Manual Reference Pages - BIO::SEQFEATURE::PRIMER (3)
Bio::SeqFeature::Primer - Primer Generic SeqFeature
# Primer object with explicitly-defined sequence object or sequence string
my $primer = Bio::SeqFeature::Primer->new( -seq => ACGTAGCT );
print "These are the details of the primer:\n".
"Tag: ".$primer->primary_tag."\n". # always Primer
"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
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
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The original concept and much of the code was written by
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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
: -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 dont know why.
This was suggested by Barry Moore (thanks!). See the discussion on
the bioperl-l with the subject "Bio::SeqFeature::Primer Calculating
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. Dont 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
primary_tag, source_tag, location, start, end, strand...
The documentation of Bio::SeqFeature::Generic describes all the methods that
Bio::SeqFeature::Primer object inherit.
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