Bio::SeqFeature::Primer - 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' ); $primer->display_name('test_id'); print "These are the details of the primer:\n". "Name: ".$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 ); $template->add_SeqFeature($primer); 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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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 http://www.idtdna.com/Scitools/Scitools.aspx 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!