Genome Explorer short help

"Search for Primers" command

    The window "Search for Primers" serves to search for primers in a sequence. In heading after a mark "#" the dropping down list serves for a choice of a sequence in which there will be a search is located.
  • The field "Change/See parameters" and button ">>" serve for change/see parameters of search (description of parameters see below).
  • The field "Command summary" displays the current adjustments of a command (the chosen parameters). The given field is only information and does not serve for adjustment of parameters of command.

Parameters description

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.

Target

    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 Region

    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.

Sequence Quality

    A list of space separated integers. There must be exactly one integer for each base in SEQUENCE if this argument is non-empty. High numbers indicate high confidence in the base call at that position and low numbers indicate low confidence in the base call at that position.

Left Input

    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.

Right Input

    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.

Mispriming Library

    This selection indicates what mispriming library (if any) Primer3 should use to screen for interspersed repeats or for other sequence to avoid as a location for primers.

Max Mispriming

    The maximum allowed weighted similarity with any sequence in Mispriming Library. Default is 12.

Product Size Range

    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 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 these pairs. Only if there are an insufficient number of primers in the first range will Primer3 return primers in a subsequent range.

Pick Internal Oligo

    If the associated value is non-0, then Primer3 will attempt to pick an internal oligo. Briefly, an "internal oligo" is intended to be used as a hybridization probe to detect the PCR product after amplification. Enter parameters at Internal Oligo Per-Sequence Inputs.

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 internal oligo if one is requested.)

Optimum Primer Size

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

Minimum Primer Size

    Minimum acceptable length of a primer.

Maximum Primer Size

    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.

Optimum Tm

    Optimum melting temperature(Celsius) for a primer oligo. Primer3 will try to pick primers with melting temperatures are close to this temperature. The oligo melting temperature formula in Primer3 is that given in Rychlik, Spencer and Rhoads, Nucleic Acids Research, vol 18, num 12, pp 6409-6412 and Breslauer, Frank, Bloeker and Marky, Proc. Natl. Acad. Sci. USA, vol 83, pp 3746-3750. Please refer to the former paper for background discussion.

Minimum Tm

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

Maximum Tm

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

Maximum Tm Difference

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

Minimum GC Content

    Minimum allowable percentage of Gs and Cs in any primer.

Maximum GC Content

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

Salt Concentration

    The millimolar concentration of salt (usually KCl) in the PCR. Primer3 uses this argument to calculate oligo melting temperatures. Default is 50.0 mM.

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 12) 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.

Maximum Ns Accepted

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

Maximum Complementarity

    The maximum allowable local alignment score when testing a single primer for (local) self-complementarity and the maximum allowable local alignment score when testing for complementarity between left and right primers. 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.

Maximum 3' Complementarity

    The maximum allowable 3'-anchored global alignment score when testing a single primer for self-complementarity, and the maximum allowable 3'-anchored global alignment score when testing for complementarity between left and right primers. 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 and primer pairs, Primer 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 because the score of a local alignment will always be at least as great as the score of a global alignment.

Maximum Poly-X

    The maximum allowable length of a mononucleotide repeat, for example AAAAAA.

Liberal Base

    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 Maximum 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.

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.

First Base Index

    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.) In the WWW interface this parameter defaults to 1.

Min Quality

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

Min End Quality

    The minimum sequence quality (as specified by PRIMER_SEQUENCE_QUALITY) allowed within the 5' pentamer of a primer.

Quality Range Min

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

Quality Range Max

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

Inside Penalty

    This experimental parameter might not be maintained in this form in the next release. Non-default values 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.

Outside Penalty

    This experimental parameter might not be maintained in this form in the next release. Non-default values 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.

Max End Stability

    The maximum stability for the five 3' bases of a left or right primer. Bigger numbers mean more stable 3' ends. The value is the maximum delta G for duplex disruption for the five 3' bases as calculated using the nearest neighbor parameters published in Breslauer, Frank, Bloeker and Marky, Proc. Natl. Acad. Sci. USA, vol 83, pp 3746-3750. Primer3 uses a completely permissive default value for backward compatibility (which we may change in the next release). Rychlik recommends a maximum value of 9 (Wojciech Rychlik, "Selection of Primers for Polymerase Chain Reaction" in BA White, Ed., "Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications", 1993, pp 31-40, Humana Press, Totowa NJ).

Internal Oligos

    Parameters governing choice of internal oligos are analogous to the parameters governing choice of primer pairs. The exception is Maximum 3' Complementarity which is meaningless when applied to internal oligos used for hybridization-based detection, since primer-dimer will not occur. We recommend that Maximum 3' Complementarity be set at least as high as Maximum Complementarity.

    What if no primers are found?

    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.

Cautions

    Some of the most important issues in primer picking can be addressed only in Primer3's input. 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://ncbi.nlm.nih.gov/repository/repbase) is an excellent source of repeat sequences and pointers to the literature. Primer3 now allows you to screen candidate oligos against a 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. When picking primers from single-pass sequence it is often best to avoid the first 20 base pairs, and to prefer shorter product sizes or shortened Included Region lengths to avoid low-quality sequence at the end of the sequence read. In addition, Primer3 takes as input a Sequence Quality list for use with those base calling programs such as Phred (http://www.mbt.washington.edu/phrap_documentation.html) Bass/Grace, and Trout that provide this output.

Copyright Notice and Disclaimer

     
     Copyright (c) 1996,1997
            Whitehead Institute for Biomedical Research. All rights reserved.
    
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            This product includes software developed by the
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    We also request that use of this software be cited in publications as 
    
       Steve Rozen and Helen J. Skaletsky (2000)
       Primer3 on the WWW for general users and for biologist programmers.
       In: Krawetz S, Misener S (eds)
       Bioinformatics Methods and Protocols: Methods in Molecular Biology.
       Humana Press, Totowa, NJ, pp 365-386
    
       (Code available at
        http://www-genome.wi.mit.edu/genome_software/other/primer3.html.)
    
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Reference

Steve Rozen and Helen J. Skaletsky (2000). Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386