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. CAUTIONS
25. WHAT TO DO IF PRIMER3 CANNOT FIND ANY PRIMERS?
26. DIFFERENCES FROM EARLIER VERSIONS
27. EXIT STATUS CODES
28. PRIMER3 WWW INTERFACES
29. ACKNOWLEDGMENTS
Copyright (c) 1996,1997,1998,1999,2000,2001,2004,2006,2007,2008,2009,2010
2011,2012,2013,2016,2017
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.
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.
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.
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.
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 reformats 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.
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
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
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 /opt/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.
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.
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.
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
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.
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.
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.
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.
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.
This argument indicates that primer3_core should generate user-oriented (rather than program-oriented) output.
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.
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.
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.
This argument is provided for backward compatibility. --io_version=4 is the only legal value and the default .
This argument specifies the file where the output should be written. If omitted, stdout is used.
This argument specifies the file where the error messages should be written. If omitted, stderr is used.
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.
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!
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
"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.
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.
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.)
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.
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.Primers and oligos may not overlap any region specified in this tag. 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.
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)
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)
Middle oligos may not overlap any region specified by this tag. 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.
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.
The sequence of an internal oligo to check and around which to design left and right primers. Must be a substring of SEQUENCE_TEMPLATE.
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.
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.
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).
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.
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.
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.
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.
"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.
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.
If the associated value = 1 (non-0), then Primer3 will attempt to pick left primers.
If the associated value = 1 (non-0), then Primer3 will attempt to pick an internal oligo (hybridization probe to detect the PCR product).
If the associated value = 1 (non-0), then Primer3 will attempt to pick a right primer.
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.
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.
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.
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 th 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.
Equivalent parameter of PRIMER_MUST_MATCH_FIVE_PRIME for the internal oligo.
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 th 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.
Equivalent parameter of PRIMER_MUST_MATCH_THREE_PRIME for the internal oligo.
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 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.
Penalty weight for products shorter than PRIMER_PRODUCT_OPT_SIZE.
Penalty weight for products longer than PRIMER_PRODUCT_OPT_SIZE.
Minimum acceptable length of a primer. Must be greater than 0 and less than or equal to PRIMER_MAX_SIZE.
Equivalent parameter of PRIMER_MIN_SIZE for the internal oligo.
Optimum length (in bases) of a primer. Primer3 will attempt to pick primers close to this length.
Equivalent parameter of PRIMER_OPT_SIZE for the internal oligo.
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.
Equivalent parameter of PRIMER_MAX_SIZE for the internal oligo.
Penalty weight for primers shorter than PRIMER_OPT_SIZE.
Equivalent parameter of PRIMER_WT_SIZE_LT for the internal oligo.
Penalty weight for primers longer than PRIMER_OPT_SIZE.
Equivalent parameter of PRIMER_INTERNAL_WT_SIZE_GT for the internal oligo.
Minimum allowable percentage of Gs and Cs in any primer.
Equivalent parameter of PRIMER_MIN_GC for the internal oligo.
Optimum GC percent. This parameter influences primer selection only if PRIMER_WT_GC_PERCENT_GT or PRIMER_WT_GC_PERCENT_LT are non-0.
Equivalent parameter of PRIMER_OPT_GC_PERCENT for the internal oligo.
Maximum allowable percentage of Gs and Cs in any primer generated by Primer.
Equivalent parameter of PRIMER_MAX_GC for the internal oligo.
Penalty weight for primers with GC percent lower than PRIMER_OPT_GC_PERCENT.
Equivalent parameter of PRIMER_WT_GC_PERCENT_LT for the internal oligo.
Penalty weight for primers with GC percent higher than PRIMER_OPT_GC_PERCENT.
Equivalent parameter of PRIMER_WT_GC_PERCENT_GT for the internal oligo.
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.)
The maximum number of Gs or Cs allowed in the last five 3' bases of a left or right primer.
Minimum acceptable melting temperature (Celsius) for a primer oligo.
Equivalent parameter of PRIMER_MIN_TM for the internal oligo.
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.
Equivalent parameter of PRIMER_OPT_TM for the internal oligo.
Maximum acceptable melting temperature (Celsius) for a primer oligo.
Equivalent parameter of PRIMER_MAX_TM for the internal oligo.
Maximum acceptable (unsigned) difference between the melting temperatures of the left and right primers.
Penalty weight for primers with Tm lower than PRIMER_OPT_TM.
Equivalent parameter of PRIMER_WT_TM_LT for the internal oligo.
Penalty weight for primers with Tm over PRIMER_OPT_TM.
Equivalent parameter of PRIMER_WT_TM_GT for the internal oligo.
Penalty weight for the TM difference between the left primer and the right primer.
The minimum allowed melting temperature of the amplicon. Please see the documentation on PRIMER_PRODUCT_MAX_TM for details.
The optimum melting temperature for the PCR product. 0 indicates that there is no optimum temperature.
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.
Penalty weight for products with a Tm lower than PRIMER_PRODUCT_OPT_TM.
Penalty weight for products with a Tm higher than PRIMER_PRODUCT_OPT_TM.
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
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).
Equivalent parameter of PRIMER_SALT_MONOVALENT for the internal oligo.
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.
Equivalent parameter of PRIMER_SALT_DIVALENT for the internal oligo.
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.
Parameter for internal oligos analogous to PRIMER_DNTP_CONC.
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.
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.
Equivalent parameter of PRIMER_DNA_CONC for the internal oligo.
If the associated value = 1, then Primer3 will use thermodynamic models to calculate the the propensity of oligos to form hairpins and dimers.
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.
If the associated value = 1, then Primer3 will print out the calculated secondary structures, for example:
t: 7.8 dG: -1724 dH: -91400 dS: -289
5' ACGCAAAGCACGCTCC-CGATC 3'
|| ||| ||| ||
3' CTAGCCC-TCGCACGAAACGCA 5'
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)/%$1;/g).
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 /opt/primer3_config/. For Windows, there is only one default location: .\primer3_config\.
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.
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:
Predicting secondary structures can improve primer design by eliminating
sequences with high possibility to form alternative secondary structures.
Equivalent parameter of PRIMER_MAX_SELF_ANY for the internal oligo.
Equivalent parameter of PRIMER_MAX_SELF_ANY_TH for the internal oligo.
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 describes the tendency of the left primer to bind to the right primer. It is similar to PRIMER_MAX_SELF_ANY_TH.
Penalty weight for the individual primer self binding value as in PRIMER_MAX_SELF_ANY.
Penalty weight for the individual primer self binding value as in PRIMER_MAX_SELF_ANY_TH.
Equivalent parameter of PRIMER_WT_SELF_ANY for the internal oligo.
Equivalent parameter of PRIMER_WT_SELF_ANY_TH for the internal oligo.
Penalty weight for the binding value of the primer pair as in PRIMER_MAX_SELF_ANY.
Penalty weight for the binding value of the primer pair as in PRIMER_MAX_SELF_ANY_TH.
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.
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 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.
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 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.
Same as PRIMER_PAIR_MAX_COMPL_END but for calculating the score (melting temperature of structure) thermodynamical approach is used.
Penalty weight for the individual primer self binding value as in PRIMER_MAX_SELF_END.
Penalty weight for the individual primer self binding value as in PRIMER_MAX_SELF_END_TH
Equivalent parameter of PRIMER_WT_SELF_END for the internal oligo.
Equivalent parameter of PRIMER_WT_SELF_END_TH for the internal oligo.
Penalty weight for the binding value of the primer pair as in PRIMER_MAX_SELF_END.
Penalty weight for the binding value of the primer pair as in PRIMER_MAX_SELF_END_TH.
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
The most stable monomer structure of internal oligo calculated by thermodynamic approach. See PRIMER_MAX_HAIRPIN_TH for details.
Penalty weight for the individual primer hairpin structure value as in PRIMER_MAX_HAIRPIN_TH.
Penalty weight for the most stable primer hairpin structure value as in PRIMER_INTERNAL_MAX_HAIRPIN_TH.
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.
Penalty factor for the calculated maximum stability for the last five 3' bases of a left or right primer.
Maximum number of unknown bases (N) allowable in any primer.
Equivalent parameter of PRIMER_MAX_NS_ACCEPTED for the internal oligo.
Penalty weight for the number of Ns in the primer.
Equivalent parameter of PRIMER_WT_NUM_NS for the internal oligo.
The maximum allowable length of a mononucleotide repeat, for example AAAAAA.
Equivalent parameter of PRIMER_MAX_POLY_X for the internal oligo.
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)
Analogous to PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE,
but for right primers.
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=3is 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.
If true use primer provided in SEQUENCE_PRIMER, SEQUENCE_PRIMER_REVCOMP, or SEQUENCE_INTERNAL_OLIGO even if it violates specific constraints.
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.
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.
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.
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.)
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).
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.
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.
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.
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.
Penalty for a single primer binding to the template sequence (thermodynamic approach, when PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT=1).
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.
Penalty for a primer pair binding to the template sequence (thermodynamic approach, when PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT=1).
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.
NOWWW
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.
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.
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.
The maximum allowed weighted similarity with any sequence in PRIMER_MISPRIMING_LIBRARY.
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.
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.
Penalty for a single primer binding to any single sequence in PRIMER_MISPRIMING_LIBRARY.
Equivalent parameter of PRIMER_WT_LIBRARY_MISPRIMING for the internal oligo.
Penalty for a primer pair binding to any single sequence in PRIMER_MISPRIMING_LIBRARY.
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.
Cutoff value of accepted failure rate for masking algorithm. Higher value gives lower stringency, meaning that fewer nucleotides in target sequence is masked.
Penalty weight for the primer failure rate.
The number of nucleotides masking algorithm should mask from 5' direction.
The number of nucleotides masking algorithm should mask from 3' direction.
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
This tag specifies the species whose k-mer lists are used for pre-masking.
The minimum sequence quality (as specified by SEQUENCE_QUALITY) allowed within a primer.
Equivalent parameter of PRIMER_MIN_QUALITY for the internal oligo.
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.
The minimum legal sequence quality (used for error checking of PRIMER_MIN_QUALITY and PRIMER_MIN_END_QUALITY).
The maximum legal sequence quality (used for error checking of PRIMER_MIN_QUALITY and PRIMER_MIN_END_QUALITY).
Penalty weight for the sequence quality of the primer.
Equivalent parameter of PRIMER_WT_SEQ_QUAL for the internal oligo.
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).
Penalty factor for the internal oligo added to the pair penalty.
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.
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.
Penalty for the primer which do not overlap the target.
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.
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.
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.
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.
"Program" input tags start with P3_... describe the parameters that deal with the behavior of the Primer3 program itself.)
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.
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.
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.
The value of this tag is ignored. It can be used to annotate input.
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:
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_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));
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));
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_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_listor
pick_sequencing_primersthe 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.
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.
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.
s describes user-correctable errors detected in the input (separated by semicolons). This tag is absent if there are no errors.
s lists warnings generated by Primer3` (separated by semicolons); this tag is absent if there are no warnings.
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.
s lists the problems (constraint violations) associated with the corresponding primer oligo.
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.
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 0The 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:
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.
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.
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.
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.
x is the product size of the PCR product.
The contribution of this individual primer or oligo to the objective function.
The value of the objective function for this pair (lower is better).
The melting TM for the selected oligo.
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.
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.
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.
The percent GC for the selected oligo (denominator is the number of non-ambiguous bases).
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.
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.
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.
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.
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.
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.
The calculated value of melting temperature of hairpin structure of primer. For details see PRIMER_MAX_HAIRPIN_TH
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.
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.
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.
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.
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.
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.
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.
f is the delta G of disruption of the five 3' bases of the primer.
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.
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.
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).
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.
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.)
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.
i is the penalty of the primer by its position.
You should run it yourself. Use the file 'example' that came with this distribution directory as input.
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.
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.
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.
The section HOW TO MIGRATE TAGS TO IO VERSION 4 describes th modified tags in detail. See also the file release_notes.txt in this directory.
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.
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.
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.