have changed in version 2.0.
There are three classes of input: "Sequence" input tags describe a particular input sequence to Primer3, and are reset after every Boulder record (now starting with SEQUENCE_). "Global" input tags describe the general parameters that Primer3 should use in its searches, and the values of these tags persist between input Boulder records until or unless they are explicitly reset (now starting with PRIMER_). "Program" parameters that deal with the behavior of the Primer3 program itself (now starting with P3_). See below for a list of the modified tags.
The handling of PRIMER_TASK changed completely. In the past we used it to tell Primer3 what task to perform. Now the task is complemented with PRIMER_PICK_RIGHT_PRIMER, PRIMER_PICK_INTERNAL_OLIGO and PRIMER_PICK_LEFT_PRIMER, which specify which primers are to be picked.
These Tags are modified:
The "per sequence" tags:

    NEW VERSION                       - OLD VERSION
    ----------------------------------------------------------------------------------------
    SEQUENCE_ID                       - PRIMER_SEQUENCE_ID
    SEQUENCE_TEMPLATE                 - SEQUENCE
    SEQUENCE_QUALITY                  - PRIMER_SEQUENCE_QUALITY
    SEQUENCE_INCLUDED_REGION          - INCLUDED_REGION
    SEQUENCE_TARGET                   - TARGET
    SEQUENCE_EXCLUDED_REGION          - EXCLUDED_REGION
    SEQUENCE_START_CODON_POSITION     - PRIMER_START_CODON_POSITION
    SEQUENCE_PRIMER                   - PRIMER_LEFT_INPUT
    SEQUENCE_PRIMER_REVCOMP           - PRIMER_RIGHT_INPUT
    SEQUENCE_INTERNAL_OLIGO           - PRIMER_INTERNAL_OLIGO_INPUT
    SEQUENCE_INTERNAL_EXCLUDED_REGION - PRIMER_INTERNAL_OLIGO_EXCLUDED_REGION
    --------------------------------------------------------------------------------
    The "global" tags:
    NEW VERSION                       - OLD VERSION
    PRIMER_TASK                       - PRIMER_TASK (modified use)
    PRIMER_PICK_RIGHT_PRIMER          - --- did not exist
    PRIMER_PICK_INTERNAL_OLIGO        - PRIMER_PICK_INTERNAL_OLIGO (modified use)
    PRIMER_PICK_LEFT_PRIMER           - --- did not exist
    PRIMER_PAIR_WT_TEMPLATE_MISPRIMING- PRIMER_PAIR_WT_TEMPLATE_MISPRIMING (modified use)
    PRIMER_WT_TEMPLATE_MISPRIMING     - PRIMER_WT_TEMPLATE_MISPRIMING (modified use)
    PRIMER_MAX_LIBRARY_MISPRIMING     - PRIMER_MAX_MISPRIMING
    PRIMER_INTERNAL_MAX_LIBRARY_MISHYB- PRIMER_INTERNAL_OLIGO_MAX_MISHYB
    PRIMER_PAIR_MAX_LIBRARY_MISPRIMING- PRIMER_PAIR_MAX_MISPRIMING
    PRIMER_WT_LIBRARY_MISPRIMING      - PRIMER_WT_REP_SIM
    PRIMER_INTERNAL_WT_LIBRARY_MISHYB - PRIMER_INTERNAL_WT_REP_SIM
    PRIMER_PAIR_WT_LIBRARY_MISPRIMING - PRIMER_PAIR_WT_REP_SIM
    PRIMER_MAX_NS_ACCEPTED            - PRIMER_NUM_NS_ACCEPTED
    PRIMER_PAIR_MAX_DIFF_TM           - PRIMER_MAX_DIFF_TM
    PRIMER_SALT_MONOVALENT            - PRIMER_SALT_CONC
    PRIMER_SALT_DIVALENT              - PRIMER_DIVALENT_CONC
    PRIMER_TM_FORMULA                 - PRIMER_TM_SANTALUCIA
    PRIMER_MAX_SELF_ANY               - PRIMER_SELF_ANY
    PRIMER_MAX_SELF_END               - PRIMER_SELF_END
    PRIMER_WT_SELF_ANY                - PRIMER_WT_COMPL_ANY
    PRIMER_WT_SELF_END                - PRIMER_WT_COMPL_END
    PRIMER_PAIR_MAX_COMPL_ANY         - PRIMER_PAIR_ANY
    PRIMER_PAIR_MAX_COMPL_END         - PRIMER_PAIR_END
    P3_FILE_FLAG                      - PRIMER_FILE_FLAG
    P3_COMMENT                        - PRIMER_COMMENT
    PRIMER_INTERNAL_SALT_MONOVALENT   - PRIMER_INTERNAL_OLIGO_SALT_CONC
    PRIMER_INTERNAL_SALT_DIVALENT     - PRIMER_INTERNAL_OLIGO_DIVALENT_CONC
    PRIMER_INTERNAL_WT_SELF_ANY       - PRIMER_IO_WT_COMPL_ANY
    PRIMER_INTERNAL_WT_SELF_END       - PRIMER_IO_WT_COMPL_END
    PRIMER_INTERNAL_MAX_NS_ACCEPTED   - PRIMER_INTERNAL_OLIGO_NUM_NS
    PRIMER_INTERNAL_MAX_SELF_ANY      - PRIMER_INTERNAL_OLIGO_SELF_ANY
    PRIMER_INTERNAL_MAX_SELF_END      - PRIMER_INTERNAL_OLIGO_SELF_END
    The following tags INTERNAL_OLIGO is replaced by INTERNAL:
    PRIMER_INTERNAL_OPT_SIZE          - PRIMER_INTERNAL_OLIGO_OPT_SIZE
    PRIMER_INTERNAL_MIN_SIZE          - PRIMER_INTERNAL_OLIGO_MIN_SIZE
    PRIMER_INTERNAL_MAX_SIZE          - PRIMER_INTERNAL_OLIGO_MAX_SIZE
    PRIMER_INTERNAL_OPT_TM            - PRIMER_INTERNAL_OLIGO_OPT_TM
    PRIMER_INTERNAL_MIN_TM            - PRIMER_INTERNAL_OLIGO_MIN_TM
    PRIMER_INTERNAL_MAX_TM            - PRIMER_INTERNAL_OLIGO_MAX_TM
    PRIMER_INTERNAL_MIN_GC            - PRIMER_INTERNAL_OLIGO_MIN_GC
    PRIMER_INTERNAL_OPT_GC_PERCENT    - PRIMER_INTERNAL_OLIGO_OPT_GC_PERCENT
    PRIMER_INTERNAL_MAX_GC            - PRIMER_INTERNAL_OLIGO_MAX_GC
    PRIMER_INTERNAL_DNTP_CONC         - PRIMER_INTERNAL_OLIGO_DNTP_CONC
    PRIMER_INTERNAL_DNA_CONC          - PRIMER_INTERNAL_OLIGO_DNA_CONC
    PRIMER_INTERNAL_MAX_POLY_X        - PRIMER_INTERNAL_OLIGO_MAX_POLY_X
    PRIMER_INTERNAL_MISHYB_LIBRARY    - PRIMER_INTERNAL_OLIGO_MISHYB_LIBRARY
    PRIMER_INTERNAL_MIN_QUALITY       - PRIMER_INTERNAL_OLIGO_MIN_QUALITY
    The following tags IO is replaced by INTERNAL:
    PRIMER_INTERNAL_WT_TM_GT          - PRIMER_IO_WT_TM_GT
    PRIMER_INTERNAL_WT_TM_LT          - PRIMER_IO_WT_TM_LT
    PRIMER_INTERNAL_WT_SIZE_LT        - PRIMER_IO_WT_SIZE_LT
    PRIMER_INTERNAL_WT_SIZE_GT        - PRIMER_IO_WT_SIZE_GT
    PRIMER_INTERNAL_WT_GC_PERCENT_LT  - PRIMER_IO_WT_GC_PERCENT_LT
    PRIMER_INTERNAL_WT_GC_PERCENT_GT  - PRIMER_IO_WT_GC_PERCENT_GT
    PRIMER_INTERNAL_WT_NUM_NS         - PRIMER_IO_WT_NUM_NS
    PRIMER_INTERNAL_WT_SEQ_QUAL       - PRIMER_IO_WT_SEQ_QUAL

--------------------------------------------------------------------------------
OUTPUT TAGS:
There are three big changes on the output:
- INTERNAL_OLIGO is now replaced by INTERNAL.
- The first version is numbered 0.
- The "PRODUCT" tags are renamed
- The errors are modified
- Errors caused by a specific primer are given as PRIMER_LEFT_4_PROBLEMS
Now all primer related output follows the rule: PRIMER_{LEFT,RIGHT,INTERNAL,PAIR}_<j>_<tag_name>. where <j> is an integer from 0 to n, where n is at most the value of PRIMER_NUM_RETURN - 1.
This allows easy scripting by using the underscores _ to split the name. The first part is PRIMER, the second the type of oligo or pair parameters, the third is always a number, starting at 0 and the rest is used by the tags.
That affects also (shown for output number 4):

    NEW VERSION                             - OLD VERSION
    ------------------------------------------------------------------------------------------------------
    PRIMER_PAIR_4_PENALTY                   - PRIMER_PAIR_PENALTY_4 (number moved behind PAIR)
    PRIMER_PAIR_4_PRODUCT_SIZE              - PRIMER_PRODUCT_SIZE_4 (grouped with PAIR)
    PRIMER_PAIR_4_PRODUCT_TM                - PRIMER_PRODUCT_TM_4 (grouped with PAIR)
    PRIMER_PAIR_4_PRODUCT_TM_OLIGO_TM_DIFF  - PRIMER_PRODUCT_TM_OLIGO_TM_DIFF_4 (grouped with PAIR)
    PRIMER_INTERNAL_EXPLAIN                 - PRIMER_INTERNAL_OLIGO_EXPLAIN
    PRIMER_LEFT_4_LIBRARY_MISPRIMING                   - PRIMER_LEFT_4_MISPRIMING_SCORE
    PRIMER_INTERNAL_4_LIBRARY_MISHYB                   - PRIMER_INTERNAL_OLIGO_4_MISHYB_SCORE
    PRIMER_RIGHT_4_LIBRARY_MISPRIMING                  - PRIMER_RIGHT_4_MISPRIMING_SCORE
    PRIMER_PAIR_4_LIBRARY_MISPRIMING                   - PRIMER_PAIR_4_MISPRIMING_SCORE

## 16. "SEQUENCE" INPUT TAGS

"Sequence" input tags start with SEQUENCE_... and describe a particular input sequence to Primer3. They are reset after every Boulder record. Errors in "Sequence" input tags invalidate the current record, but Primer3 will continue to process additional records.

SEQUENCE_EXCLUDED_REGION | SEQUENCE_INTERNAL_EXCLUDED_REGION | SEQUENCE_PRIMER_PAIR_OK_REGION_LIST
SEQUENCE_FORCE_LEFT_END | SEQUENCE_INTERNAL_OLIGO | SEQUENCE_PRIMER_REVCOMP
SEQUENCE_FORCE_LEFT_START | SEQUENCE_INTERNAL_OVERLAP_JUNCTION_LIST | SEQUENCE_QUALITY
SEQUENCE_FORCE_RIGHT_END | SEQUENCE_OVERHANG_LEFT | SEQUENCE_START_CODON_POSITION
SEQUENCE_FORCE_RIGHT_START | SEQUENCE_OVERHANG_RIGHT | SEQUENCE_START_CODON_SEQUENCE
SEQUENCE_ID | SEQUENCE_OVERLAP_JUNCTION_LIST | SEQUENCE_TARGET
SEQUENCE_INCLUDED_REGION | SEQUENCE_PRIMER | SEQUENCE_TEMPLATE

### SEQUENCE_ID (string; default empty)

Short description of the sequence. It is used as an identifier that is reproduced in the output to enable users to identify the source of the chosen primers.
This tag must be present if P3_FILE_FLAG is non-zero.

### SEQUENCE_TEMPLATE (nucleotide sequence; default empty)

The sequence from which to choose primers. The sequence must be presented 5' -> 3' (i.e, in the normal way). In general, the bases may be upper or lower case, but lower case letters are treated specially if PRIMER_LOWERCASE_MASKING is set. The entire sequence MUST be all on a single line. (In other words, the sequence cannot span several lines.)

### SEQUENCE_INCLUDED_REGION (interval list; default empty)

A sub-region of the given sequence in which to pick primers. For example, often the first dozen or so bases of a sequence are vector, and should be excluded from consideration. The value for this parameter has the form

    <start>,<length>

where <start> is the index of the first base to consider, and <length> is the number of subsequent bases in the primer-picking region.

### SEQUENCE_TARGET (interval list; default empty)

If one or more targets is specified then a legal primer pair must flank at least one of them. A target might be a simple sequence repeat site (for example a CA repeat) or a single-base-pair polymorphism, or an exon for resequencing. The value should be a space-separated list of

    <start>,<length>

pairs where <start> is the index of the first base of a target, and <length> is its length.

See also PRIMER_INSIDE_PENALTY, PRIMER_OUTSIDE_PENALTY. Has a different meaning when PRIMER_TASK=pick_sequencing_primers. See PRIMER_TASK for more information.

### SEQUENCE_EXCLUDED_REGION (interval list; default empty)

Left and Right primers and oligos may not overlap any region specified in this tag. The middle oligo may overlap, they may be limited by SEQUENCE_INTERNAL_EXCLUDED_REGION. The associated value must be a space-separated list of

    <start>,<length>

pairs where <start> is the index of the first base of the excluded region, and <length> is its length. This tag is useful for tasks such as excluding regions of low sequence quality or for excluding regions containing repetitive elements such as ALUs or LINEs.

### SEQUENCE_PRIMER_PAIR_OK_REGION_LIST (semicolon separated list of integer quadruples; default empty)

This tag allows detailed specification of possible locations of left and right primers in primer pairs.
The associated value must be a semicolon-separated list of

    <left_start>,<left_length>,<right_start>,<right_length>

quadruples. The left primer must be in the region specified by <left_start>,<left_length> and the right primer must be in the region specified by <right_start>,<right_length>. <left_start> and <left_length> specify the location of the left primer in terms of the index of the first base in the region and the length of the region. <right_start> and <right_length> specify the location of the right primer in analogous fashion. As seen in the example below, if no integers are specified for a region then the location of the corresponding primer is not constrained.
Example:

    SEQUENCE_PRIMER_PAIR_OK_REGION_LIST=100,50,300,50 ; 900,60,, ; ,,930,100

Specifies that there are three choices:
Left primer in the 50 bp region starting at position 100 AND right primer in the 50 bp region starting at position 300
OR
Left primer in the 60 bp region starting at position 900 (and right primer anywhere)
OR
Right primer in the 100 bp region starting at position 930 (and left primer anywhere)

### SEQUENCE_OVERLAP_JUNCTION_LIST (space separated integers; default empty)

If this list is not empty, then the forward OR the reverse primer must overlap one of these junction points by at least PRIMER_MIN_3_PRIME_OVERLAP_OF_JUNCTION nucleotides at the 3' end and at least PRIMER_MIN_5_PRIME_OVERLAP_OF_JUNCTION nucleotides at the 5' end.
In more detail: The junction associated with a given position is the space immediately to the right of that position in the template sequence on the strand given as input.
For example:

    SEQUENCE_OVERLAP_JUNCTION_LIST=20
    # 1-based indexes
    PRIMER_MIN_3_PRIME_OVERLAP_OF_JUNCTION=4
    template: atcataggccatgcctgagc^gctacgact
              ok           ...gagc^gcta-3'  (left primer)
              not ok       ...gagc^gct-3'   (left primer)
              ok           3'-ctcg^cgat...  (right pimer)
              not ok        3'-tcg^cgat...  (right primer)
    PRIMER_MIN_5_PRIME_OVERLAP_OF_JUNCTION=5
             ok           5'-tgagc^gccg...  (left primer)
             not ok        5'-gagc^gccg...  (left primer)
             ok           ...tgagc^gctac-5' (right primer)
             not ok       ...tgagc^gcta-5'  (right primer)

### SEQUENCE_INTERNAL_OVERLAP_JUNCTION_LIST (space separated integers; default empty)

If this list is not empty, then the internal (middle) oligo must overlap one of these junction points by at least PRIMER_INTERNAL_MIN_3_PRIME_OVERLAP_OF_JUNCTION nucleotides at the 3' (right) end and at least PRIMER_INTERNAL_MIN_5_PRIME_OVERLAP_OF_JUNCTION nucleotides at the 5' (left) end.
See SEQUENCE_OVERLAP_JUNCTION_LIST for more detail.

### SEQUENCE_INTERNAL_EXCLUDED_REGION (interval list; default empty)

Middle oligos may not overlap any region specified by this tag. Left and right primers may overlap. The associated value must be a space-separated list of

    <start>,<length>

pairs, where <start> is the index of the first base of an excluded region, and <length> is its length. Often one would make Target regions excluded regions for internal oligos.

### SEQUENCE_PRIMER (nucleotide sequence; default empty)

The sequence of a left primer to check and around which to design right primers and optional internal oligos. Must be a substring of SEQUENCE_TEMPLATE.

### SEQUENCE_INTERNAL_OLIGO (nucleotide sequence; default empty)

The sequence of an internal oligo to check and around which to design left and right primers. Must be a substring of SEQUENCE_TEMPLATE.

### SEQUENCE_PRIMER_REVCOMP (nucleotide sequence; default empty)

The sequence of a right primer to check and around which to design left primers and optional internal oligos. Must be a substring of the reverse strand of SEQUENCE_TEMPLATE.

### SEQUENCE_OVERHANG_LEFT (string; default empty)

The provided sequence is added to the 5' end of the left primer. The overhang sequences are utilized in calculating SELF_ANY, SELF_END, HAIRPIN, COMPL_ANY, COMPL_END, plus the _TH and _STRUCT versions of those outputs, as well as PRODUCT_SIZE. Internal oligos may not have an overhang.
The length of SEQUENCE_OVERHANG_LEFT and SEQUENCE_OVERHANG_RIGHT do not add to the binding product size of PRIMER_PRODUCT_SIZE_RANGE or PRIMER_PRODUCT_OPT_SIZE.
The TM anG GC_PERCENT calculations will only be based on the 3' portion of the oligo that binds to the template.
The following elements include SEQUENCE_OVERHANG_LEFT: PRIMER_LEFT_4_SELF_ANY, PRIMER_LEFT_4_SELF_ANY_TH, PRIMER_LEFT_4_SELF_ANY_STUCT, PRIMER_LEFT_4_SELF_END, PRIMER_LEFT_4_SELF_END_TH, PRIMER_LEFT_4_SELF_END_STUCT, PRIMER_LEFT_4_HAIRPIN_TH, PRIMER_LEFT_4_HAIRPIN_STUCT, PRIMER_PAIR_4_COMPL_ANY, PRIMER_PAIR_4_COMPL_ANY_TH, PRIMER_PAIR_4_COMPL_ANY_STUCT, PRIMER_PAIR_4_COMPL_END, PRIMER_PAIR_4_COMPL_END_TH, PRIMER_PAIR_4_COMPL_END_STUCT, PRIMER_PAIR_4_PRODUCT_SIZE.

### SEQUENCE_OVERHANG_RIGHT (string; default empty)

The provided sequence is added to the 5' end of the right primer. It is reverse complementary to the template sequence.
See SEQUENCE_OVERHANG_LEFT for more details.

### SEQUENCE_START_CODON_POSITION (int; default -2000000)

This parameter should be considered EXPERIMENTAL at this point. Please check the output carefully; some erroneous inputs might cause an error in Primer3.
Index of the first base of a start codon. This parameter allows Primer3 to select primer pairs to create in-frame amplicons e.g. to create a template for a fusion protein. Primer3 will attempt to select an in-frame left primer, ideally starting at or to the left of the start codon, or to the right if necessary. Negative values of this parameter are legal if the actual start codon is to the left of available sequence. If this parameter is non-negative Primer3 signals an error if the codon at the position specified by this parameter is not an ATG. A value less than or equal to -10^6 indicates that Primer3 should ignore this parameter.
Primer3 selects the position of the right primer by scanning right from the left primer for a stop codon. Ideally the right primer will end at or after the stop codon.

### SEQUENCE_START_CODON_SEQUENCE (string; default ATG)

The sequence of the start codon, by default ATG. Some bacteria use different start codons, this tag allows to specify alternative start codons.
Any triplet can be provided as start codon.

### SEQUENCE_QUALITY (space separated integers; default empty)

A list of space separated integers. There must be exactly one integer for each base in SEQUENCE_TEMPLATE if this argument is non-empty. For example, for the sequence ANNTTCA... SEQUENCE_QUALITY might be 45 10 0 50 30 34 50 67 .... High numbers indicate high confidence in the base called at that position and low numbers indicate low confidence in the base call at that position. This parameter is only relevant if you are using a base calling program that provides quality information (for example phred).

### SEQUENCE_FORCE_LEFT_START (int; default -1000000)

Forces the 5' end of the left primer to be at the indicated position. Primers are also picked if they violate certain constraints. The default value indicates that the start of the left primer can be anywhere.

### SEQUENCE_FORCE_LEFT_END (int; default -1000000)

Forces the 3' end of the left primer to be at the indicated position. Primers are also picked if they violate certain constraints. The default value indicates that the end of the left primer can be anywhere.

### SEQUENCE_FORCE_RIGHT_START (int; default -1000000)

Forces the 5' end of the right primer to be at the indicated position. Primers are also picked if they violate certain constraints. The default value indicates that the start of the right primer can be anywhere.

### SEQUENCE_FORCE_RIGHT_END (int; default -1000000)

Forces the 3' end of the right primer to be at the indicated position. Primers are also picked if they violate certain constraints. The default value indicates that the end of the right primer can be anywhere.

## 17. "GLOBAL" INPUT TAGS

"Global" input tags start with PRIMER_... and describe the general parameters that Primer3 should use in its searches. The values of these tags persist between input Boulder records until or unless they are explicitly reset. Errors in "Global" input tags are fatal because they invalidate the basic conditions under which primers are being picked.
Because the laboratory detection step using internal oligos is independent of the PCR amplification procedure, internal oligo tags have defaults that are independent of the parameters that govern the selection of PCR primers. For example, the melting temperature of an oligo used for hybridization might be considerably lower than that used as a PCR primer.

PRIMER_ANNEALING_TEMP | PRIMER_INTERNAL_WT_SIZE_LT | PRIMER_PAIR_WT_COMPL_END
PRIMER_DMSO_CONC | PRIMER_INTERNAL_WT_TM_GT | PRIMER_PAIR_WT_COMPL_END_TH
PRIMER_DMSO_FACTOR | PRIMER_INTERNAL_WT_TM_LT | PRIMER_PAIR_WT_DIFF_TM
PRIMER_DNA_CONC | PRIMER_LIBERAL_BASE | PRIMER_PAIR_WT_IO_PENALTY
PRIMER_DNTP_CONC | PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS | PRIMER_PAIR_WT_LIBRARY_MISPRIMING
PRIMER_EXPLAIN_FLAG | PRIMER_LOWERCASE_MASKING | PRIMER_PAIR_WT_PRODUCT_SIZE_GT
PRIMER_FIRST_BASE_INDEX | PRIMER_MASK_3P_DIRECTION | PRIMER_PAIR_WT_PRODUCT_SIZE_LT
PRIMER_FORMAMIDE_CONC | PRIMER_MASK_5P_DIRECTION | PRIMER_PAIR_WT_PRODUCT_TM_GT
PRIMER_GC_CLAMP | PRIMER_MASK_FAILURE_RATE | PRIMER_PAIR_WT_PRODUCT_TM_LT
PRIMER_INSIDE_PENALTY | PRIMER_MASK_KMERLIST_PATH | PRIMER_PAIR_WT_PR_PENALTY
PRIMER_INTERNAL_DMSO_CONC | PRIMER_MASK_KMERLIST_PREFIX | PRIMER_PAIR_WT_TEMPLATE_MISPRIMING
PRIMER_INTERNAL_DMSO_FACTOR | PRIMER_MASK_TEMPLATE | PRIMER_PAIR_WT_TEMPLATE_MISPRIMING_TH
PRIMER_INTERNAL_DNA_CONC | PRIMER_MAX_BOUND | PRIMER_PICK_ANYWAY
PRIMER_INTERNAL_DNTP_CONC | PRIMER_MAX_END_GC | PRIMER_PICK_INTERNAL_OLIGO
PRIMER_INTERNAL_FORMAMIDE_CONC | PRIMER_MAX_END_STABILITY | PRIMER_PICK_LEFT_PRIMER
PRIMER_INTERNAL_MAX_BOUND | PRIMER_MAX_GC | PRIMER_PICK_RIGHT_PRIMER
PRIMER_INTERNAL_MAX_GC | PRIMER_MAX_HAIRPIN_TH | PRIMER_PRODUCT_MAX_TM
PRIMER_INTERNAL_MAX_HAIRPIN_TH | PRIMER_MAX_LIBRARY_MISPRIMING | PRIMER_PRODUCT_MIN_TM
PRIMER_INTERNAL_MAX_LIBRARY_MISHYB | PRIMER_MAX_NS_ACCEPTED | PRIMER_PRODUCT_OPT_SIZE
PRIMER_INTERNAL_MAX_NS_ACCEPTED | PRIMER_MAX_POLY_X | PRIMER_PRODUCT_OPT_TM
PRIMER_INTERNAL_MAX_POLY_X | PRIMER_MAX_SELF_ANY | PRIMER_PRODUCT_SIZE_RANGE
PRIMER_INTERNAL_MAX_SELF_ANY | PRIMER_MAX_SELF_ANY_TH | PRIMER_QUALITY_RANGE_MAX
PRIMER_INTERNAL_MAX_SELF_ANY_TH | PRIMER_MAX_SELF_END | PRIMER_QUALITY_RANGE_MIN
PRIMER_INTERNAL_MAX_SELF_END | PRIMER_MAX_SELF_END_TH | PRIMER_SALT_CORRECTIONS
PRIMER_INTERNAL_MAX_SELF_END_TH | PRIMER_MAX_SIZE | PRIMER_SALT_DIVALENT
PRIMER_INTERNAL_MAX_SIZE | PRIMER_MAX_TEMPLATE_MISPRIMING | PRIMER_SALT_MONOVALENT
PRIMER_INTERNAL_MAX_TM | PRIMER_MAX_TEMPLATE_MISPRIMING_TH | PRIMER_SECONDARY_STRUCTURE_ALIGNMENT
PRIMER_INTERNAL_MIN_3_PRIME_OVERLAP_OF_JUNCTION | PRIMER_MAX_TM | PRIMER_SEQUENCING_ACCURACY
PRIMER_INTERNAL_MIN_5_PRIME_OVERLAP_OF_JUNCTION | PRIMER_MIN_3_PRIME_OVERLAP_OF_JUNCTION | PRIMER_SEQUENCING_INTERVAL
PRIMER_INTERNAL_MIN_BOUND | PRIMER_MIN_5_PRIME_OVERLAP_OF_JUNCTION | PRIMER_SEQUENCING_LEAD
PRIMER_INTERNAL_MIN_GC | PRIMER_MIN_BOUND | PRIMER_SEQUENCING_SPACING
PRIMER_INTERNAL_MIN_QUALITY | PRIMER_MIN_END_QUALITY | PRIMER_TASK
PRIMER_INTERNAL_MIN_SIZE | PRIMER_MIN_GC | PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT
PRIMER_INTERNAL_MIN_THREE_PRIME_DISTANCE | PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE | PRIMER_THERMODYNAMIC_PARAMETERS_PATH
PRIMER_INTERNAL_MIN_TM | PRIMER_MIN_QUALITY | PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT
PRIMER_INTERNAL_MISHYB_LIBRARY | PRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE | PRIMER_TM_FORMULA
PRIMER_INTERNAL_MUST_MATCH_FIVE_PRIME | PRIMER_MIN_SIZE | PRIMER_WT_BOUND_GT
PRIMER_INTERNAL_MUST_MATCH_THREE_PRIME | PRIMER_MIN_THREE_PRIME_DISTANCE | PRIMER_WT_BOUND_LT
PRIMER_INTERNAL_OPT_BOUND | PRIMER_MIN_TM | PRIMER_WT_END_QUAL
PRIMER_INTERNAL_OPT_GC_PERCENT | PRIMER_MISPRIMING_LIBRARY | PRIMER_WT_END_STABILITY
PRIMER_INTERNAL_OPT_SIZE | PRIMER_MUST_MATCH_FIVE_PRIME | PRIMER_WT_GC_PERCENT_GT
PRIMER_INTERNAL_OPT_TM | PRIMER_MUST_MATCH_THREE_PRIME | PRIMER_WT_GC_PERCENT_LT
PRIMER_INTERNAL_SALT_DIVALENT | PRIMER_NUM_RETURN | PRIMER_WT_HAIRPIN_TH
PRIMER_INTERNAL_SALT_MONOVALENT | PRIMER_OPT_BOUND | PRIMER_WT_LIBRARY_MISPRIMING
PRIMER_INTERNAL_WT_BOUND_GT | PRIMER_OPT_GC_PERCENT | PRIMER_WT_MASK_FAILURE_RATE
PRIMER_INTERNAL_WT_BOUND_LT | PRIMER_OPT_SIZE | PRIMER_WT_NUM_NS
PRIMER_INTERNAL_WT_END_QUAL | PRIMER_OPT_TM | PRIMER_WT_POS_PENALTY
PRIMER_INTERNAL_WT_GC_PERCENT_GT | PRIMER_OUTSIDE_PENALTY | PRIMER_WT_SELF_ANY
PRIMER_INTERNAL_WT_GC_PERCENT_LT | PRIMER_PAIR_MAX_COMPL_ANY | PRIMER_WT_SELF_ANY_TH
PRIMER_INTERNAL_WT_HAIRPIN_TH | PRIMER_PAIR_MAX_COMPL_ANY_TH | PRIMER_WT_SELF_END
PRIMER_INTERNAL_WT_LIBRARY_MISHYB | PRIMER_PAIR_MAX_COMPL_END | PRIMER_WT_SELF_END_TH
PRIMER_INTERNAL_WT_NUM_NS | PRIMER_PAIR_MAX_COMPL_END_TH | PRIMER_WT_SEQ_QUAL
PRIMER_INTERNAL_WT_SELF_ANY | PRIMER_PAIR_MAX_DIFF_TM | PRIMER_WT_SIZE_GT
PRIMER_INTERNAL_WT_SELF_ANY_TH | PRIMER_PAIR_MAX_LIBRARY_MISPRIMING | PRIMER_WT_SIZE_LT
PRIMER_INTERNAL_WT_SELF_END | PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING | PRIMER_WT_TEMPLATE_MISPRIMING
PRIMER_INTERNAL_WT_SELF_END_TH | PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH | PRIMER_WT_TEMPLATE_MISPRIMING_TH
PRIMER_INTERNAL_WT_SEQ_QUAL | PRIMER_PAIR_WT_COMPL_ANY | PRIMER_WT_TM_GT
PRIMER_INTERNAL_WT_SIZE_GT | PRIMER_PAIR_WT_COMPL_ANY_TH | PRIMER_WT_TM_LT

### PRIMER_TASK (string; default generic)

This tag tells Primer3 what task to perform. Legal values are:
generic
Design a primer pair (the classic Primer3 task) if the PRIMER_PICK_LEFT_PRIMER=1, and PRIMER_PICK_RIGHT_PRIMER=1. In addition, pick an internal hybridization oligo if PRIMER_PICK_INTERNAL_OLIGO=1.
NOTE: If PRIMER_PICK_LEFT_PRIMER=1, PRIMER_PICK_RIGHT_PRIMER=0 and PRIMER_PICK_INTERNAL_OLIGO=1, then behaves similarly to PRIMER_TASK=pick_primer_list.
pick_detection_primers
Deprecated alias for PRIMER_TASK=generic
check_primers
Primer3 only checks the primers provided in SEQUENCE_PRIMER, SEQUENCE_INTERNAL_OLIGO and SEQUENCE_PRIMER_REVCOMP. It is the only task that does not require a sequence. However, if SEQUENCE_TEMPLATE is provided, it is used.
pick_primer_list
Pick all primers in SEQUENCE_TEMPLATE (possibly limited by SEQUENCE_INCLUDED_REGION, SEQUENCE_EXCLUDED_REGION, SEQUENCE_PRIMER_PAIR_OK_REGION_LIST, etc.). Returns the primers sorted by quality starting with the best primers. If PRIMER_PICK_LEFT_PRIMER and PRIMER_PICK_RIGHT_PRIMER is selected Primer3 does not to pick primer pairs but generates independent lists of left primers, right primers, and, if requested, internal oligos.
pick_sequencing_primers
Pick primers suited to sequence a region. SEQUENCE_TARGET can be used to indicate several targets. The position of each primer is calculated for optimal sequencing results.
pick_cloning_primers
Pick primers suited to clone a gene were the start nucleotide and the end nucleotide of the PCR fragment must be fixed, for example to clone an ORF. SEQUENCE_INCLUDED_REGION must be used to indicate the first and the last nucleotide. Due to these limitations Primer3 can only vary the length of the primers. Set PRIMER_PICK_ANYWAY=1 to obtain primers even if they violate specific constraints.
pick_discriminative_primers
Pick primers suited to select primers which bind with their end at a specific position. This can be used to force the end of a primer to a polymorphic site, with the goal of discriminating between sequence variants. SEQUENCE_TARGET must be used to provide a single target indicating the last nucleotide of the left (nucleotide before the first nucleotide of the target) and the right primer (nucleotide following the target). The primers border the SEQUENCE_TARGET. Due to these limitations Primer3 can only vary the length of the primers. Set PRIMER_PICK_ANYWAY=1 to obtain primers even if they violate specific constraints.
pick_pcr_primers
Deprecated shortcut for the following settings:
PRIMER_TASK=generic
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_INTERNAL_OLIGO=0
PRIMER_PICK_RIGHT_PRIMER=1
WARNING: this task changes the values of PRIMER_PICK_LEFT_PRIMER, PRIMER_PICK_INTERNAL_OLIGO, and PRIMER_PICK_RIGHT_PRIMER in a way that is not obvious by looking at the input.
pick_pcr_primers_and_hyb_probe
Deprecated shortcut for the following settings:
PRIMER_TASK=generic
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_INTERNAL_OLIGO=1
PRIMER_PICK_RIGHT_PRIMER=1
WARNING: this task changes the values of PRIMER_PICK_LEFT_PRIMER, PRIMER_PICK_INTERNAL_OLIGO, and PRIMER_PICK_RIGHT_PRIMER in a way that is not obvious by looking at the input.
pick_left_only
Deprecated shortcut for the following settings:
PRIMER_TASK=generic
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_INTERNAL_OLIGO=0
PRIMER_PICK_RIGHT_PRIMER=0
WARNING: this task changes the values of PRIMER_PICK_LEFT_PRIMER, PRIMER_PICK_INTERNAL_OLIGO, and PRIMER_PICK_RIGHT_PRIMER in a way that is not obvious by looking at the input.
pick_right_only
Deprecated shortcut for the following settings:
PRIMER_TASK=generic
PRIMER_PICK_LEFT_PRIMER=0
PRIMER_PICK_INTERNAL_OLIGO=0
PRIMER_PICK_RIGHT_PRIMER=1
WARNING: this task changes the values of PRIMER_PICK_LEFT_PRIMER, PRIMER_PICK_INTERNAL_OLIGO, and PRIMER_PICK_RIGHT_PRIMER in a way that is not obvious by looking at the input.
pick_hyb_probe_only
Deprecated shortcut for the following settings:
PRIMER_TASK=generic
PRIMER_PICK_LEFT_PRIMER=0
PRIMER_PICK_INTERNAL_OLIGO=1
PRIMER_PICK_RIGHT_PRIMER=0
WARNING: this task changes the values of PRIMER_PICK_LEFT_PRIMER, PRIMER_PICK_INTERNAL_OLIGO, and PRIMER_PICK_RIGHT_PRIMER in a way that is not obvious by looking at the input.

### PRIMER_PICK_LEFT_PRIMER (boolean; default 1)

If the associated value = 1 (non-0), then Primer3 will attempt to pick left primers.

### PRIMER_PICK_INTERNAL_OLIGO (boolean; default 0)

If the associated value = 1 (non-0), then Primer3 will attempt to pick an internal oligo (hybridization probe to detect the PCR product).

### PRIMER_PICK_RIGHT_PRIMER (boolean; default 1)

If the associated value = 1 (non-0), then Primer3 will attempt to pick a right primer.

### PRIMER_NUM_RETURN (int; default 5)

The maximum number of primer (pairs) to return. Primer pairs returned are sorted by their "quality", in other words by the value of the objective function (where a lower number indicates a better primer pair). Caution: setting this parameter to a large value will increase running time.

### PRIMER_MIN_3_PRIME_OVERLAP_OF_JUNCTION (int; default 4)

The 3' end of the left OR the right primer must overlap one of the junctions in SEQUENCE_OVERLAP_JUNCTION_LIST by this amount. See details in SEQUENCE_OVERLAP_JUNCTION_LIST.

### PRIMER_INTERNAL_MIN_3_PRIME_OVERLAP_OF_JUNCTION (int; default 4)

The 3' end of the middle oligo / probe must overlap one of the junctions in SEQUENCE_INTERNAL_OVERLAP_JUNCTION_LIST by this amount. See details in SEQUENCE_OVERLAP_JUNCTION_LIST.

### PRIMER_MIN_5_PRIME_OVERLAP_OF_JUNCTION (int; default 7)

The 5' end of the left OR the right primer must overlap one of the junctions in SEQUENCE_OVERLAP_JUNCTION_LIST by this amount. See details in SEQUENCE_OVERLAP_JUNCTION_LIST.

### PRIMER_INTERNAL_MIN_5_PRIME_OVERLAP_OF_JUNCTION (int; default 7)

The 5' end of the middle oligo / probe must overlap one of the junctions in SEQUENCE_INTERNAL_OVERLAP_JUNCTION_LIST by this amount. See details in SEQUENCE_OVERLAP_JUNCTION_LIST.

### PRIMER_MUST_MATCH_FIVE_PRIME (ambiguous nucleotide sequence; default empty)

Discards all primers which do not match this match sequence at the 5' end. (New in v. 2.3.6, added by A. Untergasser.)
The match sequence must be 5 nucletides long and can contain the following letters:

        N   Any nucleotide
        A   Adenine
        G   Guanine
        C   Cytosine
        T   Thymine
        R   Purine (A or G)
        Y   Pyrimidine (C or T)
        W   Weak (A or T)
        S   Strong (G or C)
        M   Amino (A or C)
        K   Keto (G or T)
        B   Not A (G or C or T)
        H   Not G (A or C or T)
        D   Not C (A or G or T)
        V   Not T (A or G or C)

Any primer which will not match the entire match sequence at the 5' end will be discarded and not evaluated. Setting strict requirements here will result in low quality primers due to the high numbers of primers discarded at this step.
Example 1:
PRIMER_MUST_MATCH_FIVE_PRIME=tgnnn
Could result in the following matching:

        tgcatgattggatacgtttga
        |||||
        tgnnn
     -> This primer would be used.
        attcgattctccccggtatc
          |||
        tgnnn
     -> This primer would be discarded.

Example 2:
PRIMER_MUST_MATCH_FIVE_PRIME=hnnnn
Could result in the following matching:

        tgcatgattggatacgtttga
        |||||
        hnnnn
     -> This primer would be used.
        ggctgatgaaggaaagcaag
         ||||
        hnnnn
     -> This primer would be discarded.

This parameter would force all primers selected by Primer3 to not have guanosine at the 5' end of any primer which could be useful to avoid quenching of flourochromes.

### PRIMER_INTERNAL_MUST_MATCH_FIVE_PRIME (ambiguous nucleotide sequence; default empty)

Equivalent parameter of PRIMER_MUST_MATCH_FIVE_PRIME for the internal oligo.

### PRIMER_MUST_MATCH_THREE_PRIME (ambiguous nucleotide sequence; default empty)

Discards all primers which do not match this match sequence at the 3' end. Similar parameter to PRIMER_MUST_MATCH_FIVE_PRIME, but limits the 3' end. (New in v. 2.3.6, added by A. Untergasser.)
The match sequence must be 5 nucletides long and can contain the following letters:

        N   Any nucleotide
        A   Adenine
        G   Guanine
        C   Cytosine
        T   Thymine
        R   Purine (A or G)
        Y   Pyrimidine (C or T)
        W   Weak (A or T)
        S   Strong (G or C)
        M   Amino (A or C)
        K   Keto (G or T)
        B   Not A (G or C or T)
        H   Not G (A or C or T)
        D   Not C (A or G or T)
        V   Not T (A or G or C)

Any primer which will not match the entire match sequence at the 3' end will be discarded and not evaluated. Setting strict requirements here will result in low quality primers due to the high numbers of primers discarded at this step.
Example 1:
PRIMER_MUST_MATCH_FIVE_PRIME=nnnga
Could result in the following matching:

        tgcatgattggatacgtttga
                        |||||
                        nnnga
     -> This primer would be used.
        attcgattctccccggtatc
                       |||
                       nnnga
     -> This primer would be discarded.

### PRIMER_INTERNAL_MUST_MATCH_THREE_PRIME (ambiguous nucleotide sequence; default empty)

Equivalent parameter of PRIMER_MUST_MATCH_THREE_PRIME for the internal oligo.

### PRIMER_PRODUCT_SIZE_RANGE (size range list; default 100-300)

The associated values specify the lengths of the product that the user wants the primers to create, and is a space separated list of elements of the form

    <x>-<y>

where an <x>-<y> pair is a legal range of lengths for the product. For example, if one wants PCR products to be between 100 to 150 bases (inclusive) then one would set this parameter to 100-150. If one desires PCR products in either the range from 100 to 150 bases or in the range from 200 to 250 bases then one would set this parameter to 100-150 200-250.
Primer3 favors product-size ranges to the left side of the parameter string. Primer3 will return legal primers pairs in the first range regardless the value of the objective function for pairs in subsequent ranges. Only if there are an insufficient number of primers in the first range will Primer3 return primers in a subsequent range.
For those with primarily a computational background, the PCR product size is the size (in base pairs) of the DNA fragment that would be produced by the PCR reaction on the given sequence template. This would, of course, include the primers themselves.
The length of SEQUENCE_OVERHANG_LEFT and SEQUENCE_OVERHANG_RIGHT do not add to the binding product size of PRIMER_PRODUCT_SIZE_RANGE or PRIMER_PRODUCT_OPT_SIZE.

### PRIMER_PRODUCT_OPT_SIZE (int; default 0)

The optimum size for the PCR product. 0 indicates that there is no optimum product size. This parameter influences primer pair selection only if PRIMER_PAIR_WT_PRODUCT_SIZE_GT or PRIMER_PAIR_WT_PRODUCT_SIZE_LT is non-0.
A non-0 value for this parameter will likely increase calculation time, so set this only if a product size near a specific value is truly important.
The length of SEQUENCE_OVERHANG_LEFT and SEQUENCE_OVERHANG_RIGHT do not add to the binding product size of PRIMER_PRODUCT_SIZE_RANGE or PRIMER_PRODUCT_OPT_SIZE.

### PRIMER_PAIR_WT_PRODUCT_SIZE_LT (float; default 0.0)

Penalty weight for products shorter than PRIMER_PRODUCT_OPT_SIZE.

### PRIMER_PAIR_WT_PRODUCT_SIZE_GT (float; default 0.0)

Penalty weight for products longer than PRIMER_PRODUCT_OPT_SIZE.

### PRIMER_MIN_SIZE (int; default 18)

Minimum acceptable length of a primer. Must be greater than 0 and less than or equal to PRIMER_MAX_SIZE.

### PRIMER_INTERNAL_MIN_SIZE (int; default 18)

Equivalent parameter of PRIMER_MIN_SIZE for the internal oligo.

### PRIMER_OPT_SIZE (int; default 20)

Optimum length (in bases) of a primer. Primer3 will attempt to pick primers close to this length.

### PRIMER_INTERNAL_OPT_SIZE (int; default 20)

Equivalent parameter of PRIMER_OPT_SIZE for the internal oligo.

### PRIMER_MAX_SIZE (int; default 27)

Maximum acceptable length (in bases) of a primer. Currently this parameter cannot be larger than 35. This limit is governed by maximum oligo size for which Primer3's melting-temperature is valid.

### PRIMER_INTERNAL_MAX_SIZE (int; default 27)

Equivalent parameter of PRIMER_MAX_SIZE for the internal oligo.

### PRIMER_WT_SIZE_LT (float; default 1.0)

Penalty weight for primers shorter than PRIMER_OPT_SIZE.

### PRIMER_INTERNAL_WT_SIZE_LT (float; default 1.0)

Equivalent parameter of PRIMER_WT_SIZE_LT for the internal oligo.

### PRIMER_WT_SIZE_GT (float; default 1.0)

Penalty weight for primers longer than PRIMER_OPT_SIZE.

### PRIMER_INTERNAL_WT_SIZE_GT (float; default 1.0)

Equivalent parameter of PRIMER_INTERNAL_WT_SIZE_GT for the internal oligo.

### PRIMER_MIN_GC (float; default 20.0)

Minimum allowable percentage of Gs and Cs in any primer.

### PRIMER_INTERNAL_MIN_GC (float; default 20.0)

Equivalent parameter of PRIMER_MIN_GC for the internal oligo.

### PRIMER_OPT_GC_PERCENT (float; default 50.0)

Optimum GC percent. This parameter influences primer selection only if PRIMER_WT_GC_PERCENT_GT or PRIMER_WT_GC_PERCENT_LT are non-0.

### PRIMER_INTERNAL_OPT_GC_PERCENT (float; default 50.0)

Equivalent parameter of PRIMER_OPT_GC_PERCENT for the internal oligo.

### PRIMER_MAX_GC (float; default 80.0)

Maximum allowable percentage of Gs and Cs in any primer generated by Primer.

### PRIMER_INTERNAL_MAX_GC (float; default 80.0)

Equivalent parameter of PRIMER_MAX_GC for the internal oligo.

### PRIMER_WT_GC_PERCENT_LT (float; default 0.0)

Penalty weight for primers with GC percent lower than PRIMER_OPT_GC_PERCENT.

### PRIMER_INTERNAL_WT_GC_PERCENT_LT (float; default 0.0)

Equivalent parameter of PRIMER_WT_GC_PERCENT_LT for the internal oligo.

### PRIMER_WT_GC_PERCENT_GT (float; default 0.0)

Penalty weight for primers with GC percent higher than PRIMER_OPT_GC_PERCENT.

### PRIMER_INTERNAL_WT_GC_PERCENT_GT (float; default 0.0)

Equivalent parameter of PRIMER_WT_GC_PERCENT_GT for the internal oligo.

### PRIMER_GC_CLAMP (int; default 0)

Require the specified number of consecutive Gs and Cs at the 3' end of both the left and right primer. (This parameter has no effect on the internal oligo if one is requested.)

### PRIMER_MAX_END_GC (int; default 5)

The maximum number of Gs or Cs allowed in the last five 3' bases of a left or right primer.

### PRIMER_MIN_TM (float; default 57.0)

Minimum acceptable melting temperature (Celsius) for a primer oligo.

### PRIMER_INTERNAL_MIN_TM (float; default 57.0)

Equivalent parameter of PRIMER_MIN_TM for the internal oligo.

### PRIMER_OPT_TM (float; default 60.0)

Optimum melting temperature (Celsius) for a primer. Primer3 will try to pick primers with melting temperatures are close to this temperature. The oligo melting temperature formula used can be specified by user. Please see PRIMER_TM_FORMULA for more information.

### PRIMER_INTERNAL_OPT_TM (float; default 60.0)

Equivalent parameter of PRIMER_OPT_TM for the internal oligo.

### PRIMER_MAX_TM (float; default 63.0)

Maximum acceptable melting temperature (Celsius) for a primer oligo.

### PRIMER_INTERNAL_MAX_TM (float; default 63.0)

Equivalent parameter of PRIMER_MAX_TM for the internal oligo.

### PRIMER_PAIR_MAX_DIFF_TM (float; default 100.0)

Maximum acceptable (unsigned) difference between the melting temperatures of the left and right primers.

### PRIMER_WT_TM_LT (float; default 1.0)

Penalty weight for primers with Tm lower than PRIMER_OPT_TM.

### PRIMER_INTERNAL_WT_TM_LT (float; default 1.0)

Equivalent parameter of PRIMER_WT_TM_LT for the internal oligo.

### PRIMER_WT_TM_GT (float; default 1.0)

Penalty weight for primers with Tm over PRIMER_OPT_TM.

### PRIMER_INTERNAL_WT_TM_GT (float; default 1.0)

Equivalent parameter of PRIMER_WT_TM_GT for the internal oligo.

### PRIMER_PAIR_WT_DIFF_TM (float; default 0.0)

Penalty weight for the TM difference between the left primer and the right primer.

### PRIMER_PRODUCT_MIN_TM (float; default -1000000.0)

The minimum allowed melting temperature of the amplicon. Please see the documentation on PRIMER_PRODUCT_MAX_TM for details.

### PRIMER_PRODUCT_OPT_TM (float; default 0.0)

The optimum melting temperature for the PCR product. 0 indicates that there is no optimum temperature.

### PRIMER_PRODUCT_MAX_TM (float; default 1000000.0)

The maximum allowed melting temperature of the amplicon. Primer3 calculates product Tm calculated using the formula from Bolton and McCarthy, PNAS 84:1390 (1962) as presented in Sambrook, Fritsch and Maniatis, Molecular Cloning, p 11.46 (1989, CSHL Press).

       Tm = 81.5 + 16.6(log10([Na+])) + .41*(%GC) - 600/length

Where [Na+] is the molar sodium concentration, (%GC) is the percent of Gs and Cs in the sequence, and length is the length of the sequence.
A similar formula is used by the prime primer selection program in GCG (http://www.gcg.com), which instead uses 675.0 / length in the last term (after F. Baldino, Jr, M.-F. Chesselet, and M.E. Lewis, Methods in Enzymology 168:766 (1989) eqn (1) on page 766 without the mismatch and formamide terms). The formulas here and in Baldino et al. assume Na+ rather than K+. According to J.G. Wetmur, Critical Reviews in BioChem. and Mol. Bio. 26:227 (1991) 50 mM K+ should be equivalent in these formulae to .2 M Na+. Primer3 uses the same salt concentration value for calculating both the primer melting temperature and the oligo melting temperature. If you are planning to use the PCR product for hybridization later this behavior will not give you the Tm under hybridization conditions.

### PRIMER_PAIR_WT_PRODUCT_TM_LT (float; default 0.0)

Penalty weight for products with a Tm lower than PRIMER_PRODUCT_OPT_TM.

### PRIMER_PAIR_WT_PRODUCT_TM_GT (float; default 0.0)

Penalty weight for products with a Tm higher than PRIMER_PRODUCT_OPT_TM.

### PRIMER_TM_FORMULA (int; default 1)

Specifies details of melting temperature calculation. (New in v. 1.1.0, added by Maido Remm and Triinu Koressaar.)
A value of 0 directs Primer3 to a backward compatible calculation (in other words, the only calculation available in previous version of Primer3).
This backward compatible calculation uses the table of thermodynamic parameters in the paper [Breslauer KJ, Frank R, Blöcker H and Marky LA (1986) "Predicting DNA duplex stability from the base sequence" Proc Natl Acad Sci 83:4746-50 http://dx.doi.org/10.1073/pnas.83.11.3746], and the method in the paper [Rychlik W, Spencer WJ and Rhoads RE (1990) "Optimization of the annealing temperature for DNA amplification in vitro", Nucleic Acids Res 18:6409-12 http://dx.doi.org/10.1093/nar/18.21.6409].
A value of 1 (*RECOMMENDED*) directs Primer3 to use the table of thermodynamic values and the method for melting temperature calculation suggested in the paper [SantaLucia JR (1998) "A unified view of polymer, dumbbell and oligonucleotide DNA nearest-neighbor thermodynamics", Proc Natl Acad Sci 95:1460-65 http://dx.doi.org/10.1073/pnas.95.4.1460].
Use tag PRIMER_SALT_CORRECTIONS, to specify the salt correction method for melting temperature calculation.
Example of calculating the melting temperature of an oligo if PRIMER_TM_FORMULA=1 and PRIMER_SALT_CORRECTIONS=1 recommended values):
primer=CGTGACGTGACGGACT
Using default salt and DNA concentrations we have

    Tm = deltaH/(deltaS + R*ln(C/4))

where R is the gas constant (1.987 cal/K mol) and C is the DNA concentration.

    deltaH(predicted) =
      = dH(CG) + dH(GT) + dH(TG) + .. + dH(CT) +
         + dH(init.w.term.GC) + dH(init.w.term.AT) =
      = -10.6 + (-8.4) + (-8.5) + .. + (-7.8) + 0.1 + 2.3  =
      = -128.8 kcal/mol

where 'init.w.term GC' and 'init.w.term AT' are two initiation parameters for duplex formation: 'initiation with terminal GC' and 'initiation with terminal AT'

    deltaS(predicted) =
      = dS(CG) + dS(GT) + dS(TG) + .. + dS(CT) +
        + dS(init.w.term.GC) + dS(init.w.term.AT) =
      = -27.2 + (-22.4) + (-22.7) + .. + (-21.0) + (-2.8) + 4.1 =
      = -345.2 cal/k*mol
    deltaS(salt corrected) =
      = deltaS(predicted) + 0.368*15(NN pairs)*ln(0.05M monovalent cations) =
      = -361.736
    Tm = -128.800/(-361.736+1.987*ln((5*10^(-8))/4)) =
       = 323.704 K
    Tm(C) = 323.704 - 273.15 = 50.554 C

### PRIMER_SALT_MONOVALENT (float; default 50.0)

The millimolar (mM) concentration of monovalent salt cations (usually KCl) in the PCR. Primer3 uses this argument to calculate oligo and primer melting temperatures. Use tag PRIMER_SALT_DIVALENT and PRIMER_INTERNAL_SALT_DIVALENT to specify the concentrations of divalent cations (in which case you also should also set tag PRIMER_DNTP_CONC to a reasonable value).

### PRIMER_INTERNAL_SALT_MONOVALENT (float; default 50.0)

Equivalent parameter of PRIMER_SALT_MONOVALENT for the internal oligo.

### PRIMER_SALT_DIVALENT (float; default 1.5)

The millimolar concentration of divalent salt cations (usually MgCl^(2+)) in the PCR. (New in v. 1.1.0, added by Maido Remm and Triinu Koressaar)
Primer3 converts concentration of divalent cations to concentration of monovalent cations using formula suggested in the paper [Ahsen von N, Wittwer CT, Schutz E (2001) "Oligonucleotide Melting Temperatures under PCR Conditions: Nearest-Neighbor Corrections for Mg^(2+), Deoxynucleotide Triphosphate, and Dimethyl Sulfoxide Concentrations with Comparison to Alternative Empirical Formulas", Clinical Chemistry 47:1956-61 http://www.clinchem.org/cgi/content/full/47/11/1956].

    [Monovalent cations] = [Monovalent cations] + 120*(([divalent cations] - [dNTP])^0.5)

In addition, if the specified concentration of dNTPs (PRIMER_DNTP_CONC) is larger than the concentration of divalent cations (PRIMER_SALT_DIVALENT) then the effect of the divalent cations is not considered. The concentration of dNTPs is considered in the formula above because of some magnesium is bound by the dNTP. The adjusted concentration of monovalent cations is used in the calculation of oligo/primer melting temperature, PCR product melting temperature, the stability of oligo dimers and secondary structures (when PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT is 1), and the stability of ectopic annealing of oligos to template (when PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT is 1). If PRIMER_SALT_DIVALENT > 0.0, be sure to set tag PRIMER_DNTP_CONC to specify the concentration of dNTPs.

### PRIMER_INTERNAL_SALT_DIVALENT (float; default 0.0)

Equivalent parameter of PRIMER_SALT_DIVALENT for the internal oligo.

### PRIMER_DNTP_CONC (float; default 0.6)

The millimolar concentration of the sum of all deoxyribonucleotide triphosphates. A reaction mix containing 0.2 mM ATP, 0.2 mM CTP, 0.2 mM GTP and 0.2 mM TTP would have a PRIMER_DNTP_CONC=0.8. This argument is considered for oligo and primer melting temperatures, for PCR product melting temperature, or for secondary structure calculations only if PRIMER_SALT_DIVALENT is > 0.0. See PRIMER_SALT_DIVALENT.

### PRIMER_INTERNAL_DNTP_CONC (float; default 0.0)

Parameter for internal oligos analogous to PRIMER_DNTP_CONC.

### PRIMER_SALT_CORRECTIONS (int; default 1)

Specifies the salt correction formula for the melting temperature calculation. (New in v. 1.1.0, added by Maido Remm and Triinu Koressaar)
A value of 0 directs Primer3 to use the the salt correction formula in the paper [Schildkraut, C, and Lifson, S (1965) "Dependence of the melting temperature of DNA on salt concentration", Biopolymers 3:195-208 (not available on-line)]. This was the formula used in older versions of Primer3.
A value of 1 (*RECOMMENDED*) directs Primer3 to use the salt correction formula in the paper [SantaLucia JR (1998) "A unified view of polymer, dumbbell and oligonucleotide DNA nearest-neighbor thermodynamics", Proc Natl Acad Sci 95:1460-65 http://dx.doi.org/10.1073/pnas.95.4.1460]
A value of 2 directs Primer3 to use the salt correction formula in the paper [Owczarzy, R., Moreira, B.G., You, Y., Behlke, M.A., and Walder, J.A. (2008). Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations. Biochemistry 47, 5336-5353 http://dx.doi.org/10.1021/bi702363u] following recommendations in the paper [Ahsen, v.N., Wittwer, C.T., and Schütz, E. (2010). Monovalent and divalent salt correction algorithms for Tm prediction-recommendations for Primer3 usage. Brief Bioinform 12, 514 http://dx.doi.org/10.1093/bib/bbq081].
For all values of PRIMER_SALT_CORRECTIONS, Primer3 also considers the values of the tags PRIMER_SALT_DIVALENT, PRIMER_INTERNAL_SALT_DIVALENT, PRIMER_DNTP_CONC, and PRIMER_INTERNAL_DNTP_CONC.

### PRIMER_DNA_CONC (float; default 50.0)

A value to use as nanomolar (nM) concentration of each annealing oligo over the course the PCR. Primer3 uses this argument to esimate oligo melting temperatures. This parameter corresponds to 'c' in equation (ii) of the paper [SantaLucia (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci 95:1460-1465 http://www.pnas.org/content/95/4/1460.full.pdf+html], where a suitable value (for a lower initial concentration of template) is "empirically determined".
The default (50nM) works well with the standard protocol used at the Whitehead/MIT Center for Genome Research--0.5 microliters of 20 micromolar concentration for each primer in a 20 microliter reaction with 10 nanograms template, 0.025 units/microliter Taq polymerase in 0.1 mM each dNTP, 1.5mM MgCl2, 50mM KCl, 10mM Tris-HCL (pH 9.3) using 35 cycles with an annealing temperature of 56 degrees Celsius.
The value of this parameter is less than the actual concentration of oligos in the initial reaction mix because it is the concentration of annealing oligos, which in turn depends on the amount of template (including PCR product) in a given cycle. This concentration increases a great deal during a PCR; fortunately PCR seems quite robust for a variety of oligo melting temperatures.
See ADVICE FOR PICKING PRIMERS.

### PRIMER_INTERNAL_DNA_CONC (float; default 50.0)

Equivalent parameter of PRIMER_DNA_CONC for the internal oligo.

### PRIMER_DMSO_CONC (float; default 0.0)

The concentration of DMSO in percent. See PRIMER_DMSO_FACTOR for details of Tm correction.

### PRIMER_INTERNAL_DMSO_CONC (float; default 0.0)

Equivalent parameter of PRIMER_DMSO_CONC for the internal oligo.

### PRIMER_DMSO_FACTOR (float; default 0.6)

The melting temperature of primers can be approximately corrected for DMSO:
Tm = Tm (without DMSO) - PRIMER_DMSO_FACTOR * PRIMER_DMSO_CONC
The PRIMER_DMSO_CONC concentration must be given in %. By default the PRIMER_DMSO_FACTOR for correction is 0.6 as suggested by Musielski et al (H Musielski, W Mann, R Laue and S Michel. Z allg Microbiol, 21:447–456, 1981). Ahsen et al. propose a factor of 0.75 (N von Ahsen, C T Wittwer and E Schutz. Clinical Chemistry, 47:1956–1961, 2001), Cullen et al. a factor of 0.5 (B Cullen and M Bick. Nucleic acids research, 3:49–62, 1976) and Escara et al. a factor of 0.675 (J Escara and J Hutton. Biopolymers, 19:1315–1327, 1980).

### PRIMER_INTERNAL_DMSO_FACTOR (float; default 0.6)

Equivalent parameter of PRIMER_DMSO_FACTOR for the internal oligo.

### PRIMER_FORMAMIDE_CONC (float; default 0.0)

The concentration od DMSO in mol/l. The melting temperature of primers can be approximately corrected for formamide:
Tm = Tm (without formamide) +(0.453 * PRIMER_[LEFT/INTERNAL/RIGHT]_4_GC_PERCENT / 100 - 2.88) * PRIMER_FORMAMIDE_CONC
The PRIMER_FORMAMIDE_CONC correction was suggested by Blake and Delcourt (R D Blake and S G Delcourt. Nucleic Acids Research, 24, 11:2095–2103, 1996).
Convert % into mol/l:
[DMSO in mol/l] = [DMSO in % weight] * 10 / 45.04 g/mol
[DMSO in mol/l] = [DMSO in % volume] * 10 * 1.13 g/cm3 / 45.04 g/mol
Casey, Davidson and Hutton suggest an alternative formula (N Casey and J Davidson. Nucleic acids research, 4:1539–1532, 1977; JR Hutton. Nucleic acids research, 4:3537–3555, 1977):
Tm = Tm (without formamide) - 0.65 * formamide_conc (in %)
To apply this formula in Primer3, PRIMER_FORMAMIDE_CONC could be set to 0.0, PRIMER_DMSO_FACTOR set to 1.0. Then the formamide_conc can be multiplied by 0.65 and added to the PRIMER_DMSO_CONC multiplied by PRIMER_DMSO_FACTOR. The resulting value is given in PRIMER_DMSO_CONC.

### PRIMER_INTERNAL_FORMAMIDE_CONC (float; default 0.0)

Equivalent parameter of PRIMER_FORMAMIDE_CONC for the internal oligo.

### PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT (boolean; default 1)

If the associated value = 1, then Primer3 will use thermodynamic models to calculate the the propensity of oligos to form hairpins and dimers.

### PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT (boolean; default 0)

If the associated value = 1, then Primer3 will use thermodynamic models to calculate the the propensity of oligos to anneal to undesired sites in the template sequence.

### PRIMER_SECONDARY_STRUCTURE_ALIGNMENT (boolean; default 0)

If the associated value = 1, then Primer3 will print out the calculated secondary structures, for example:

#### Dimers:

    t: 7.8  dG: -1724  dH: -91400  dS: -289
      5' ACGCAAAGCACGCTCC-CGATC 3'
          ||   |||  |||   ||
    3' CTAGCCC-TCGCACGAAACGCA 5'

#### Hairpins:

    t: 36.4  dG: 42  dH: -22500  dS: -73
    5' CGCAAAGCACGCT┐
                ||  │
            3' AGCCC┘

The tags PRIMER_LEFT_4_SELF_ANY_STUCT, PRIMER_LEFT_4_SELF_END_STUCT, PRIMER_LEFT_4_HAIRPIN_STUCT (these tags are also present for RIGHT and INTERNAL primers), PRIMER_PAIR_4_COMPL_ANY_STUCT and PRIMER_PAIR_4_COMPL_END_STUCT are only present if a secondary structure could be calculated.
As the string has to fit on one line, the newlines are indicated by the two characters '\' and 'n' and have to be replaced (regex: /\\n/\n/g). Hairpins use unicode characters for the turn. For html they have to be replaced (regex: /U\+25(\d\d)/&#x25$1;/g).

### PRIMER_THERMODYNAMIC_PARAMETERS_PATH (string; default ./primer3_config)

This tag specifies the path to the directory that contains all the parameter files used by the thermodynamic approach. In Linux, there are two default locations that are tested if this tag is not defined: ./primer3_config/ and /etc/primer3_config/. For Windows, there is only one default location: .\primer3_config\.

### PRIMER_ANNEALING_TEMP (float; default -10.0)

The annealing temperature (Celsius) used in the PCR reaction. Usually it is chosen up to 10°C below the melting temperature of the primers. If provided, Primer3 will calculate the fraction of primers bound at the provided annealing temperature for each oligo. By default not active, see "GENERAL THOUGHTS ON PRIMER BINDING" for more details.

### PRIMER_MIN_BOUND (float; default -10.0)

Minimum acceptable fraction of primer bound at PRIMER_ANNEALING_TEMP for a primer oligo in percent. By default not active, see "GENERAL THOUGHTS ON PRIMER BINDING" for more details.

### PRIMER_INTERNAL_MIN_BOUND (float; default -10.0)

Equivalent parameter of PRIMER_MIN_BOUND for the internal oligo.

### PRIMER_OPT_BOUND (float; default 97.0)

Optimum fraction of primer bound at PRIMER_ANNEALING_TEMP for a primer oligo in percent. By default not active, see "GENERAL THOUGHTS ON PRIMER BINDING" for more details.

### PRIMER_INTERNAL_OPT_BOUND (float; default 97.0)

Equivalent parameter of PRIMER_OPT_BOUND for the internal oligo.

### PRIMER_MAX_BOUND (float; default 110.0)

Maximum acceptable fraction of primer bound at PRIMER_ANNEALING_TEMP for a primer oligo in percent. By default not active, see "GENERAL THOUGHTS ON PRIMER BINDING" for more details.

### PRIMER_INTERNAL_MAX_BOUND (float; default 110.0)

Equivalent parameter of PRIMER_MAX_BOUND for the internal oligo.

### PRIMER_WT_BOUND_LT (float; default 0.0)

Penalty weight for primers with a fraction of primer bound lower than PRIMER_OPT_BOUND. By default not active, see "GENERAL THOUGHTS ON PRIMER BINDING" for more details.

### PRIMER_INTERNAL_WT_BOUND_LT (float; default 0.0)

Equivalent parameter of PRIMER_WT_BOUND_LT for the internal oligo.

### PRIMER_WT_BOUND_GT (float; default 0.0)

Penalty weight for primers with a fraction of primer bound over PRIMER_OPT_BOUND. By default not active, see "GENERAL THOUGHTS ON PRIMER BINDING" for more details.

### PRIMER_INTERNAL_WT_BOUND_GT (float; default 0.0)

Equivalent parameter of PRIMER_WT_BOUND_GT for the internal oligo.

### PRIMER_MAX_SELF_ANY (decimal, 9999.99; default 8.00)

PRIMER_MAX_SELF_ANY describes the tendency of a primer to bind to itself (interfering with target sequence binding). It will score ANY binding occurring within the entire primer sequence.
It is the maximum allowable local alignment score when testing a single primer for (local) self-complementarity. Local self-complementarity is taken to predict the tendency of primers to anneal to each other without necessarily causing self-priming in the PCR. The scoring system gives 1.00 for complementary bases, -0.25 for a match of any base (or N) with an N, -1.00 for a mismatch, and -2.00 for a gap. Only single-base-pair gaps are allowed. For example, the alignment

       5' ATCGNA 3'
          || | |
       3' TA-CGT 5'

is allowed (and yields a score of 1.75), but the alignment

       5' ATCCGNA 3'
          ||  | |
       3' TA--CGT 5'

is not considered. Scores are non-negative, and a score of 0.00 indicates that there is no reasonable local alignment between two oligos.

### PRIMER_MAX_SELF_ANY_TH (decimal, 9999,99; default 47.00)

The same as PRIMER_MAX_SELF_ANY but all calculations are based on thermodynamical approach. The melting temperature of the most stable structure is calculated. To calculate secondary structures nearest-neighbor parameters for perfect matches, single internal mismatches, terminal mismatches, dangling ends have been used. Also parameters for increments for length dependence of bulge and internal loops have been used. This parameter is calculated only if PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=1. The default value is 10 degrees lower than the default value of PRIMER_MIN_TM. For example, the alignment width length 15nt

      5' ATTAGATAGAGCATC 3'
      3' TAATCTATCTCGTAG 5'

is allowed (and yields a melting temperature of 32.1493 width by default Primer3 parameters), but the alignment

         T        C
      5'  GCGGCCGC GCGC 3'
      3'  CGCCGGCG CGCG 5'
         A        A

is not considered (Tm=57.0997 and the length of oligo is 14nt). Thermodynamical parameters and methods for finding the most stable structure are described in following papers:

- [SantaLucia JR (1998) "A unified view of polymer, dumbbell and oligonucleotide DNA nearest-neighbor thermodynamics", Proc Natl Acad Sci 95:1460-65 http://dx.doi.org/10.1073/pnas.95.4.1460]
- [SantaLucia JR and Hicks D (2004) "The thermodynamics of DNA structural motifs", Annu Rev Biophys Biomol Struct 33:415-40 http://dx.doi.org/10.1146/annurev.biophys.32.110601.141800]
- [Bommarito S, Peyret N and SantaLucia J Jr (2000) "Thermodynamic parameters for DNA sequences with dangling ends", Nucleic Acids Res 28(9):1929-34 http://dx.doi.org/10.1093/nar/28.9.1929]
- [Peyret N, Seneviratne PA, Allawi HT, SantaLucia J Jr. (1999) "Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches", Biochemistry 38(12):3468-77 http://dx.doi.org/10.1021/bi9825091]
- [Allawi HT and SantaLucia J Jr. (1998) "Nearest-Neighbor Thermodynamics of Internal A·C Mismatches in DNA: Sequence Dependence and pH Effects", Biochemistry 37(26):9435-44 http://dx.doi.org/10.1021/bi9803729
- [Allawi HT and SantaLucia J Jr. (1998) "Thermodynamics of internal C.T mismatches in DNA." Nucleic Acids Res 26(11):2694-701http://dx.doi.org/10.1093/nar/26.11.2694]
- [Allawi HT and SantaLucia J Jr. (1998) "Nearest neighbor thermodynamic parameters for internal G.A mismatches in DNA." Biochemistry 37(8):2170-9 http://dx.doi.org/10.1021/bi9724873]
- [Allawi HT and SantaLucia J Jr. (1997) "Thermodynamics and NMR of internal G.T mismatches in DNA." Biochemistry 36(34):10581-94 http://dx.doi.org/10.1021/bi962590c]
- [SantaLucia J Jr and Peyret N. (2001) "Method and system for predicting nucleic acid hybridization thermodynamics and computer-readable storage medium for use therein" World Intellectual Property Organization, WO 01/94611 http://www.wipo.int/pctdb/en/wo.jsp?wo=2001094611]

Predicting secondary structures can improve primer design by eliminating sequences with high possibility to form alternative secondary structures.

### PRIMER_INTERNAL_MAX_SELF_ANY (decimal, 9999.99; default 12.00)

Equivalent parameter of PRIMER_MAX_SELF_ANY for the internal oligo.

### PRIMER_INTERNAL_MAX_SELF_ANY_TH (decimal, 9999.99; default 47.00)

Equivalent parameter of PRIMER_MAX_SELF_ANY_TH for the internal oligo.

### PRIMER_PAIR_MAX_COMPL_ANY (decimal, 9999.99; default 8.00)

PRIMER_PAIR_MAX_COMPL_ANY describes the tendency of the left primer to bind to the right primer. It is the maximum allowable local alignment score when testing for complementarity between left and right primers. It is similar to PRIMER_MAX_SELF_ANY.

### PRIMER_PAIR_MAX_COMPL_ANY_TH (decimal, 9999.99; default 47.00)

PRIMER_PAIR_MAX_COMPL_ANY_TH describes the tendency of the left primer to bind to the right primer. It is similar to PRIMER_MAX_SELF_ANY_TH.

### PRIMER_WT_SELF_ANY (float; default 0.0)

Penalty weight for the individual primer self binding value as in PRIMER_MAX_SELF_ANY.

### PRIMER_WT_SELF_ANY_TH (float; default 0.0)

Penalty weight for the individual primer self binding value as in PRIMER_MAX_SELF_ANY_TH.

### PRIMER_INTERNAL_WT_SELF_ANY (float; default 0.0)

Equivalent parameter of PRIMER_WT_SELF_ANY for the internal oligo.

### PRIMER_INTERNAL_WT_SELF_ANY_TH (float; default 0.0)

Equivalent parameter of PRIMER_WT_SELF_ANY_TH for the internal oligo.

### PRIMER_PAIR_WT_COMPL_ANY (float; default 0.0)

Penalty weight for the binding value of the primer pair as in PRIMER_MAX_SELF_ANY.

### PRIMER_PAIR_WT_COMPL_ANY_TH (float; default 0.0)

Penalty weight for the binding value of the primer pair as in PRIMER_MAX_SELF_ANY_TH.

### PRIMER_MAX_SELF_END (decimal, 9999.99; default 3.00)

PRIMER_MAX_SELF_END tries to bind the 3'-END to a identical primer and scores the best binding it can find. This is critical for primer quality because it allows primers use itself as a target and amplify a short piece (forming a primer-dimer). These primers are then unable to bind and amplify the target sequence.
PRIMER_MAX_SELF_END is the maximum allowable 3'-anchored global alignment score when testing a single primer for self-complementarity. The 3'-anchored global alignment score is taken to predict the likelihood of PCR-priming primer-dimers, for example

       5' ATGCCCTAGCTTCCGGATG 3'
                    ||| |||||
                 3' AAGTCCTACATTTAGCCTAGT 5'

or

       5` AGGCTATGGGCCTCGCGA 3'
                      ||||||
                   3' AGCGCTCCGGGTATCGGA 5'

The scoring system is as for the Maximum Complementarity argument. In the examples above the scores are 7.00 and 6.00 respectively. Scores are non-negative, and a score of 0.00 indicates that there is no reasonable 3'-anchored global alignment between two oligos. In order to estimate 3'-anchored global alignments for candidate primers, Primer3 assumes that the sequence from which to choose primers is presented 5'->3'. It is nonsensical to provide a larger value for this parameter than for the Maximum (local) Complementarity parameter (PRIMER_MAX_SELF_ANY) because the score of a local alignment will always be at least as great as the score of a global alignment.

### PRIMER_MAX_SELF_END_TH (decimal 9999.99; default 47.00)

Same as PRIMER_MAX_SELF_END but is based on thermodynamical approach - the stability of structure is analyzed. The value of tag is expressed as melting temperature. See PRIMER_MAX_SELF_ANY_TH for details.

### PRIMER_INTERNAL_MAX_SELF_END (decimal 9999.99; default 12.00)

PRIMER_INTERNAL_MAX_SELF_END is meaningless when applied to internal oligos used for hybridization-based detection, since primer-dimer will not occur. We recommend that PRIMER_INTERNAL_MAX_SELF_END be set at least as high as PRIMER_INTERNAL_MAX_SELF_ANY.

### PRIMER_INTERNAL_MAX_SELF_END_TH (decimal 9999.99; default 47.00)

Same as PRIMER_INTERNAL_MAX_SELF_END but for calculating the score (melting temperature of structure) thermodynamical approach is used.

### PRIMER_PAIR_MAX_COMPL_END (decimal, 9999.99; default 3.00)

PRIMER_PAIR_MAX_COMPL_END tries to bind the 3'-END of the left primer to the right primer and scores the best binding it can find. It is similar to PRIMER_MAX_SELF_END.

### PRIMER_PAIR_MAX_COMPL_END_TH (decimal, 9999.99; default 47.00)

Same as PRIMER_PAIR_MAX_COMPL_END but for calculating the score (melting temperature of structure) thermodynamical approach is used.

### PRIMER_WT_SELF_END (float; default 0.0)

Penalty weight for the individual primer self binding value as in PRIMER_MAX_SELF_END.

### PRIMER_WT_SELF_END_TH (float; default 0.0)

Penalty weight for the individual primer self binding value as in PRIMER_MAX_SELF_END_TH

### PRIMER_INTERNAL_WT_SELF_END (float; default 0.0)

Equivalent parameter of PRIMER_WT_SELF_END for the internal oligo.

### PRIMER_INTERNAL_WT_SELF_END_TH (float; default 0.0)

Equivalent parameter of PRIMER_WT_SELF_END_TH for the internal oligo.

### PRIMER_PAIR_WT_COMPL_END (float; default 0.0)

Penalty weight for the binding value of the primer pair as in PRIMER_MAX_SELF_END.

### PRIMER_PAIR_WT_COMPL_END_TH (float; default 0.0)

Penalty weight for the binding value of the primer pair as in PRIMER_MAX_SELF_END_TH.

### PRIMER_MAX_HAIRPIN_TH (float; default 47.0)

This is the most stable monomer structure of internal oligo calculated by thermodynamic approach. The hairpin loops, bulge loops, internal loops, internal single mismatches, dangling ends, terminal mismatches have been considered. This parameter is calculated only if PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=1. The default value is 10 degrees lower than the default value of PRIMER_MIN_TM. For example the structure:

          -///------\\\-
       5' ACGCTGTGCTGCGA 3'

with melting temperature 53.7263 (calculated according to by default values of Primer3) and

          //////----\\\\\\
       5' CCGCAGTAAGCTGCGG 3'

with melting temperature 71.0918 (calculated according to by default values of Primer3) For details about papers used for calculating hairpins see PRIMER_MAX_SELF_ANY_TH

### PRIMER_INTERNAL_MAX_HAIRPIN_TH (float; default 47.0)

The most stable monomer structure of internal oligo calculated by thermodynamic approach. See PRIMER_MAX_HAIRPIN_TH for details.

### PRIMER_WT_HAIRPIN_TH (float; default 0.0)

Penalty weight for the individual primer hairpin structure value as in PRIMER_MAX_HAIRPIN_TH.

### PRIMER_INTERNAL_WT_HAIRPIN_TH (float; default 0.0)

Penalty weight for the most stable primer hairpin structure value as in PRIMER_INTERNAL_MAX_HAIRPIN_TH.

### PRIMER_MAX_END_STABILITY (float, 999.9999; default 100.0)

The maximum stability for the last five 3' bases of a left or right primer. Bigger numbers mean more stable 3' ends. The value is the maximum delta G (kcal/mol) for duplex disruption for the five 3' bases as calculated using the nearest-neighbor parameter values specified by the option of PRIMER_TM_FORMULA
For example if the table of thermodynamic parameters suggested by SantaLucia 1998, DOI:10.1073/pnas.95.4.1460 is used the deltaG values for the most stable and for the most labile 5mer duplex are 6.86 kcal/mol (GCGCG) and 0.86 kcal/mol (TATAT) respectively.
If the table of thermodynamic parameters suggested by Breslauer et al. 1986, 10.1073/pnas.83.11.3746 is used the deltaG values for the most stable and for the most labile 5mer are 13.4 kcal/mol (GCGCG) and 4.6 kcal/mol (TATAC) respectively.

### PRIMER_WT_END_STABILITY (float; default 0.0)

Penalty factor for the calculated maximum stability for the last five 3' bases of a left or right primer.

### PRIMER_MAX_NS_ACCEPTED (int; default 0)

Maximum number of unknown bases (N) allowable in any primer.

### PRIMER_INTERNAL_MAX_NS_ACCEPTED (int; default 0)

Equivalent parameter of PRIMER_MAX_NS_ACCEPTED for the internal oligo.

### PRIMER_WT_NUM_NS (float; default 0.0)

Penalty weight for the number of Ns in the primer.

### PRIMER_INTERNAL_WT_NUM_NS (float; default 0.0)

Equivalent parameter of PRIMER_WT_NUM_NS for the internal oligo.

### PRIMER_MAX_POLY_X (int; default 5)

The maximum allowable length of a mononucleotide repeat, for example AAAAAA, 'GGGNNN' violates MAX_POLY_X=5. It is based on the worst possible case (all 3 Ns could be Gs).

### PRIMER_INTERNAL_MAX_POLY_X (int; default 5)

Equivalent parameter of PRIMER_MAX_POLY_X for the internal oligo.

### PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE (int; default -1)

When returning multiple primer pairs, the minimum number of base pairs between the 3' ends of any two left primers.
Primers with 3' ends at positions e.g. 30 and 31 in the template sequence have a three-prime distance of 1.
In addition to positive values, the values -1 and 0 are acceptable and have special interpretations:
-1 indicates that a given left primer can appear in multiple primer pairs returned by Primer3. This is the default behavior.
0 indicates that a left primer is acceptable if it was not already used. In other words, two left primers are allowed to have the same 3' position provided their 5' positions differ.
For n > 0: A left primer is acceptable if:
NOT(3' end of left primer closer than n to the 3' end of a previously used left primer)

### PRIMER_INTERNAL_MIN_THREE_PRIME_DISTANCE (int; default -1)

Analogous to PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE, but for internal primer / probe.

### PRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE (int; default -1)

Analogous to PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE, but for right primers.

### PRIMER_MIN_THREE_PRIME_DISTANCE (int; default -1)

A "convenience" tag that simultaneously sets PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE and PRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE
For example

    PRIMER_MIN_THREE_PRIME_DISTANCE=3

is equivalent to

    PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE=3
    PRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE=3

It is an error to specify both PRIMER_MIN_THREE_PRIME_DISTANCE and either PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE or PRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE in the same input record.

### PRIMER_PICK_ANYWAY (boolean; default 0)

If true use primer provided in SEQUENCE_PRIMER, SEQUENCE_PRIMER_REVCOMP, or SEQUENCE_INTERNAL_OLIGO even if it violates specific constraints.

### PRIMER_LOWERCASE_MASKING (int; default 0)

This option allows for intelligent design of primers in sequence in which masked regions (for example repeat-masked regions) are lower-cased. (New in v. 1.1.0, added by Maido Remm and Triinu Koressaar)
A value of 1 directs Primer3 to reject primers overlapping lowercase a base exactly at the 3' end.
This property relies on the assumption that masked features (e.g. repeats) can partly overlap primer, but they cannot overlap the 3'-end of the primer. In other words, lowercase bases at other positions in the primer are accepted, assuming that the masked features do not influence the primer performance if they do not overlap the 3'-end of primer.

### PRIMER_EXPLAIN_FLAG (boolean; default 0)

If this flag is 1 (non-0), produce PRIMER_LEFT_EXPLAIN, PRIMER_RIGHT_EXPLAIN, PRIMER_INTERNAL_EXPLAIN and/or PRIMER_PAIR_EXPLAIN output tags as appropriate. These output tags are intended to provide information on the number of oligos and primer pairs that Primer3 examined and counts of the number discarded for various reasons. If -format_output is set similar information is produced in the user-oriented output.

### PRIMER_LIBERAL_BASE (boolean; default 0)

This parameter provides a quick-and-dirty way to get Primer3 to accept IUB / IUPAC codes for ambiguous bases (i.e. by changing all unrecognized bases to N). If you wish to include an ambiguous base in an oligo, you must set PRIMER_MAX_NS_ACCEPTED to a 1 (non-0) value.
Perhaps '-' and '* ' should be squeezed out rather than changed to 'N', but currently they simply get converted to N's. The authors invite user comments.

### PRIMER_FIRST_BASE_INDEX (int; default 0)

This parameter is the index of the first base in the input sequence. For input and output using 1-based indexing (such as that used in GenBank and to which many users are accustomed) set this parameter to 1. For input and output using 0-based indexing set this parameter to 0. (This parameter also affects the indexes in the contents of the files produced when the primer file flag is set.)

### PRIMER_MAX_TEMPLATE_MISPRIMING (decimal, 9999.99; default -1.00)

The maximum allowed similarity to ectopic sites in the template. A negative value means do not check. The scoring system is the same as used for PRIMER_MAX_LIBRARY_MISPRIMING, except that an ambiguity code in the template is never treated as a consensus (see PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS).

### PRIMER_MAX_TEMPLATE_MISPRIMING_TH (decimal, 9999.99; default -1.00)

Similar to PRIMER_MAX_TEMPLATE_MISPRIMING but assesses alternative binding sites in the template using thermodynamic models (when PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT=1). This parameter specifies the maximum allowed melting temperature of an oligo (primer) at an "ectopic" site within the template sequence; 47.0 would be a reasonable choice if PRIMER_MIN_TM is 57.0.

### PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING (decimal, 9999.99; default -1.00)

The maximum allowed summed similarity of both primers to ectopic sites in the template. A negative value means do not check. The scoring system is the same as used for PRIMER_PAIR_MAX_LIBRARY_MISPRIMING, except that an ambiguity code in the template is never treated as a consensus (see PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS). Primer3 does not check the similarity of hybridization oligos (internal oligos) to locations outside of the amplicon.

### PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH (decimal, 9999.99; default -1.00)

The maximum allowed summed melting temperatures of both primers at ectopic sites within the template (with the two primers in an orientation that would allow PCR amplification.) The melting temperatures are calculated as for PRIMER_MAX_TEMPLATE_MISPRIMING_TH.

### PRIMER_WT_TEMPLATE_MISPRIMING (float; default 0.0)

Penalty for a single primer binding to the template sequence.
The use of this Tag is modified from Primer3 version 2.0 on: The values used with the older versions have to be multiplied by the factor 100 to have the same effect.

### PRIMER_WT_TEMPLATE_MISPRIMING_TH (float; default 0.0)

Penalty for a single primer binding to the template sequence (thermodynamic approach, when PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT=1).

### PRIMER_PAIR_WT_TEMPLATE_MISPRIMING (float; default 0.0)

Penalty for a primer pair binding to the template sequence.
The use of this Tag is modified from Primer3 version 2.0 on: The values used with the older versions have to be multiplied by the factor 100 to have the same effect.

### PRIMER_PAIR_WT_TEMPLATE_MISPRIMING_TH (float; default 0.0)

Penalty for a primer pair binding to the template sequence (thermodynamic approach, when PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT=1).

### PRIMER_MISPRIMING_LIBRARY (string; default empty)

The name of a file containing a nucleotide sequence library of sequences to avoid amplifying (for example repetitive sequences, or possibly the sequences of genes in a gene family that should not be amplified.) The file must be in (a slightly restricted) FASTA format (W. B. Pearson and D.J. Lipman, PNAS 85:8 pp 2444-2448 [1988]); we briefly discuss the organization of this file below. If this parameter is specified then Primer3 locally aligns each candidate primer against each library sequence and rejects those primers for which the local alignment score times a specified weight (see below) exceeds PRIMER_MAX_LIBRARY_MISPRIMING. (The maximum value of the weight is arbitrarily set to 100.0.)
Each sequence entry in the FASTA-format file must begin with an "id line" that starts with '>'. The contents of the id line is "slightly restricted" in that Primer3 parses everything after any optional asterisk ('*') as a floating point number to use as the weight mentioned above. If the id line contains no asterisk then the weight defaults to 1.0. The alignment scoring system used is the same as for calculating complementarity among oligos (e.g. PRIMER_MAX_SELF_ANY), except for the handling of IUB/IUPAC ambiguity codes (discussed below).
The remainder of an entry contains the sequence as lines following the id line up until a line starting with '>' or the end of the file. Whitespace and newlines are ignored. Characters 'A', 'T', 'G', 'C', 'a', 't', 'g', 'c' and IUB/IUPAC 'ambiguity' codes ('R, 'Y', 'K', 'M', 'S', 'W', 'N', including lower case) are retained. For technical reasons the length of the sequence must be >= 3. Of course, sequences of length < 10 or so are probably useless, but will be accepted without complaint.
WARNING: always set PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS=0 if any sequence in the library contains strings of 'N's: NNNNNNNNNNNNNNNNNNNN.
There are no restrictions on line length.
An empty value for this parameter indicates that no repeat library should be used and "turns off" the use of a previously specified library.
Repbase (J. Jurka, A.F.A. Smit, C. Pethiyagoda, and others, 1995-1996, ftp://ftp.ncbi.nih.gov/repository/repbase/) is an excellent source of repeat sequences and pointers to the literature. (The Repbase files need to be converted to Fasta format before they can be used by Primer3.)
See providedMisprimingLibs for the sequence libraries available on this server.

### PRIMER_INTERNAL_MISHYB_LIBRARY (string; default empty)

Similar to PRIMER_MISPRIMING_LIBRARY, except that the event we seek to avoid is hybridization of the internal oligo to sequences in this library rather than priming from them.

### PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS (boolean; default 0)

If set to 1, treat ambiguity codes as if they were consensus codes when matching oligos to mispriming or mishyb libraries. For example, if this flag is set, then a C in an oligo will be scored as a perfect match to an S in a library sequence, as will a G in the oligo. More importantly, though, any base in an oligo will be scored as a perfect match to an N in the library. This is very bad if the library contains strings of Ns, as no oligo will be legal (and it will take a long time to find this out). So unless you know for sure that your library does not have runs of Ns (or Xs), then set this flag to 0.

### PRIMER_MAX_LIBRARY_MISPRIMING (decimal, 9999.99; default 12.00)

The maximum allowed weighted similarity with any sequence in PRIMER_MISPRIMING_LIBRARY.

### PRIMER_INTERNAL_MAX_LIBRARY_MISHYB (decimal,9999.99; default 12.00)

Similar to PRIMER_MAX_LIBRARY_MISPRIMING except that this parameter applies to the similarity of candidate internal oligos to the library specified in PRIMER_INTERNAL_MISHYB_LIBRARY.

### PRIMER_PAIR_MAX_LIBRARY_MISPRIMING (decimal, 9999.99; default 24.00)

The maximum allowed sum of similarities of a primer pair (one similarity for each primer) with any single sequence in PRIMER_MISPRIMING_LIBRARY. Library sequence weights are not used in computing the sum of similarities.

### PRIMER_WT_LIBRARY_MISPRIMING (float; default 0.0)

Penalty for a single primer binding to any single sequence in PRIMER_MISPRIMING_LIBRARY.

### PRIMER_INTERNAL_WT_LIBRARY_MISHYB (float; default 0.0)

Equivalent parameter of PRIMER_WT_LIBRARY_MISPRIMING for the internal oligo.

### PRIMER_PAIR_WT_LIBRARY_MISPRIMING (float; default 0.0)

Penalty for a primer pair binding to any single sequence in PRIMER_MISPRIMING_LIBRARY.

### PRIMER_MASK_TEMPLATE (boolean; default 0)

This feature helps to prevent designing primers to template regions that are repetitive. Primers with more binding sites tend to have higher failure rates. The masking is based on statistical model, which calculates the probability of failure Pf as follows:

    Pf= em / (1 + em),

where m = 0.1772 * K11 + 0.239 * K16 - 4.336
and K11 and K16 are frequencies of 11-mers and 16-mers in given genome. The frequencies are stored in species-specific k-mer list files. Users can build their own k-mer lists for species of interest. GenomeTester4 software for making properly formatted k-mer lists can be downloaded from GitHub: https://github.com/bioinfo-ut/GenomeTester4.

### PRIMER_MASK_FAILURE_RATE (float; default 0.1)

Cutoff value of accepted failure rate for masking algorithm. Higher value gives lower stringency, meaning that fewer nucleotides in target sequence is masked.

### PRIMER_WT_MASK_FAILURE_RATE (float; default 0.0)

Penalty weight for the primer failure rate.

### PRIMER_MASK_5P_DIRECTION (int; default 1)

The number of nucleotides masking algorithm should mask from 5' direction.

### PRIMER_MASK_3P_DIRECTION (int; default 0)

The number of nucleotides masking algorithm should mask from 3' direction.

### PRIMER_MASK_KMERLIST_PATH (string; default ../kmer_lists/)

This tag specifies the path to the directory that contains k-mer list files for masking algorithm. Required for command-line execution. On web interface the species is selected from drop-down menu

### PRIMER_MASK_KMERLIST_PREFIX (string; default homo_sapiens)

This tag specifies the species whose k-mer lists are used for pre-masking.

### PRIMER_MIN_QUALITY (int; default 0)

The minimum sequence quality (as specified by SEQUENCE_QUALITY) allowed within a primer.

### PRIMER_INTERNAL_MIN_QUALITY (int; default 0)

Equivalent parameter of PRIMER_MIN_QUALITY for the internal oligo.

### PRIMER_MIN_END_QUALITY (int; default 0)

The minimum sequence quality (as specified by SEQUENCE_QUALITY) allowed within the 5' pentamer of a primer. Note that there is no PRIMER_INTERNAL_MIN_END_QUALITY.

### PRIMER_QUALITY_RANGE_MIN (int; default 0)

The minimum legal sequence quality (used for error checking of PRIMER_MIN_QUALITY and PRIMER_MIN_END_QUALITY).

### PRIMER_QUALITY_RANGE_MAX (int; default 100)

The maximum legal sequence quality (used for error checking of PRIMER_MIN_QUALITY and PRIMER_MIN_END_QUALITY).

### PRIMER_WT_SEQ_QUAL (float; default 0.0)

Penalty weight for the sequence quality of the primer.

### PRIMER_INTERNAL_WT_SEQ_QUAL (float; default 0.0)

Equivalent parameter of PRIMER_WT_SEQ_QUAL for the internal oligo.

### PRIMER_PAIR_WT_PR_PENALTY (float; default 1.0)

Penalty factor for the sum of the left and the right primer added to the pair penalty. Setting this value below 1.0 will increase running time.
As PRIMER_PAIR_WT_PR_PENALTY or the per-primer penalties it multiplies become lower with respect to various pair penalties (for example PRIMER_PAIR_WT_PRODUCT_SIZE_LT PRIMER_PAIR_WT_PRODUCT_SIZE_GT PRIMER_PAIR_WT_DIFF_TM, etc.) the running time of the search for primer pairs is likely to grow substantially. The reason is that the search algorithm must calculate the penalty for more primer pairs (as opposed to excluding them based on the penalties of the individual oligos).

### PRIMER_PAIR_WT_IO_PENALTY (float; default 0.0)

Penalty factor for the internal oligo added to the pair penalty.

### PRIMER_INSIDE_PENALTY (float; default -1.0)

Non-default values are valid only for sequences with 0 or 1 target regions. If the primer is part of a pair that spans a target and overlaps the target, then multiply this value times the number of nucleotide positions by which the primer overlaps the (unique) target to get the 'position penalty'. The effect of this parameter is to allow Primer3 to include overlap with the target as a term in the objective function.

### PRIMER_OUTSIDE_PENALTY (float; default 0.0)

Non-default values are valid only for sequences with 0 or 1 target regions. If the primer is part of a pair that spans a target and does not overlap the target, then multiply this value times the number of nucleotide positions from the 3' end to the (unique) target to get the 'position penalty'. The effect of this parameter is to allow Primer3 to include nearness to the target as a term in the objective function.

### PRIMER_WT_POS_PENALTY (float; default 1.0)

Penalty for the primer which do not overlap the target.

### PRIMER_SEQUENCING_LEAD (int; default 50)

Defines the space from the 3'end of the primer to the point were the trace signals are readable. Value only used if PRIMER_TASK=pick_sequencing_primers.

### PRIMER_SEQUENCING_SPACING (int; default 500)

Defines the space from the 3'end of the primer to the 3'end of the next primer on the same strand. Value only used if PRIMER_TASK=pick_sequencing_primers.

### PRIMER_SEQUENCING_INTERVAL (int; default 250)

Defines the space from the 3'end of the primer to the 3'end of the next primer on the reverse strand. Value only used if PRIMER_TASK=pick_sequencing_primers.

### PRIMER_SEQUENCING_ACCURACY (int; default 20)

Defines the space from the calculated position of the 3'end to both sides in which Primer3Plus picks the best primer. Value only used if PRIMER_TASK=pick_sequencing_primers.

### PRIMER_WT_END_QUAL (float; default 0.0)

### PRIMER_INTERNAL_WT_END_QUAL (float; default 0.0)

## 18. "PROGRAM" INPUT TAGS

"Program" input tags start with P3_... describe the parameters that deal with the behavior of the Primer3 program itself.)

### P3_FILE_ID (string; default empty)

This tag is only valid in Primer3 setting files. It should be used to identify the purpose of the settings files it appears in. It is always printed out on the output of Primer3. See also also the command line flag -echo_settings_file, which causes the entire settings file to be echoed in primer3_core's output.

### P3_FILE_TYPE (string; default sequence)

This tag is only valid in the header of Primer3 setting files. It should be used in the second line to identify the type files it appears in. Valid options are sequence, settings or all. In Primer3Plus it can control which parameters are loaded.

### P3_FILE_FLAG (boolean; default 0)

If the associated value = 1 (non-0), then Primer3 creates one or more output files for each input SEQUENCE_TEMPLATE. File <sequence_id>.for lists all acceptable left primers for <sequence_id>, and <sequence_id>.rev lists all acceptable right primers for <sequence_id>, where <sequence_id> is the value of the SEQUENCE_ID tag (which must be supplied). If internal oligos are requested, Primer3 produces a file <sequence_id>.int, which lists all acceptable internal oligos. Obviously, <sequence_id> needs to be a string that will work as a file name.
See also the `pick_primer_list` argument to PRIMER_TASK, which offers similar functionality on stdout.

### P3_COMMENT (string; default empty)

The value of this tag is ignored. It can be used to annotate input.

## 19. HOW PRIMER3 CALCULATES THE PENALTY VALUE

In essense, the penalty values define what is the best primer pair. The calculation of penalty values takes into consideration penalty weights, which allow one to fine-tune the selection of primers to specific needs.
This section will explain the selection process of primers by Primer3. In general the selection is a multi step process:
In the first step, Primer3 evaluates every primer that can be picked in the region of interest, possibly subject to constraints due to target regions, product size ranges, and so forth, that might preclude the use of primers in the eventually selected primer pairs. In this pass the hard limits are tested like PRIMER_MAX_GC or PRIMER_MIN_TM. Primers with a GC lower than PRIMER_MAX_GC or a Tm higher than PRIMER_MIN_TM are memorized, the primers which fail in one of these tests are excluded. Primer3 can be forced to use primers failing to pass this test by setting PRIMER_PICK_ANYWAY to one (only available for primers provided by the user).
In the second step, Primer3 calculates a penalty for each primer. This penalty is the only score by which Primer3 evaluates the primers It is also provided as output PRIMER_LEFT_4_PENALTY, PRIMER_INTERNAL_4_PENALTY and PRIMER_RIGHT_4_PENALTY (shown for the primer set 4). For each primer, it is calculated like that:

### Left Primers:

    PRIMER_LEFT_4_PENALTY =
       If PRIMER_LEFT_4_TM > PRIMER_OPT_TM then this is added (+):
           + PRIMER_WT_TM_GT * ( PRIMER_LEFT_4_TM - PRIMER_OPT_TM )
       If PRIMER_LEFT_4_TM < PRIMER_OPT_TM then this is added (+):
           + PRIMER_WT_TM_LT * ( PRIMER_OPT_TM - PRIMER_LEFT_4_TM )
       If PRIMER_LEFT_4_BOUND > PRIMER_OPT_BOUND then this is added (+):
           + PRIMER_WT_BOUND_GT * ( PRIMER_LEFT_4_BOUND - PRIMER_OPT_BOUND )
       If PRIMER_LEFT_4_BOUND < PRIMER_OPT_BOUND then this is added (+):
           + PRIMER_WT_BOUND_LT * ( PRIMER_OPT_BOUND - PRIMER_LEFT_4_BOUND )
       If PRIMER_LEFT_4_GC_PERCENT > PRIMER_OPT_GC_PERCENT then
           this is added (+):
           + PRIMER_WT_GC_PERCENT_GT *
                ( PRIMER_LEFT_4_GC_PERCENT - PRIMER_OPT_GC_PERCENT )
       If PRIMER_LEFT_4_GC_PERCENT < PRIMER_OPT_GC_PERCENT then
           this is added (+):
           + PRIMER_WT_GC_PERCENT_LT *
                ( PRIMER_OPT_GC_PERCENT - PRIMER_LEFT_4_GC_PERCENT )
       If masking is used (PRIMER_MASK_TEMPLATE=1), then this is added (+):
           + PRIMER_WT_MASK_FAILURE_RATE * PRIMER_LEFT_4_MASK_FAILURE_RATE
       The following section uses <primer length> as part of
       the term which is given as output in
       PRIMER_LEFT_4=position,<primer length>
       If <primer length> > PRIMER_OPT_SIZE then
           this is added (+):
           + PRIMER_WT_SIZE_GT *
                ( <primer length> - PRIMER_OPT_SIZE )
       If <primer length> < PRIMER_OPT_SIZE then
           this is added (+):
           + PRIMER_WT_SIZE_LT *
                ( PRIMER_OPT_SIZE - <primer length> )
       If the primer does not overlap a target then
           this is added (+):
           + PRIMER_WT_POS_PENALTY * PRIMER_LEFT_4_POSITION_PENALTY
       These are allways added (+) to the penalty
       (if the thermodynamic approach is used then the part in italic
          is substituted with text below this calculation):
        + PRIMER_WT_SELF_ANY * PRIMER_LEFT_4_SELF_ANY
           + PRIMER_WT_SELF_END * PRIMER_LEFT_4_SELF_END
           + PRIMER_WT_TEMPLATE_MISPRIMING *
                PRIMER_LEFT_4_TEMPLATE_MISPRIMING
           + PRIMER_WT_END_STABILITY * PRIMER_LEFT_4_END_STABILITY
           + PRIMER_WT_NUM_NS * <numbers of N in the selected primer>
           + PRIMER_WT_LIBRARY_MISPRIMING * PRIMER_LEFT_4_LIBRARY_MISPRIMING
           + PRIMER_WT_SEQ_QUAL *
                ( PRIMER_QUALITY_RANGE_MAX -
                  PRIMER_LEFT_4_MIN_SEQ_QUALITY )
          If the thermodynamic approach is used then the part of italic in
              the above calculation is replaced by this:

         If ((PRIMER_LEFT_4_TM - 5) ≤ PRIMER_LEFT_4_SELF_ANY_TH) then is added (+):
          + PRIMER_WT_SELF_ANY_TH *
                      (PRIMER_LEFT_4_SELF_ANY_TH - (PRIMER_LEFT_4_TM - 5 - 1))
        else if ((PRIMER_LEFT_4_TM - 5) > PRIMER_LEFT_4_SELF_ANY_TH) then is added (+):
          + PRIMER_WT_SELF_ANY_TH *
                (1/(PRIMER_LEFT_4_TM - 5 + 1 - PRIMER_LEFT_4_SELF_ANY_TH));
        If ((PRIMER_LEFT_4_TM - 5) ≤ PRIMER_LEFT_4_SELF_END_TH) then is added (+):
         + PRIMER_WT_SELF_END_TH *
                   (PRIMER_LEFT_4_SELF_END_TH - (PRIMER_LEFT_4_TM - 5 - 1))
        else if ((PRIMER_LEFT_4_TM - 5) > PRIMER_LEFT_4_SELF_END_TH) then is added (+):
         + PRIMER_WT_SELF_END_TH *
           (1/(PRIMER_LEFT_4_TM - 5 + 1 - PRIMER_LEFT_4_SELF_ANY_TH));
        If ((PRIMER_LEFT_4_TM - 5) ≤ PRIMER_LEFT_4_TEMPLATE_MISPRIMING_TH) then is added (+):
         + PRIMER_WT_TEMPLATE_MISPRIMING_TH *
           (PRIMER_LEFT_4_TEMPLATE_MISPRIMING_TH - (PRIMER_LEFT_4_TM - 5 - 1))
        else if ((PRIMER_LEFT_4_TM - 5) > PRIMER_LEFT_4_TEMPLATE_MISPRIMING_TH) then is added (+):
         + PRIMER_WT_TEMPLATE_MISPRIMING_TH *
          (1/(PRIMER_LEFT_4_TM - 5 + 1 - PRIMER_LEFT_4_TEMPLATE_MISPRIMING_TH));
        If ((PRIMER_LEFT_4_TM - 5) ≤ PRIMER_LEFT_4_HAIRPIN_TH) then is added (+):
          + PRIMER_WT_HAIRPIN_TH *
                 (PRIMER_LEFT_4_HAIRPIN_TH - (PRIMER_LEFT_4_TM - 5 - 1))
        else if ((PRIMER_LEFT_4_TM - 5) > PRIMER_LEFT_4_HAIRPIN_TH) then is added (+):
          + PRIMER_WT_HAIRPIN_TH *
                (1/(PRIMER_LEFT_4_TM - 5 + 1 - PRIMER_LEFT_4_HAIRPIN_TH));

### Right Primers (identical to Left Primers):

    PRIMER_RIGHT_4_PENALTY =
       If PRIMER_RIGHT_4_TM > PRIMER_OPT_TM then
           this is added (+):
           + PRIMER_WT_TM_GT * ( PRIMER_RIGHT_4_TM - PRIMER_OPT_TM )
       If PRIMER_RIGHT_4_TM < PRIMER_OPT_TM then
           this is added (+):
           + PRIMER_WT_TM_LT * ( PRIMER_OPT_TM - PRIMER_RIGHT_4_TM )
       If PRIMER_RIGHT_4_GC_PERCENT > PRIMER_OPT_GC_PERCENT then
           this is added (+):
           + PRIMER_WT_GC_PERCENT_GT *
                ( PRIMER_RIGHT_4_GC_PERCENT - PRIMER_OPT_GC_PERCENT )
       If PRIMER_RIGHT_4_GC_PERCENT < PRIMER_OPT_GC_PERCENT then
           this is added (+):
           + PRIMER_WT_GC_PERCENT_LT *
                ( PRIMER_OPT_GC_PERCENT - PRIMER_RIGHT_4_GC_PERCENT )
       If masking is used (PRIMER_MASK_TEMPLATE=1), then this is added (+):
           + PRIMER_WT_MASK_FAILURE_RATE * PRIMER_RIGHT_4_MASK_FAILURE_RATE
       The following section uses <primer length> as part of
       the term which is given as output in
       PRIMER_RIGHT_4=position,<primer length>
       If <primer length> > PRIMER_OPT_SIZE then
           this is added (+):
           + PRIMER_WT_SIZE_GT *
                ( <primer length> - PRIMER_OPT_SIZE )
       If <primer length> < PRIMER_OPT_SIZE then
           this is added (+):
           + PRIMER_WT_SIZE_LT *
                ( PRIMER_OPT_SIZE - <primer length> )
       If the primer does not overlap a target then
           this is added (+):
           + PRIMER_WT_POS_PENALTY * PRIMER_RIGHT_4_POSITION_PENALTY
       These are allways added (+) to the penalty
       (if the thermodynamic approach is used then the part in italic
          is substituted with text below this calculation):
        + PRIMER_WT_SELF_ANY * PRIMER_RIGHT_4_SELF_ANY
           + PRIMER_WT_SELF_END * PRIMER_RIGHT_4_SELF_END
           + PRIMER_WT_TEMPLATE_MISPRIMING *
                PRIMER_RIGHT_4_TEMPLATE_MISPRIMING
           + PRIMER_WT_END_STABILITY * PRIMER_RIGHT_4_END_STABILITY
           + PRIMER_WT_NUM_NS * <numbers of N in the selected primer>
           + PRIMER_WT_LIBRARY_MISPRIMING * PRIMER_RIGHT_4_LIBRARY_MISPRIMING
           + PRIMER_WT_SEQ_QUAL *
                ( PRIMER_QUALITY_RANGE_MAX -
                  PRIMER_RIGHT_4_MIN_SEQ_QUALITY )
         If the thermodynamic approach is used then the part of italic in
             the above calculation is replaced by this:

        If ((PRIMER_RIGHT_4_TM - 5) ≤ PRIMER_RIGHT_4_SELF_ANY_TH) then is added (+):
          + PRIMER_WT_SELF_ANY_TH *
                      (PRIMER_RIGHT_4_SELF_ANY_TH - (PRIMER_RIGHT_4_TM - 5 - 1))
        else if ((PRIMER_RIGHT_4_TM - 5) > PRIMER_RIGHT_4_SELF_ANY_TH) then is added (+):
          + PRIMER_WT_SELF_ANY_TH *
                (1/(PRIMER_RIGHT_4_TM - 5 + 1 - PRIMER_RIGHT_4_SELF_ANY_TH));
        If ((PRIMER_RIGHT_4_TM - 5) ≤ PRIMER_RIGHT_4_SELF_END_TH) then is added (+):
         + PRIMER_WT_SELF_END_TH *
                   (PRIMER_RIGHT_4_SELF_END_TH - (PRIMER_RIGHT_4_TM - 5 - 1))
        else if ((PRIMER_RIGHT_4_TM - 5) > PRIMER_RIGHT_4_SELF_END_TH) then is added (+):
         + PRIMER_WT_SELF_END_TH *
                   (1/(PRIMER_RIGHT_4_TM - 5 + 1 - PRIMER_RIGHT_4_SELF_ANY_TH));
        If ((PRIMER_RIGHT_4_TM - 5) ≤ PRIMER_RIGHT_4_TEMPLATE_MISPRIMING_TH) then is added (+):
          + PRIMER_WT_TEMPLATE_MISPRIMING_TH *
           (PRIMER_RIGHT_4_TEMPLATE_MISPRIMING_TH - (PRIMER_RIGHT_4_TM - 5 - 1))
        else if ((PRIMER_RIGHT_4_TM - 5) > PRIMER_RIGHT_4_TEMPLATE_MISPRIMING_TH) then is added (+):
          + PRIMER_WT_TEMPLATE_MISPRIMING_TH *
          (1/(PRIMER_RIGHT_4_TM - 5 + 1 - PRIMER_RIGHT_4_TEMPLATE_MISPRIMING_TH));
        If ((PRIMER_RIGHT_4_TM - 5) ≤ PRIMER_RIGHT_4_HAIRPIN_TH) then is added (+):
          + PRIMER_WT_HAIRPIN_TH *
                 (PRIMER_RIGHT_4_HAIRPIN_TH - (PRIMER_RIGHT_4_TM - 5 - 1))
        else if ((PRIMER_RIGHT_4_TM - 5) > PRIMER_RIGHT_4_HAIRPIN_TH) then is added (+):
          + PRIMER_WT_HAIRPIN_TH *
                (1/(PRIMER_RIGHT_4_TM - 5 + 1 - PRIMER_RIGHT_4_HAIRPIN_TH));

### Internal Oligos:

    PRIMER_INTERNAL_4_PENALTY =
       If PRIMER_INTERNAL_4_TM > PRIMER_INTERNAL_OPT_TM then
           this is added (+):
           + PRIMER_INTERNAL_WT_TM_GT *
                ( PRIMER_INTERNAL_4_TM - PRIMER_INTERNAL_OPT_TM )
       If PRIMER_INTERNAL_4_TM < PRIMER_INTERNAL_OPT_TM then
           this is added (+):
           + PRIMER_INTERNAL_WT_TM_LT *
                ( PRIMER_INTERNAL_OPT_TM - PRIMER_INTERNAL_4_TM )
       If PRIMER_INTERNAL_4_BOUND > PRIMER_INTERNAL_OPT_BOUND then
           this is added (+):
           + PRIMER_INTERNAL_WT_BOUND_GT *
                ( PRIMER_INTERNAL_4_BOUND - PRIMER_INTERNAL_OPT_BOUND )
       If PRIMER_INTERNAL_4_BOUND < PRIMER_INTERNAL_OPT_BOUND then
           this is added (+):
           + PRIMER_INTERNAL_WT_BOUND_LT *
                ( PRIMER_INTERNAL_OPT_BOUND - PRIMER_INTERNAL_4_BOUND )
       If PRIMER_INTERNAL_4_GC_PERCENT > PRIMER_INTERNAL_OPT_GC_PERCENT
           then this is added (+):
           + PRIMER_INTERNAL_WT_GC_PERCENT_GT *
                ( PRIMER_INTERNAL_4_GC_PERCENT -
                  PRIMER_INTERNAL_OPT_GC_PERCENT )
       If PRIMER_INTERNAL_4_GC_PERCENT < PRIMER_INTERNAL_OPT_GC_PERCENT
           then this is added (+):
           + PRIMER_INTERNAL_WT_GC_PERCENT_LT *
                ( PRIMER_INTERNAL_OPT_GC_PERCENT -
                  PRIMER_INTERNAL_4_GC_PERCENT )
       The following section uses <primer length> as part of
       the term which is given as output in
       PRIMER_INTERNAL_4=position,<primer length>
       If <primer length> > PRIMER_INTERNAL_OPT_SIZE then
           this is added (+):
           + PRIMER_INTERNAL_WT_SIZE_GT *
                ( <primer length> - PRIMER_INTERNAL_OPT_SIZE )
       If <primer length> < PRIMER_INTERNAL_OPT_SIZE then
           this is added (+):
           + PRIMER_INTERNAL_WT_SIZE_LT *
                ( PRIMER_INTERNAL_OPT_SIZE - <primer length> )
       These are always added (+) to the penalty:
       (if the thermodynamic approach is used then the part in italic
          is substituted with text below this calculation):
      + PRIMER_INTERNAL_WT_SELF_ANY * PRIMER_INTERNAL_4_SELF_ANY
         + PRIMER_INTERNAL_WT_SELF_END * PRIMER_INTERNAL_4_SELF_END
         + PRIMER_INTERNAL_WT_NUM_NS *
              <numbers of N in the selected primer>
         + PRIMER_INTERNAL_WT_LIBRARY_MISHYB *
              PRIMER_INTERNAL_4_LIBRARY_MISHYB
         + PRIMER_INTERNAL_WT_SEQ_QUAL *
              ( PRIMER_QUALITY_RANGE_MAX -
                PRIMER_INTERNAL_4_MIN_SEQ_QUALITY )
       If the thermodynamic approach is used then the part of italic in
       the above calculation is replaced by this:

       If ((PRIMER_INTERNAL_4_TM - 5) ≤ PRIMER_INTERNAL_4_SELF_ANY_TH) then is added (+):
        + PRIMER_INTERNAL_WT_SELF_ANY_TH *
            (PRIMER_INTERNAL_4_SELF_ANY_TH - (PRIMER_INTERNAL_4_TM - 5 - 1))
        else if ((PRIMER_INTERNAL_4_TM - 5) > PRIMER_INTERNAL_4_SELF_ANY_TH) then is added (+):
        + PRIMER_INTERNAL_WT_SELF_ANY_TH *
            (1/(PRIMER_INTERNAL_4_TM - 5 + 1 - PRIMER_INTERNAL_4_SELF_ANY_TH));
        If ((PRIMER_INTERNAL_4_TM - 5) ≤ PRIMER_INTERNAL_4_SELF_END_TH) then is added (+):
         + PRIMER_INTERNAL_WT_SELF_END_TH *
             (PRIMER_INTERNAL_4_SELF_END_TH - (PRIMER_INTERNAL_4_TM - 5 - 1))
        else if ((PRIMER_INTERNAL_4_TM - 5) > PRIMER_INTERNAL_4_SELF_END_TH) then is added (+):
          + PRIMER_INTERNAL_WT_SELF_END_TH *
              (1/(PRIMER_INTERNAL_4_TM - 5 + 1 - PRIMER_INTERNAL_4_SELF_ANY_TH));
        If ((PRIMER_INTERNAL_4_TM - 5) ≤ PRIMER_INTERNAL_4_HAIRPIN_TH) then is added (+):
          + PRIMER_INTERNAL_WT_HAIRPIN_TH *
            (PRIMER_INTERNAL_4_HAIRPIN_TH - (PRIMER_INTERNAL_4_TM - 5 - 1))
        else if ((PRIMER_INTERNAL_4_TM - 5) > PRIMER_INTERNAL_4_HAIRPIN_TH) then is added (+):
          + PRIMER_INTERNAL_WT_HAIRPIN_TH *
            (1/(PRIMER_INTERNAL_4_TM - 5 + 1 - PRIMER_INTERNAL_4_HAIRPIN_TH));

The primers are then sorted by penalty and Primer3 tries to pick the primers with the lowest penalty. For the PRIMER_TASK

    pick_primer_list

or

    pick_sequencing_primers

the selection ends at this point. If primer pairs have to be selected, a PRIMER_PAIR_4_PENALTY is calculated:

    PRIMER_PAIR_4_PENALTY =
       To the pair penalty are at first the single primer penalties
       added (+):
           + PRIMER_PAIR_WT_PR_PENALTY *
                ( PRIMER_LEFT_4_PENALTY + PRIMER_RIGHT_4_PENALTY )
       If internal oligo is picked then this is added (+):
           + PRIMER_PAIR_WT_IO_PENALTY * PRIMER_INTERNAL_4_PENALTY
       If PRIMER_PAIR_4_PRODUCT_TM > PRIMER_PRODUCT_OPT_TM then
           this is added (+):
           + PRIMER_PAIR_WT_PRODUCT_TM_GT *
                ( PRIMER_PAIR_4_PRODUCT_TM - PRIMER_PRODUCT_OPT_TM )
       If PRIMER_PAIR_4_PRODUCT_TM < PRIMER_PRODUCT_OPT_TM then
           this is added (+):
           + PRIMER_PAIR_WT_PRODUCT_TM_LT *
                ( PRIMER_PRODUCT_OPT_TM - PRIMER_PAIR_4_PRODUCT_TM )
       If PRIMER_PAIR_4_PRODUCT_SIZE > PRIMER_PRODUCT_OPT_SIZE then
           this is added (+):
           + PRIMER_PAIR_WT_PRODUCT_SIZE_GT *
                ( PRIMER_PAIR_4_PRODUCT_SIZE - PRIMER_PRODUCT_OPT_SIZE )
       If PRIMER_PAIR_4_PRODUCT_SIZE < PRIMER_PRODUCT_OPT_SIZE then
           this is added (+):
           + PRIMER_PAIR_WT_PRODUCT_SIZE_LT *
                ( PRIMER_PRODUCT_OPT_SIZE - PRIMER_PAIR_4_PRODUCT_SIZE )
       These are allways added (+) to the penalty:
       (if the thermodynamic approach is used then the part in italic
           is substituted with text below this calculation):
         + PRIMER_PAIR_WT_DIFF_TM *
              <difference in Tm between the left and the right primer>
      + PRIMER_PAIR_WT_COMPL_ANY * PRIMER_PAIR_4_COMPL_ANY
         + PRIMER_PAIR_WT_COMPL_END * PRIMER_PAIR_4_COMPL_END
         + PRIMER_PAIR_WT_LIBRARY_MISPRIMING * PRIMER_PAIR_4_LIBRARY_MISPRIMING
         + PRIMER_PAIR_WT_TEMPLATE_MISPRIMING *
              PRIMER_PAIR_4_TEMPLATE_MISPRIMING
         If the thermodynamic approach is used then the part of italic in
         the above calculation is replaced by this:

       If ((min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM) - 5) ≤ PRIMER_PAIR_4_COMPL_ANY_TH) then is added (+):
       + PRIMER_PAIR_WT_COMPL_ANY_TH *
           (PRIMER_PAIR_4_COMPL_ANY_TH - (min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM) - 5 - 1))
      else if ((min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM) - 5) > PRIMER_PAIR_4_COMPL_ANY_TH) then is added (+):
       + PRIMER_PAIR_WT_COMPL_ANY_TH *
          (1/(min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM - 5 + 1 - PRIMER_PAIR_4_COMPL_ANY_TH));
       If ((min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM - 5) ≤ PRIMER_PAIR_4_COMPL_END_TH) then is added (+):
       + PRIMER_PAIR_WT_COMPL_END_TH *
           (PRIMER_PAIR_4_COMPL_END_TH - (min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM) - 5 - 1))
      else if ((min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM - 5) > PRIMER_PAIR_4_COMPL_END_TH) then is added (+):
        + PRIMER_PAIR_WT_COMPL_END_TH *
             (1/(min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM - 5 + 1 - PRIMER_PAIR_4_COMPL_ANY_TH));
      If ((min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM - 5) ≤ PRIMER_PAIR_4_TEMPLATE_MISPRIMING_TH) then is added (+):
       + PRIMER_PAIR_WT_TEMPLATE_MISPRIMING_TH *
        (PRIMER_PAIR_4_TEMPLATE_MISPRIMING_TH - (min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM - 5 - 1))
      else if ((min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM - 5) > PRIMER_PAIR_4_TEMPLATE_MISPRIMING_TH) then is added (+):
       + PRIMER_PAIR_WT_TEMPLATE_MISPRIMING_TH *
         (1/(min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM - 5 + 1 - PRIMER_PAIR_4_TEMPLATE_MISPRIMING_TH));

Primer3 tries to select pairs with the lowest penalty which still fulfill all necessary requirements like non-redundancy or product size limits.

## 20. PRIMER3 SETTINGS FILE FORMAT

Primer3 can read global settings from a text file at program start up. This allows the user to save and exchange settings tailored to special applications.
Such a Primer3 settings file is a text file. The first three lines of the file have to be as described below followed by tags as they would be provided at standard input:

    Primer3 File - http://primer3.org
    P3_FILE_TYPE=settings
    P3_FILE_ID=Description of the settings
    SEQUENCE_TEMPLATE=ATG...
    ...
    ...
    ...
    =

In the first line "Primer3 File - http://primer3.org" without tailing space. In the second line "P3_FILE_TYPE=settings". Valid values for P3_FILE_TYPE are all_parameters, sequence and settings. Up to now, only settings is supported. The third line has to be empty. It is strongly advised to describe the settings using the P3_FILE_ID tag. It will print the description of the settings on the output. From the fourth line on regular Boulder-IO can be used as it is used in regular input. It also has to be terminated with a single "=". There can be only one input per file.

## 21. OUTPUT TAGS

For each Boulder-IO record passed into Primer3 via stdin, exactly one Boulder-IO record comes out of Primer3 on stdout. If a settings file is provided and the option to echo the settings file is given on the command line, then the contents of the settings file will also be part of the output. Two additional tags are used to indicate where the records of the settings file begin and end: P3_SETTINGS_FILE_USED specifies the path to the settings file that was provided, P3_SETTINGS_FILE_END does not have any value and it just indicates the end of the settings records.
The output records contain everything that the input record contains, plus a subset of the following tag/value pairs. Unless noted by (*), each tag appears for each primer pair returned.
Tags are of the form PRIMER_{LEFT,RIGHT,INTERNAL,PAIR}_<j>_<tag_name> where <j> is an integer from 0 to n, where n is at most the value of PRIMER_NUM_RETURN. In the documentation the output number 4 is shown as for example: PRIMER_LEFT_4_TM.
In the descriptions below, 'i,n' represents a start/length pair, 's' represents a string, x represents an arbitrary integer, and f represents a float.

### PRIMER_ERROR=s (*)

s describes user-correctable errors detected in the input (separated by semicolons). This tag is absent if there are no errors.

### PRIMER_WARNING=s (*)

s lists warnings generated by Primer3` (separated by semicolons); this tag is absent if there are no warnings.

### PRIMER_LEFT_NUM_RETURNED=i

### PRIMER_RIGHT_NUM_RETURNED=i

### PRIMER_INTERNAL_NUM_RETURNED=i

### PRIMER_PAIR_NUM_RETURNED=i

i is the number of primers or primer pairs returned on standard output. These tags are always generated under IO version 4 if there are no internal errors and if PRIMER_ERROR is not present.
If primer pairs were requested, PRIMER_LEFT_NUM_RETURNED and PRIMER_RIGHT_NUM_RETURNED will be equal to the number of pairs returned, even if the actual number of distinct left or right primers was lower than the number of pairs. If primer pairs with internal oligos were requested, PRIMER_INTERNAL_NUM_RETURNED will also be set to the number of pairs returned.
If only left or right primers or hybridization (internal) oligos were requested, PRIMER_PAIR_NUM_RETURNED will be 0 and only the relevant tag will have a non-zero value. For example, if only left primers were requested, PRIMER_RIGHT_NUM_RETURNED, PRIMER_INTERNAL_NUM_RETURNED and PRIMER_PAIR_NUM_RETURNED will be 0.
Some tasks, such as pick_sequencing_primers or pick_primer_list, return left and right primers that are not parts of primer pairs. In this case PRIMER_PAIR_NUM_RETURNED will be 0.

### PRIMER_LEFT_4_PROBLEMS=s (*)

### PRIMER_INTERNAL_4_PROBLEMS=s (*)

### PRIMER_RIGHT_4_PROBLEMS=s (*)

s lists the problems (constraint violations) associated with the corresponding primer oligo.

### PRIMER_LEFT_EXPLAIN=s (*)

### PRIMER_INTERNAL_EXPLAIN=s (*)

### PRIMER_RIGHT_EXPLAIN=s (*)

s is a (more or less) self-documenting string containing statistics on the possibilities that Primer3 considered in selecting a single oligo. For example

    PRIMER_LEFT_EXPLAIN=considered 62, too many Ns 53, ok 9
    PRIMER_RIGHT_EXPLAIN=considered 62, too many Ns 53, ok 9
    PRIMER_INTERNAL_OLIGO_EXPLAIN=considered 87, too many Ns 39, overlap excluded region 40, ok 8

All the categories are exclusive, except the 'considered' category. In some cases the ok count may be higher than the actual number of ok oligos. This is because a primer can be considered as part of pair before all of the primer's characteristics have been computed and checked. If a primer is never in a legal pair or never in a pair with a fully evaluated penalty, then this may occur. This situation never results in a primer pair that contains an illegal primer.

### PRIMER_PAIR_EXPLAIN=s (*)

s is a self-documenting string containing statistics on picking a primer pair (plus internal oligo if requested). For example

    PRIMER_PAIR_EXPLAIN=considered 81, unacceptable product size 49, no internal oligo 32, ok 0

The purpose of this string is to provide information in the case that not enough primer pairs are returned. This information can be used, for example, to decide which constraints to relax. In some cases the information in this string can also give insight into the causes of long running time. The counts in the string are only approximate, because of several reasons:

- When there are multiple PRIMER_PRODUCT_SIZE_RANGEs, a primer pair may not be ok in one size range but ok in another. Approximately, the counts for each statistic are summed over all product size ranges.
- When PRIMER_MIN_THREE_PRIME_DISTANCE is > -1, the number of ok primer pairs reported may be larger than the actual number. The discrepancy is due to a primer in the pair that would overlap a primer in a 'better' (lower penalty) pair. (The search algorithm does not record state that would allow it to detect when a pair that was formerly ok becomes not ok as the result of another primer being inserted into the output list before it is.)
- In some instances, Primer3 will examine a primer pair before it discovers that one of the individual primers in the pair violates specified constraints. In this case PRIMER_PAIR_EXPLAIN might have a non-0 number 'considered', even though one or more of PRIMER_LEFT_EXPLAIN, PRIMER_RIGHT_EXPLAIN, or PRIMER_INTERNAL_EXPLAIN has 'ok 0'.

### PRIMER_LEFT_4=i,n

The selected left primer (the primer to the left in the input sequence). i is the 0-based index of the start base of the primer, and n is t its length.

### PRIMER_INTERNAL_4=i,n

The selected internal oligo. Primer3 outputs this tag if PRIMER_PICK_INTERNAL_OLIGO was non-0. If Primer3 fails to pick a middle oligo upon request, this tag will not be output. i is the 0-based index of start base of the internal oligo, and n is its length.

### PRIMER_RIGHT_4=i,n

The selected right primer (the primer to the right in the input sequence). i is the 0-based index of the last base of the primer, and n is its length.

### PRIMER_LEFT_4_SEQUENCE=s

### PRIMER_INTERNAL_4_SEQUENCE=s

### PRIMER_RIGHT_4_SEQUENCE=s

The actual sequence of the oligo. The sequence of left primer and internal oligo is presented 5' -> 3' on the same strand as the input SEQUENCE_TEMPLATE (which must be presented 5' -> 3'). The sequence of the right primer is presented 5' -> 3' on the opposite strand from the input SEQUENCE_TEMPLATE.

### PRIMER_PAIR_4_PRODUCT_SIZE=x

x is the product size of the PCR product.

### PRIMER_LEFT_4_PENALTY=f

### PRIMER_INTERNAL_4_PENALTY=f

### PRIMER_RIGHT_4_PENALTY=f

The contribution of this individual primer or oligo to the objective function.

### PRIMER_PAIR_4_PENALTY=f

The value of the objective function for this pair (lower is better).

### PRIMER_LEFT_4_TM=f

### PRIMER_INTERNAL_4_TM=f

### PRIMER_RIGHT_4_TM=f

The melting TM for the selected oligo.

### PRIMER_LEFT_4_BOUND=f

### PRIMER_INTERNAL_4_BOUND=f

### PRIMER_RIGHT_4_BOUND=f

The calculated fraction of primers bound to a template at the PRIMER_ANNEALING_TEMP in percent if the primer and template would be at equal concentration. See "GENERAL THOUGHTS ON PRIMER BINDING" for more details.

### PRIMER_PAIR_4_PRODUCT_TM=f

f is the melting temperature of the product. Calculated using equation (iii) from the paper [Rychlik W, Spencer WJ and Rhoads RE (1990) "Optimization of the annealing temperature for DNA amplification in vitro", Nucleic Acids Res 18:6409-12 http://dx.doi.org/10.1093/nar/18.21.6409]. Printed only if a non-default value of PRIMER_PRODUCT_MAX_TM or PRIMER_PRODUCT_MIN_TM is specified.

### PRIMER_PAIR_4_PRODUCT_TM_OLIGO_TM_DIFF=f

f is the difference between the melting temperature of the product and the melting temperature of the less stable primer. Printed only if PRIMER_PRODUCT_MAX_TM or PRIMER_PRODUCT_MIN_TM is specified.

### PRIMER_PAIR_4_T_OPT_A=f

f is T sub a super OPT from equation (i) in [Rychlik W, Spencer WJ and Rhoads RE (1990) "Optimization of the annealing temperature for DNA amplification in vitro", Nucleic Acids Res 18:6409-12. http://dx.doi.org/10.1093/nar/18.21.6409]. Printed only if PRIMER_PRODUCT_MAX_TM or PRIMER_PRODUCT_MIN_TM is specified.

### PRIMER_LEFT_4_GC_PERCENT=f

### PRIMER_INTERNAL_4_GC_PERCENT=f

### PRIMER_RIGHT_4_GC_PERCENT=f

The percent GC for the selected oligo (denominator is the number of non-ambiguous bases).

### PRIMER_LEFT_4_SELF_ANY=f

### PRIMER_INTERNAL_4_SELF_ANY=f

### PRIMER_RIGHT_4_SELF_ANY=f

The calculated value for the tendency of a primer to bind to itself (interfering with target sequence binding). It will score ANY binding occurring within the entire primer sequence. For details see PRIMER_MAX_SELF_ANY.
The self-complementarity measures for the selected oligo.

### PRIMER_LEFT_4_SELF_ANY_TH=f

### PRIMER_INTERNAL_4_SELF_ANY_TH=f

### PRIMER_RIGHT_4_SELF_ANY_TH=f

The calculated value for the tendency of a primer to bind to itself (interfering with target sequence binding). It will calculate the melting temperature for ANY binding occurring within the entire primer sequence. For details see PRIMER_MAX_SELF_ANY_TH. The self-complementarity measures for the selected oligo.

### PRIMER_LEFT_4_SELF_ANY_STUCT=s

### PRIMER_INTERNAL_4_SELF_ANY_STUCT=s

### PRIMER_RIGHT_4_SELF_ANY_STUCT=s

A string representation of the calculated secondary structure. The tag is only present if a secondary structure could be calculated and PRIMER_SECONDARY_STRUCTURE_ALIGNMENT=1.
See PRIMER_SECONDARY_STRUCTURE_ALIGNMENT for examples and notes on the necessary reformating of the string.

### PRIMER_LEFT_4_SELF_END=f

### PRIMER_INTERNAL_4_SELF_END=f

### PRIMER_RIGHT_4_SELF_END=f

The calculated value for the tendency of the 3'-END to bind to a identical primer. This is critical for primer quality because it allows primers use itself as a target and amplify a short piece (forming a primer-dimer). These primer are then unable to bind and amplify the target sequence. For details see PRIMER_MAX_SELF_END.
The self-complementarity measures for the ends of selected oligo.

### PRIMER_LEFT_4_SELF_END_TH=f

### PRIMER_INTERNAL_4_SELF_END_TH=f

### PRIMER_RIGHT_4_SELF_END_TH=f

The calculated value for the tendency of the 3'-END to bind to a identical primer. This is critical for primer quality because it allows primers use itself as a target and amplify a short piece (forming a primer-dimer). These primer are then unable to bind and amplify the target sequence. For details see PRIMER_MAX_SELF_END_TH. The self-complementarity measures for the ends of selected oligo.

### PRIMER_LEFT_4_SELF_END_STUCT=s

### PRIMER_INTERNAL_4_SELF_END_STUCT=s

### PRIMER_RIGHT_4_SELF_END_STUCT=s

A string representation of the calculated secondary structure. The tag is only present if a secondary structure could be calculated and PRIMER_SECONDARY_STRUCTURE_ALIGNMENT=1.
See PRIMER_SECONDARY_STRUCTURE_ALIGNMENT for examples and notes on the necessary reformating of the string.

### PRIMER_LEFT_4_HAIRPIN_TH=f

### PRIMER_INTERNAL_4_HAIRPIN_TH=f

### PRIMER_RIGHT_4_HAIRPIN_TH=f

The calculated value of melting temperature of hairpin structure of primer. For details see PRIMER_MAX_HAIRPIN_TH

### PRIMER_LEFT_4_HAIRPIN_STUCT=s

### PRIMER_INTERNAL_4_HAIRPIN_STUCT=s

### PRIMER_RIGHT_4_HAIRPIN_STUCT=s

A string representation of the calculated secondary structure. The tag is only present if a secondary structure could be calculated and PRIMER_SECONDARY_STRUCTURE_ALIGNMENT=1.
See PRIMER_SECONDARY_STRUCTURE_ALIGNMENT for examples and notes on the necessary reformating of the string.

### PRIMER_PAIR_4_COMPL_ANY=f

The calculated value for the tendency of a primer pair to bind to each other (interfering with target sequence binding). It will score ANY binding occurring within the entire primer sequence. For details see PRIMER_MAX_SELF_ANY.
The inter-pair complementarity measures over the complete primer for selected left and right primer.

### PRIMER_PAIR_4_COMPL_ANY_TH=f

The calculated value for the tendency of a primer pair to bind to each other (interfering with target sequence binding). It will calculate the melting temperature of ANY binding occurring within the entire primer sequence. For details see PRIMER_MAX_SELF_ANY_TH. The inter-pair complementarity measures over the complete primer for selected left and right primer.

### PRIMER_PAIR_4_COMPL_ANY_STUCT=s

A string representation of the calculated secondary structure. The tag is only present if a secondary structure could be calculated and PRIMER_SECONDARY_STRUCTURE_ALIGNMENT=1.
See PRIMER_SECONDARY_STRUCTURE_ALIGNMENT for examples and notes on the necessary reformating of the string.

### PRIMER_PAIR_4_COMPL_END=f

The calculated value for the tendency of the 3'-ENDs of a primer pair to bind to each other. This is critical for primer quality because it allows primers use itself as a target and amplify a short piece (forming a primer-dimer). These primer are then unable to bind and amplify the target sequence. For details see PRIMER_MAX_SELF_END.
The inter-pair complementarity measures for the ends of selected left and right primer.

### PRIMER_PAIR_4_COMPL_END_TH=f

The calculated value for the tendency of the 3'-ENDs of a primer pair to bind to each other. This is critical for primer quality because it allows primers use itself as a target and amplify a short piece (forming a primer-dimer). These primer are then unable to bind and amplify the target sequence. For details see PRIMER_MAX_SELF_END_TH. The inter-pair complementarity measures for the ends of selected left and right primer.

### PRIMER_PAIR_4_COMPL_END_STUCT=s

A string representation of the calculated secondary structure. The tag is only present if a secondary structure could be calculated and PRIMER_SECONDARY_STRUCTURE_ALIGNMENT=1.
See PRIMER_SECONDARY_STRUCTURE_ALIGNMENT for examples and notes on the necessary reformating of the string.

### PRIMER_LEFT_4_END_STABILITY=f

### PRIMER_RIGHT_4_END_STABILITY=f

f is the delta G of disruption of the five 3' bases of the primer.

### PRIMER_LEFT_4_TEMPLATE_MISPRIMING=f

### PRIMER_RIGHT_4_TEMPLATE_MISPRIMING=f

### PRIMER_PAIR_4_TEMPLATE_MISPRIMING=f

Analogous to PRIMER_{LEFT,RIGHT,PAIR}_LIBRARY_MISPRIMING, except that these output tags apply to mispriming within the template sequence. This often arises, for example, in genes with repeated exons. For backward compatibility, these tags only appear if the corresponding input tags have defined values.

### PRIMER_LEFT_4_TEMPLATE_MISPRIMING_TH=f

### PRIMER_RIGHT_4_TEMPLATE_MISPRIMING_TH=f

### PRIMER_PAIR_4_TEMPLATE_MISPRIMING_TH=f

These output tags apply to mispriming within the template sequence and the calculation method is based on thermodynamical approach. This often arises, for example, in genes with repeated exons.

### PRIMER_LEFT_4_LIBRARY_MISPRIMING=f, s

### PRIMER_RIGHT_4_LIBRARY_MISPRIMING=f, s

### PRIMER_PAIR_4_LIBRARY_MISPRIMING=f, s

f is the maximum mispriming score for the right primer against any sequence in the given PRIMER_MISPRIMING_LIBRARY; s is the id of corresponding library sequence. PRIMER_PAIR_MAX_LIBRARY_MISPRIMING is the maximum sum of mispriming scores in any single library sequence (perhaps a more reasonable estimator of the likelihood of mispriming).

### PRIMER_INTERNAL_4_LIBRARY_MISHYB=f, s

f is the maximum mishybridization score for the right primer against any sequence in the given PRIMER_INTERNAL_MISHYB_LIBRARY; s is the id of corresponding library sequence.

### PRIMER_LEFT_4_MIN_SEQ_QUALITY=i

### PRIMER_INTERNAL_4_MIN_SEQ_QUALITY=i

### PRIMER_RIGHT_4_MIN_SEQ_QUALITY=i

i is the minimum _sequence_ quality within the primer or oligo (not to be confused with the PRIMER_PAIR_4_PENALTY output tag, which is really the value of the objective function.)

### PRIMER_STOP_CODON_POSITION=i

i is the position of the first base of the stop codon, if Primer3 found one, or -1 if Primer3 did not. Printed only if the input tag SEQUENCE_START_CODON_POSITION with a non-default value is supplied.

### PRIMER_LEFT_4_POSITION_PENALTY=i

### PRIMER_RIGHT_4_POSITION_PENALTY=i

i is the penalty of the primer by its position.

## 22. EXAMPLE OUTPUT

You should run it yourself. Use the file 'example' that came with this distribution directory as input.

## 23. ADVICE FOR PICKING PRIMERS

We suggest consulting: Wojciech Rychlik (1993) "Selection of Primers for Polymerase Chain Reaction" in BA White, Ed., "Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications", pp 31-40, Humana Press, Totowa NJ.

## 24. GENERAL THOUGHTS ON PRIMER BINDING

The binding of primers to a template DNA is depending on the primer sequence and the concentration of primer and template DNA, monovalent, divalent ions and dNTPs. Also other components like DMSO or contaminants influence the binding of primers. In primer design, the melting temperature is a critical factor and is calculated using thermodynamic equations (see PRIMER_TM_FORMULA for more information). The melting temperature is defined as the temperature where half of the primers are bound to target and it can be measured by heating up a double stranded DNA while monitoring the UV absorbance. In this situation, the double stranded DNA [AB] melts into two single strands ([A] and [B]) of equal concentration. The situation at the start of a PCR reaction is quite different. Usually the concentration of primer [A] in a typical PCR reaction (see PRIMER_DNA_CONC) is with 0.5 micromolar high, the concentration of template DNA [B] with 10 nanograms low. The actual micromolar concentration of the primer binding sites which would be required for the thermodynamic calculations depends on the type of DNA and will be very different in genomic DNA (few binding sites per nanogram) or plasmid and cDNA (many binding sites per nanogram). As the concentration of [AB], [A] and [B] changes dramatically during a PCR run (this is the aim of the PCR reaction), so does the melting temperature. For the thermodynamic calculations in Primer3 we use PRIMER_DNA_CONC as an "empirically determined" concentration of annealing oligo over the course the PCR (see PRIMER_DNA_CONC for details), which is less than the actual concentration of oligos in the initial reaction mix because of its dependence on the amount of template (including PCR product) in a given cycle. The melting temperature is calculated based on:

    Tm = deltaH / ( deltaS + R * ln ( [PRIMER_DNA_CONC] / 4 ) )

Primers are rarely used at melting temperature. Usually, the annealing temperature in a PCR reaction is usually chosen 6-10°C below the melting temperature of the primers and can be indicated to Primer3 with the PRIMER_ANNEALING_TEMP parameter. The idea behind this reduction in temperature is to increase the fraction of primers bound to target. While at the melting temperature 50% of the primers are bound to target, at the reduced annealing temperature 95-98% should be bound. The fraction of bound primers depends on dH and dS and can be calculated based on (see PRIMER_LEFT_4_BOUND for details):

    dG = dH - T * dS
    K = e^[-dG / ( R * T )]
    K = e^[( dS / R ) - ( dH / ( R * T ) ) ]
    fract = ( 1 / ( 1 + sqrt( 1 / ( ( PRIMER_DNA_CONC / 4000000000.0 ) * K)))) * 100

Unfortunately, the effect of dH on the fraction of bound primers is depending on the temperature, while the effect of dS is not. Therefore, primers which do have the same melting temperature and bind to 50% at melting temperature, may differ in binding at annealing temperature. Or on the other hand, primers which are dG matched to have identical binding at annealing temperature may differ in melting temperature. This dates back to the third myth described by SantaLucia on page 18 in [SantaLucia (2007) Physical principles and visual-OMP software for optimal PCR design. Methods Mol Biol. 2007;402:3-34. doi: 10.1007/978-1-59745-528-2_1.] SantaLucia argues, that primers should not be matched on melting temperature (PRIMER_OPT_TM) but on the fraction of primers bound at annealing temperature (PRIMER_OPT_BOUND). Especially multiplex primers should profit from thermodynamic parameters where the individual primers match better to each other.
It may come as a surprise that the calculated melting temperature and bound fractions do not reflect the actual situation in the final PCR reaction and you could wonder if this parameters are at all useful. Although this parameters are representations of the primers thermodynamic properties under ideal conditions, they are useful to select primers with matching thermodynamic properties that perform well together in a PCR reaction. The melting temperature and the fraction of primer bound at annealing temperature are intuitive parameters that are easier to evaluate than the dG, dH and dS values they are based on.
In Primer3 dG matching can be archived by setting a group of parameters as described below. Please consider this feature experimental. Primer selection based on melting temperature proved successful over the last decades. The selection based on deltaG by setting a bound fraction at melting temperature as target value is new and the optimal parameters still have to be found. As a start the fraction of bound primers may be calculated with no big impact on primer selection by solely providing an annealing temperature (PRIMER_ANNEALING_TEMP). For true dG matching the selection on melting temperature has to be switched off.

    # Activate statistics
    PRIMER_EXPLAIN_FLAG=1
    # Activate the SantaLucia Tm calculation and salt correction
    PRIMER_TM_FORMULA=1
    PRIMER_SALT_CORRECTIONS=1
    # Provide the annealing temperature
    PRIMER_ANNEALING_TEMP=50.0
    # Set the fraction of bound primers
    PRIMER_MIN_BOUND=96.5
    PRIMER_OPT_BOUND=97.0
    PRIMER_MAX_BOUND=97.5
    PRIMER_INTERNAL_MIN_BOUND=96.5
    PRIMER_INTERNAL_OPT_BOUND=97.0
    PRIMER_INTERNAL_MAX_BOUND=97.5
    # Activate selection by penalty values
    PRIMER_WT_BOUND_LT=1.0
    PRIMER_WT_BOUND_GT=1.0
    PRIMER_INTERNAL_WT_BOUND_LT=1.0
    PRIMER_INTERNAL_WT_BOUND_GT=1.0
    # Do not exclude primers based on Tm
    PRIMER_MAX_TM=80.0
    PRIMER_MIN_TM=40.0
    PRIMER_INTERNAL_MAX_TM=80.0
    PRIMER_INTERNAL_MIN_TM=40.0
    # Inactivate selection by penalty values
    PRIMER_WT_TM_LT=0.0
    PRIMER_WT_TM_GT=0.0
    PRIMER_INTERNAL_WT_TM_LT=0.0
    PRIMER_INTERNAL_WT_TM_GT=0.0

The fraction of primers bound to a template at the PRIMER_ANNEALING_TEMP can be calculated for the situation where the primer and template would be at equal concentration.
The thermodynamic parameter dG of the primer are used to calculate the equilibrium constant:

       R = 1.9872
       T = (PRIMER_ANNEALING_TEMP) + 273.15
       dG = dH - T * dS
       K = e^[-dG / ( R * T )]
       K = e^[( dS / R ) - ( dH / ( R * T ) ) ]

The equilibrium constant for the reaction is based on the concentration of primer [A], template[B] and primer bound to template [AB]:

       A + B <==> AB
       K = c[AB] / ( c[A] * c[B] )

c[AB] of this equation is the "empirically determined", the molar concentration of each annealing oligo over the course the PCR. It is provided in PRIMER_DNA_CONC.
c[A] is the molar concentration of oligos in the initial reaction mix.
c[B] is the molar concentration of template in the initial reaction mix.
The molar concentration of template in a PCR reaction is usually not known and low. The molar concentration of oligos is high. Therefore the fraction of primers bound is calculated for a situation, where primer and template are at an equal concentration. This simplifies the equation:

       c[A] = c[B]
       K = c[AB] / ( c[A] * c[A] )

The faction of primers bound to a template can be calculated:

       frac = c[AB] / ( c[A] + c[AB] )
       c[A] = ( 1 / frac - 1 ) * c[AB]
       K = c[AB] / ( c[A] * c[A] )
       K = c[AB] / [ ( 1 / frac - 1 ) * c[AB] * ( 1 / frac - 1 ) * c[AB] ]
       K = 1 / [ ( 1 / frac - 1 ) ^ 2 * c[AB] ]
       ( 1 / frac - 1 ) ^ 2 = 1 / ( K * c[AB] )
       1 / frac - 1 = sqrt[ 1 / ( K * c[AB] ) ]
       frac = 1 / ( 1 + sqrt[ 1 / ( K * c[AB] ) ] )

As the fraction should be in percent and the PRIMER_DNA_CONC is in nanomolar, the final calculation is for symmetrical oligos:

       fract = ( 1 / ( 1 + sqrt( 1 / ( ( PRIMER_DNA_CONC / 1000000000.0 ) * K)))) * 100

and for unsymmetrical oligos:

       fract = ( 1 / ( 1 + sqrt( 1 / ( ( PRIMER_DNA_CONC / 4000000000.0 ) * K)))) * 100

with

       K = e^[( dS / R ) - ( dH / ( R * T ) ) ]

These calculations are not available if PRIMER_SALT_CORRECTIONS=2 (Owczarzy, R), as this empirical salt correction corrects the melting temperature of primers and does not allow the correction of the thermodynamic parameters required for the bound fraction calculation.

## 25. CAUTIONS

Some of the most important issues in primer picking can be addressed only before using Primer3. These are sequence quality (including making sure the sequence is not vector and not chimeric) and avoiding repetitive elements.
Techniques for avoiding problems include a thorough understanding of possible vector contaminants and cloning artifacts coupled with database searches using blast, Fasta, or other similarity searching program to screen for vector contaminants and possible repeats. Repbase (J. Jurka, A.F.A. Smit, C. Pethiyagoda, and others, 1995-1996, ftp://ftp.ncbi.nih.gov/repository/repbase/) is an excellent source of repeat sequences and pointers to the literature. (The Repbase files need to be converted to Fasta format before they can be used by Primer3.) Primer3 now allows you to screen candidate oligos against a Mispriming Library (or a Mishyb Library in the case of internal oligos).
Sequence quality can be controlled by manual trace viewing and quality clipping or automatic quality clipping programs. Low- quality bases should be changed to N's or can be made part of Excluded Regions. The beginning of a sequencing read is often problematic because of primer peaks, and the end of the read often contains many low-quality or even meaningless called bases. Therefore, when picking primers from single-pass sequence it is often best to use the SEQUENCE_INCLUDED_REGION parameter to ensure that Primer3 chooses primers in the high quality region of the read.
In addition, Primer3 takes as input a SEQUENCE_QUALITY list for use with those base calling programs (e.g. Phred) that output this information.

## 26. WHAT TO DO IF PRIMER3 CANNOT FIND ANY PRIMERS?

Try relaxing various parameters, including the self-complementarity parameters and max and min oligo melting temperatures. For example, for very A-T-rich regions you might have to increase maximum primer size or decrease minimum melting temperature. It is usually unwise to reduce the minimum primer size if your template is complex (e.g. a mammalian genome), since small primers are more likely to be non-specific. Make sure that there are adequate stretches of non-Ns in the regions in which you wish to pick primers. If necessary you can also allow an N in your primer and use an oligo mixture containing all four bases at that position.
Try setting the PRIMER_EXPLAIN_FLAG input tag.

## 27. DIFFERENCES FROM EARLIER VERSIONS

The section HOW TO MIGRATE TAGS TO IO VERSION 4 describes the modified tags in detail. See also the file release_notes.txt in this directory.

## 28. EXIT STATUS CODES

     0 on normal operation
    -1 under the following conditions:
       illegal command-line arguments.
       unable to fflush stdout.
       unable to open (for writing and creating) a .for, .rev
         or .int file (probably due to a protection problem).
    -2 on out-of-memory
    -3 empty input
    -4 error in a "Global" input tag (message in PRIMER_ERROR).

Primer3 calls abort() and dumps core (if possible) if a programming error is detected by an assertion violation.
SIGINT and SIGTERM are handled essentially as empty input, except the signal received is returned as the exit status and printed to stderr.
In all of the error cases above Primer3 prints a message to stderr.

## 29. PRIMER3 WWW INTERFACES

There are two web interfaces available :
The Bioinformatics workgroup at University of Tartu provides a basic web-based interface to Primer3 named Primer3Web at http://primer3.ut.ee/
A Primer3Plus web services is at http://primer3plus.com
Web interface code is available on GitHub: https://github.com/primer3-org.

## 30. ACKNOWLEDGMENTS

Initial development of Primer3 was funded by Howard Hughes Medical Institute and by the National Institutes of Health, National Human Genome Research Institute under grants R01-HG00257 (to David C. Page) and P50-HG00098 (to Eric S. Lander), but ongoing development and maintenance are not currently funded.
Primer3 was originally written by Helen J. Skaletsky (Howard Hughes Medical Institute, Whitehead Institute) and Steve Rozen (Duke-NUS Graduate Medical School Singapore, formerly at Whitehead Institute) based on the design of earlier versions, notably Primer 0.5 (Steve Lincoln, Mark Daly, and Eric S. Lander). The original web interface was designed by Richard Resnick. Lincoln Stein designed the Boulder-IO format in the days before XML and RDF, and championed the idea of making Primer3 a software component, which has been key to its wide utility.
In addition, among others, Ernst Molitor, Carl Foeller, and James Bonfield contributed to the early design of Primer3. Brant Faircloth has helped with ensuring that Primer3 runs on Windows and MacOS and with the Primer3 web site. Triinu Koressaar and Maido Remm modernized the melting temperature calculations in 2008. Triinu Koressaar added secondary structure, primer-dimer, and template mispriming based on a thermodynamic model in 2.2.0. Ioana Cutcutache is responsible for most of the remaining improvements in 2.2.0, including performance enhancements, modern command line arguments, and new input tags to control primer location (with the "overlap junction" tags initially implemented by Andreas Untergasser). Jian Ye patiently provided new requirements.
Harm Nijveen and Andreas Untergasser developed the webinterface Primer3Plus in 2006-2009. Currently Primer3Plus is maintained by Andreas Untergasser.
Primer3 is an open software development project hosted on GitHub: https://github.com/primer3-org.