For Generating Quickchange Mutagenesis Primers

#!perl.exe
#
# For generating QuickChange Mutagenesis Primers
#
#
#                    seq.txt            integer              A or ALA, &c.             integer     integer
#./quickchanger.pl <sequence file> <# of residue to mutate> <residue to mutate to> <sequence offset> <num codons on each side>
# Optional:< number of codons on each side of mutation>
#
#
# Sequence file should contain only the nucleotide sequence no header or bullshit
#
# My quickchange primers from IDT have always worked by adding 1uL of a 1:20,000 dilution to a 50uL PCR reaction
# I always use 7 codons or 21 bases on each side of the mutation this is the default also for the program
# You can change this by a command line argument but make sure if you do you also specify
# a sequence offset or 0 as the program reads in the arguments in a specific order
# If you choose a mutation 6 codons from the end it will use only 6 codons
# If you chose a mutation 5 codons from the end it will not work unless you specifically specify <number of codons on each side of mutation>
# I run the quickchange at 56.5C with pfu and have 99% efficiency
#
#
#
#


use Bio::Tools::CodonTable;
$d = 0;
$e = 0;
$x = 0;
my %val = ();
my %seqer = ();

if(!$ARGV[0] || !$ARGV[1] || !$ARGV[2]) { print "Usage: ./quickchanger.pl <sequence file> <number of residue to mutate> <residue to mutate to> <sequence offset>\n\n"; die;}


open(FILE, $ARGV[0]);

@file=<FILE>;

#
#load up sequence and separate it by codons
#
@seq = $file[0] =~ /(...)/g;

$numt = @seq;
#print "$numt\n\n"; die;
#$seq = $ARGV[0];



$num = $ARGV[1];
$aa2 = $ARGV[2];
if($ARGV[4]){ $primer = $ARGV[4];} else { $primer = 7; }

$offset = $ARGV[3] + 0;

if($primer > 5){
if(($num - $offset) < 6 || ($num - $offset) > (@seq - 7)){ print "Mutation to close to ends need more sequence\n";die; }
elsif(($num - $offset) eq 6 || (($num - $offset) - (@seq)) eq -7) { $primer = 6; }
}

#
# If we need to define a sequence offset
#
foreach $seqcod (@seq) {
  $seqer{$offset + $x} = $seqcod;
  #print "$seqer{$offset + $x}\n";
  $x++;
}

#
# Find our mutation codons
#
$table = Bio::Tools::CodonTable->new();
$mut = $table->translate($seqer{$num});
$unmut = $table->translate($seqer{$num});
@codons = $table->revtranslate($aa2);

@orig = $seqer{$num} =~ /(.)/g;

#
# Find best mutation codon match to
# original sequence
#
foreach $cod (@codons) {
$val{$d} = 0;
@co = $cod =~ /(.)/g;
for($c=0;$c<3;$c++) {
   if($orig[$c] eq $co[$c]) {
   $val{$d}++;
   }

}

#print "@cod $val{$d}\n";
$d++;
}

#
# Sort to find best mutation codon match
#
foreach $codonsers (sort {$val{$b} <=> $val{$a}} keys %val){
$final[$e] = $codons[$codonsers];

$e++;

}
#print "@orig\n@codons\n$final[0]\n";

#
# print out forward primer and mutation
#
print "$num $aa2\n";
for($z=$primer;$z>=1;$z--) {
print $seqer{$num-$z};
}
print " $final[0] ";
for($z=1;$z<=$primer;$z++) {
print $seqer{$num+$z};
}
print "\n";