MultiMPrimer3 Input Help

Source Sequence
The sequence from which to select primers or hybridization oligos.
Sequence Id
An identifier that is reproduced in the output to enable you to identify the chosen primers.
Targets
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. 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.
Excluded Regions
Primer 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.
Product Size Range
A list of product size ranges, for example 
150-250 100-300 301-400
Primer3 first tries to pick primers in the first range. If that is not possible, it goes to the next range and tries again. It continues in this way until it has either picked all necessary primers or until there are no more ranges. For technical reasons this option makes much lighter computational demands than the Product Size option.
Product Size
Minimum, Optimum, and Maximum lengths (in bases) of the PCR product. Primer3 will not generate primers with products shorter than Min or longer than Max, and with default arguments Primer3 will attempt to pick primers producing products close to the Optimum length.
Number To Return
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.
Max 3' Stability
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 'Table of thermodynamic parameters'. 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.
Max Mispriming
The maximum allowed weighted similarity with any sequence in Mispriming Library. Default is 12.
Max Template Mispriming
The maximum allowed similarity to ectopic sites in the sequence from which you are designing the primers. The similarity is based on thermodynamic approach. A negative value means do not check. To consider this argument you have to provide the maximum value of melting temperature which could be achieved when primer hybridizes to alternative site of template.
Pair Max Mispriming
The maximum allowed sum of similarities of a primer pair (one similarity for each primer) with any single sequence in Mispriming Library. Library sequence weights are not used in computing the sum of similarities.
Pair Max Template Mispriming
The maximum allowed summed similarity of both primers to ectopic sites in the sequence from which you are designing the primers. The principle is the same as Max Template Mispriming.
Primer Size
Minimum, Optimum, and Maximum lengths (in bases) of a primer oligo. Primer3 will not pick primers shorter than Min or longer than Max, and with default arguments will attempt to pick primers close with size close to Opt. Min cannot be smaller than 1. Max cannot be larger than 36. (This limit is governed by maximum oligo size for which melting-temperature calculations are valid.) Min cannot be greater than Max.
Primer Tm
Minimum, Optimum, and Maximum melting temperatures (Celsius) for a primer oligo. Primer3 will not pick oligos with temperatures smaller than Min or larger than Max, and with default conditions will try to pick primers with melting temperatures close to Opt.

By default Primer3 uses the oligo melting temperature formula and the table of thermodynamic parameters given in Breslauer et al. 1986, DOI:10.1073/pnas.83.11.3746 For more information see caption Table of thermodynamic parameters

Maximum Tm Difference
Maximum acceptable (unsigned) difference between the melting temperatures of the left and right primers.
Table of thermodynamic parameters
Option for the table of Nearest-Neighbor thermodynamic parameters and for the method of melting temperature calculation. Two different tables of thermodynamic parameters are available:
  1. Breslauer et al. 1986, DOI:10.1073/pnas.83.11.3746 In that case the formula for melting temperature calculation suggested by Rychlik et al. 1990 is used (this is used until Primer3 version 1.0.1). This is the default value of Primer3 (for backward compatibility).
  2. SantaLucia 1998, DOI:10.1073/pnas.95.4.1460 This is the recommended value.

For specifying the salt correction method for melting temperature calculation see Salt correction formula

Product Tm
The minimum, optimum, and maximum melting temperature of the amplicon. Primer3 will not pick a product with melting temperature less than min or greater than max. If Opt is supplied and the Penalty Weights for Product Size are non-0 Primer3 will attempt to pick an amplicon with melting temperature close to Opt.

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.accelrys.com/products/gcg/), which instead uses 675.0 / length in the last term (after F. Baldino, Jr, M.-F. Chesselet, and M.E. Lewis, Methods in Enzymology 168:766 (1989) eqn (1) on page 766 without the mismatch and formamide terms). The formulas here and in Baldino et al. assume Na+ rather than K+. According to J.G. Wetmur, Critical Reviews in BioChem. and Mol. Bio. 26:227 (1991) 50 mM K+ should be equivalent in these formulae to .2 M Na+. Primer3 uses the same salt concentration value for calculating both the primer melting temperature and the oligo melting temperature. If you are planning to use the PCR product for hybridization later this behavior will not give you the Tm under hybridization conditions.

Primer GC% Minimum, Optimum, and Maximum percentage of Gs and Cs in any primer or oligo.
Max Complementarity

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. The by default value is 10 degrees lower than the default value of primer minimum melting temperature. 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.

Max 3' Complementarity
The maximum allowable 3'-anchored melting temperature when testing a single primer for self-complementarity, and the maximum allowable 3'-anchored melting temperature when testing for complementarity between left and right primers. For example 
5' ATGCCCTAGCTTCCGGATG 3'
             ||| |||||
          3' AAGTCCTACATTTAGCCTAGT 5'
or 
5` AGGCTATGGGCCTCGCGA 3'
               ||||||
            3' AGCGCTCCGGGTATCGGA 5'
The thermodynamic approach used is as for the Max Complementarity.
Primer hairpin stability

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. The by default value is 10 degrees lower than the default value of primer minimum melting temperature. 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)

Max Poly-X
The maximum allowable length of a mononucleotide repeat, for example AAAAAA.
Included Region
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.
CG Clamp
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 hybridization oligo if one is requested.)
Concentration of monovalent cations
The millimolar concentration of salt (usually KCl) in the PCR. Primer3 uses this argument to calculate oligo melting temperatures.
Concentration of divalent cations
The millimolar concentration of divalent salt cations (usually MgCl2+ in the PCR). Primer3 converts concentration of divalent cations to concentration of monovalent cations using formula suggested in the paper Ahsen et al., 2001 
                     [Monovalent cations] = [Monovalent cations] + 120*(√([divalent cations] - [dNTP]))
According to the formula concentration of desoxynucleotide triphosphate [dNTP] must be smaller than concentration of divalent cations. The concentration of dNTPs is included to the formula beacause of some magnesium is bound by the dNTP. Attained concentration of monovalent cations is used to calculate oligo/primer melting temperature. See Concentration of dNTPs to specify the concentration of dNTPs.
Concentration of dNTPs
The millimolar concentration of deoxyribonucleotide triphosphate. This argument is considered only if Concentration of divalent cations is specified.
Salt correction formula
Option for specifying the salt correction formula for the melting temperature calculation.

    There are three different options available:
  1. Schildkraut and Lifson 1965, DOI:10.1002/bip.360030207 (this is used until the version 1.0.1 of Primer3).The default value of Primer3 version 1.1.0 (for backward compatibility)
  2. SantaLucia 1998, DOI:10.1073/pnas.95.4.1460 This is the recommended value.
  3. Owczarzy et al. 2004, DOI:10.1021/bi034621r


Annealing Oligo Concentration
The nanomolar concentration of annealing oligos in the PCR. Primer3 uses this argument to calculate oligo melting temperatures. 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 oligo 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. This parameter corresponds to 'c' in Rychlik, Spencer and Rhoads' equation (ii) (Nucleic Acids Research, vol 18, num 21) where a suitable value (for a lower initial concentration of template) is "empirically determined". The value of this parameter is less than the actual concentration of oligos in the reaction 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.

Length of alternative product
The maximal length of alternative product (in bp) to be calculated. Alternative products are looked from genomes of non-target species, including human genome.

FASTA
Primer design for a sequence. Maximally "Number To Return" primer pairs will be returned per one FASTA format file. Example input file may be found from HERE. 
Example of the sequence in FASTA format:

>Name of the sequence
ACCGATGCACGACGATCGACACGATCGACATCATATATATTTTGCGCGCATCGCGGCGCGCGATATC
GATCGACTGATCGATACGCGACGACCACGCACGCGGATATACGATCGACTCGACATCGACATCGATC
GATAGCTAGCCTA
CLUSTALW
Primer design for a multiple alignment. Maximally "Number To Return" primer pairs will be returned per one multiple alignment (or a file).

Example of the sequence in CLUSTALW format:
CLUSTAL W (1.83) multiple sequence alignment


L43967.390305.390804.500.1.+ AGAAAAAGTTGTTTGGTCTTGTTGATCTGTATCAATTGGTAATTGGTAAC
L43967.389393.389845.500.2.+ AGAAAAAGTTGTTTGGTCTTGTTGATCTGTATCAATTGGTAATTGGTAAC
                             **************************************************

L43967.390305.390804.500.1.+ TATCAACTACTTGGGCTTGTTTACTTTCAAAATCAGTAGTTACTTTTTTT
L43967.389393.389845.500.2.+ TATCAACTACTTGGGCTTGTTTACTTTCAAAATCAGTAGTTACTTCTTTT
                             ********************************************* ****
Max Ns Accepted
Maximum number of unknown bases (N) allowable in any primer. Max Ns Accepted to a 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.

MultiMPrimer3 is the modified version of widely used program Primer3.

Copyright Notice and Disclaimer

Copyright (c) 1996,1997,1998,1999,2000,2001,2004,2006,2007
Whitehead Institute for Biomedical Research, Steve Rozen, and Helen Skaletsky
All rights reserved. 

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Acknowledgments

The development of Primer3 and the Primer3 web site 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).

We thank Centerline Software, Inc., for use of their TestCenter memory-error, -leak, and test-coverage checker.

Web interface by Steve Rozen.
Release 0.4.0
Last modified: February 07, 2007