User:Russell Durrett/Notebook/Perl/BiobrickingPrimers

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  1. perl;
  2. biobrickprimers script
  1. This script promts the user for a DNA sequence, then outputs forward and reverse primer sequences with biobrick restriction site extensions. It does not, as yet, calculate possible primer dimers but it does output the Tm of the complementary region of each oligo.
  2. Designed by Russell Durrett, June 2010. Email russell (at) durrett.org
  1. UNRESOLVED : primer dimers,

use 5.010;

print "This script will take a DNA sequence and design primers for 'biobricking' the sequence. It automatically adds Biobricking primer extensions - if you don't want those then consult the script 'primerdesign' instead. \n\n";


say "DNA sequence to biobrick: "; my $DNA = <STDIN>;


  1. Defines forward primer as first 15 bases

my $fprimer = substr ($DNA, 0, 15);

  1. Defines reverse primer as last 15 bases

sub rprimer { print "taking reverse...\n\n"; $_ = reverse $DNA; tr /AGCTagct/TCGAtcga/d; $DNArc = $_; }

&rprimer; my $rprimer = substr ($DNArc, 1, 15);

print "Forward primer: "; print &Tm($fprimer), "\n" ; print "$fprimer\n"; print "\n";

print "Reverse primer: " ; print &Tm($rprimer), "\n" ; print "$rprimer\n"; print "\n";


$fext = "CGCGCGAGAATTCGCGGCCGCTTCTAGA"; $rext = "GCGCGCACTGCAGCGGCCGCTACTAGT";


my $fprimer_wext = $fext . $fprimer; my $rprimer_wext = $rext . $rprimer;

say ""; say "Forward primer with Biobrick Prefix Extension:"; print "$fprimer_wext\n\n"; say "Reverse primer with Biobrick Suffix R.C. Extension:"; print "$rprimer_wext\n"; say "";

say "This script now calculates the Tm of the complementary region of the primers, but does not check for primer dimers (yet - as soon as I figure how to do that in Perl it will) - so ALWAYS TEST YOUR PRIMERS BEFORE ORDERING!!!";


    1. Perl script to calculate Tm of oligos
    2. Nearest Neighbor Tm subroutines were courtesy of a script written by David Gresham

sub Tm {

my $seq = shift @_;

$seq = uc($seq);

print "\n"; print "Tm: ", &calcTm(&calcNnEnthalpy($seq), &calcNnEntropy($seq));

}

              1. subroutine to calculate Tm based on total enthalpy and entropy

sub calcTm {

   my ($totalEnthalpy, $totalEntropy) = @_;
   my $R = 1.9872; #gas constant cal/K-mol
   my $x = 4; #equals 4 for nonself-complementary duplex
   my $Ct = 0.0000000000006; #$Ct is strand concentration 
   
   my $Tm = ($totalEnthalpy * 1000) / ($totalEntropy + $R * log($Ct/$x)) - 273.15;
   return $Tm;

}


              1. subroutine to calculate enthalpy by summing over nearest neighbors using the unified nearest neighbor values

sub calcNnEnthalpy {

  1. print "Calculating Enthalpy..\n";
   my $dna = shift(@_);
   my %enthalpies = (
       'AA' => -7.9,
       'TT' => -7.9,                                                                                                        
       'AT' => -7.2,                                                                                                        
       'TA' => -7.2,                                                                                                        
       'CA' => -8.5,                                                                                                        
       'TG' => -8.5,                                                                                                        
       'GT' => -8.4,                                                                                                        
       'AC' => -8.4,                                                                                                        
       'CT' => -7.8,                                                                                                        
       'AG' => -7.8,                                                                                                        
       'GA' => -8.2,                                                                                                        
       'TC' => -8.2,                                                                                                        
       'CG' => -10.6,                                                                                                      
       'GC' => -9.8,                                                                                                       
       'GG' => -8.0,                                                                                                        
       'CC' => -8.0                                                                                                          
       );
  1. select initialization value depending on whether terminal bases are A/T
  2. initialization and terminal penalty are not relevant for array hybridization
   my $totalEnthalpy;
   if((substr($dna,0,1) eq 'A' || substr($dna,0,1) eq 'T') && (substr($dna,-1) eq 'A'|| substr($dna,-1) eq 'T'))
   {$totalEnthalpy = 0;} # 4.6;}                                                                                                   
   elsif((substr($dna,-1) eq 'A'|| substr($dna,-1) eq 'T') && (substr($dna,-1) ne 'A'|| substr($dna,-1) ne 'T'))
   {$totalEnthalpy = 0;} # 2.4;}                                                                                                   
   elsif((substr($dna,-1) ne 'A' || substr($dna,-1) ne 'T') && (substr($dna,-1) eq 'A' || substr($dna,-1) eq 'T'))
   {$totalEnthalpy = 0;} #2.4;}                                                                                                    
   else{$totalEnthalpy = 0;} #= 0.2};                                                                                              
  1. calculate total enthalpy based on each NN for oligo
   for(my $i=0; $i< (length($dna) - 1); $i++) {
       $totalEnthalpy += $enthalpies{substr($dna,$i,2)};
   }
   return $totalEnthalpy;

}

          1. subroutine to calculate entropy by summing over nearest neighbors using the nearest neighbor parameters
  1. initialization and terminal penalty are not relevant for array hybridization

sub calcNnEntropy {

  1. print "Calculating Entropy..\n";
   my $dna = shift(@_);
   my %entropies = (
       'AA' => -22.2,
       'TT' => -22.2,
       'AT' => -20.4,
       'TA' => -21.3,
       'CA' => -22.7,
       'TG' => -22.7,
       'GT' => -22.4,
       'AC' => -22.4,
       'CT' => -21.0,
       'AG' => -21.0,
       'GA' => -22.2,
       'TC' => -22.2,
       'CG' => -27.2,
       'GC' => -24.4,
       'GG' => -19.9,
       'CC' => -19.9
       );
  1. initialize total entropy depending on whether there are terminal A/T
   my $totalEntropy;
   if((substr($dna,0,1) eq 'A' || substr($dna,0,1) eq 'T') && (substr($dna,-1) eq 'A'|| substr($dna,-1) eq 'T'))
   {$totalEntropy = 0;} #8.1;}                                                                                                                          
   elsif((substr($dna,-1) eq 'A'|| substr($dna,-1) eq 'T') && (substr($dna,-1) ne 'A'|| substr($dna,-1) ne 'T'))
   {$totalEntropy = 0;} #1.2;}                                                                                                                          
   elsif((substr($dna,-1) ne 'A' || substr($dna,-1) ne 'T') && (substr($dna,-1) eq 'A' || substr($dna,-1) eq 'T'))
   {$totalEntropy = 0;} #1.2;}                                                                                                                          
   else {$totalEntropy = 0;} #-5.7;}                                                                                                                    
  1. add together entropies for each NN in string
   for(my $i=0; $i < (length($dna) - 1); $i++) {
       $totalEntropy += $entropies{substr($dna,$i,2)};
   }
   return $totalEntropy;

}

              1. subroutine to calculate deltaG at 37C by summing over nearest neighbors using the unified nearest neighbors values

sub calcDeltaG37 {

   my $dna = shift(@_);
   my %deltaG37 = (
       'AA' => -1.0,
       'TT' => -1.0,
       'AT' => -0.88,
       'TA' => -0.58,
       'CA' => -1.45,
       'TG' => -1.45,
       'GT' => -1.44,
       'AC' => -1.44,
       'CT' => -1.28,
       'AG' => -1.28,
       'GA' => -1.30,
       'TC' => -1.30,
       'CG' => -2.17,
       'GC' => -2.24,
       'GG' => -1.84,
       'CC' => -1.84
       );
   my $totalDeltaG37;
   if((substr($dna,0,1) eq 'A' || substr($dna,0,1) eq 'T') && (substr($dna,-1) eq 'A'|| substr($dna,-1) eq 'T'))
   {$totalDeltaG37 = 2.06;}
   elsif((substr($dna,-1) eq 'A'|| substr($dna,-1) eq 'T') && (substr($dna,-1) ne 'A'|| substr($dna,-1) ne 'T'))
   {$totalDeltaG37 = 2.01;}
   elsif((substr($dna,-1) ne 'A' || substr($dna,-1) ne 'T') && (substr($dna,-1) eq 'A' || substr($dna,-1) eq 'T'))
   {$totalDeltaG37 = 2.01;}
   else {$totalDeltaG37 = 1.96;}
   for(my $i=0; $i < (length($dna) - 1); $i++) {
 
       $totalDeltaG37 += $deltaG37{substr($dna,$i,2)};
   }
   return $totalDeltaG37;

}

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