Bio::SeqFeature::Primer(3) Primer Generic SeqFeature


use Bio::SeqFeature::Primer;
# Primer object with explicitly-defined sequence object or sequence string
my $primer = Bio::SeqFeature::Primer->new( -seq => 'ACGTAGCT' );
print "These are the details of the primer:\n".
"Name: ".$primer->display_name."\n".
"Tag: ".$primer->primary_tag."\n". # always 'Primer'
"Sequence: ".$primer->seq->seq."\n".
"Tm: ".$primer->Tm."\n\n"; # melting temperature
# Primer object with implicit sequence object
# It is a lighter approach for when the primer location on a template is known
use Bio::Seq;
my $template = Bio::Seq->new( -seq => 'ACGTAGCTCTTTTCATTCTGACTGCAACG' );
$primer = Bio::SeqFeature::Primer->new( -start => 1, -end =>5, -strand => 1 );
print "Primer sequence is: ".$primer->seq->seq."\n";
# Primer sequence is 'ACGTA'


This module handles PCR primer sequences. The Bio::SeqFeature::Primer object is a Bio::SeqFeature::Subseq object that can additionally contain a primer sequence and its coordinates on a template sequence. The primary_tag() for this object is 'Primer'. A method is provided to calculate the melting temperature Tm of the primer. Bio::SeqFeature::Primer objects are useful to build Bio::Seq::PrimedSeq amplicon objects such as the ones returned by Bio::Tools::Primer3.


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Rob Edwards, [email protected]

The original concept and much of the code was written by Chad Matsalla, [email protected]


The rest of the documentation details each of the object methods. Internal methods are usually preceded with a _


 Title   : new()
 Usage   : my $primer = Bio::SeqFeature::Primer( -seq => $seq_object );
 Function: Instantiate a new Bio::SeqFeature::Primer object
 Returns : A Bio::SeqFeature::Primer object
 Args    : -seq , a sequence object or a sequence string (optional)
           -id  , the ID to give to the primer sequence, not feature (optional)


 Title   : Tm()
 Usage   : my $tm = $primer->Tm(-salt => 0.05, -oligo => 0.0000001);
 Function: Calculate the Tm (melting temperature) of the primer
 Returns : A scalar containing the Tm.
 Args    : -salt  : set the Na+ concentration on which to base the calculation
                    (default=0.05 molar).
         : -oligo : set the oligo concentration on which to base the
                    calculation (default=0.00000025 molar).
 Notes   : Calculation of Tm as per Allawi et. al Biochemistry 1997
           36:10581-10594. Also see documentation at
  as they use this
           formula and have a couple nice help pages. These Tm values will be
           about are about 0.5-3 degrees off from those of the idtdna web tool.
           I don't know why.
           This was suggested by Barry Moore (thanks!). See the discussion on
           the bioperl-l with the subject "Bio::SeqFeature::Primer Calculating
           the PrimerTM"


 Title   : Tm_estimate
 Usage   : my $tm = $primer->Tm_estimate(-salt => 0.05);
 Function: Estimate the Tm (melting temperature) of the primer
 Returns : A scalar containing the Tm.
 Args    : -salt set the Na+ concentration on which to base the calculation.
 Notes   : This is only an estimate of the Tm that is kept in for comparative
           reasons. You should probably use Tm instead!
           This Tm calculations are taken from the Primer3 docs: They are
           based on 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
           %G+C of the sequence, and length is the length of the sequence.
           However.... I can never get this calculation to give me the same result
           as primer3 does. Don't ask why, I never figured it out. But I did 
           want to include a Tm calculation here because I use these modules for 
           other things besides reading primer3 output.
           The primer3 calculation is saved as 'PRIMER_LEFT_TM' or 'PRIMER_RIGHT_TM'
           and this calculation is saved as $primer->Tm so you can get both and
           average them!

primary_tag, source_tag, location, start, end, strand...

The documentation of Bio::SeqFeature::Generic describes all the methods that Bio::SeqFeature::Primer object inherit.