#! /usr/bin/perl -w

# NCBI_Primer_Blast - automate primer validation v. 0.001

use strict;
use WWW::Mechanize;
use HTTP::Cookies;

my $cj = HTTP::Cookies->new();

my $mech = WWW::Mechanize->new(
								timeout => '300',
								cookie_jar => $cj
								);
$mech->agent( 'Mozilla/5.0 (Macintosh; Intel Mac OS X 10.6; rv:2.0.1) Gecko/20100101 Firefox/4.0.1' );


my $url = "http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome";

my $accession = "NM_199168.3";

my $left_primer = "GTGGTCGTGCTGGTCCTC";
my $right_primer = "AGATGCTTGACGTTGGCTCT";

my $product_min = "70";
my $product_max = "150";

my $max_number_to_return = "5";

my $min_tm = "57";
my $opt_tm = "60";
my $max_tm = "63";
my $max_tm_diff = "3";

my $span_intron = "on";
my $min_intron = "1000";
my $max_intron = "100000000";

my $search_specific = "on";
my $organism = "Homo sapiens";
my $primer_specificity = "refseq_mrna";

my $min_primer_size = "18";
my $max_primer_size = "22";
my $opt_primer_size = "20";

my $primer_min_gc = "20";
my $primer_max_gc = "80";

my $gc_clamp = "0";
my $max_poly_x = "5";
my $max_self_comp = "8";
my $max_3prime_comp = "3";

my $monovalent_cations = "50";
my $divalent_cations = "0";
my $dNTPs = "0";

my $salt_formula = "0";
my $tm_method = "0";

my $annealing_oligo_conc = "50";

my $result = $mech->get($url);
die "Opening NCBI Primer Blast Failed....are you connected to the internet??\n"
	unless $result->is_success;

print "Opened NCBI PrimerBLAST website...\n";

$mech->submit_form(
	form_name => 'searchForm',
	fields => {
				INPUT_SEQUENCE => $accession,
				PRIMER_LEFT_INPUT => $left_primer,
				PRIMER_RIGHT_INPUT => $right_primer,
				PRIMER_PRODUCT_MIN => $product_min,
				PRIMER_PRODUCT_MAX => $product_max,
				PRIMER_NUM_RETURN => $max_number_to_return,
				PRIMER_MIN_TM => $min_tm,
				PRIMER_OPT_TM => $opt_tm,
				PRIMER_MAX_TM => $max_tm,
				PRIMER_MAX_DIFF_TM => $max_tm_diff,
				SPAN_INTRON => $span_intron,
				MIN_INTRON_SIZE => $min_intron,
				MAX_INTRON_SIZE => $max_intron,
				SEARCH_SPECIFIC_PRIMER => $search_specific,
				ORGANISM => $organism,
				PRIMER_SPECIFICITY_DATABASE => $primer_specificity,
				PRIMER_MIN_SIZE => $min_primer_size,
				PRIMER_OPT_SIZE => $opt_primer_size,
				PRIMER_MAX_SIZE => $max_primer_size,
				PRIMER_MIN_GC => $primer_min_gc,
				PRIMER_MAX_GC => $primer_max_gc,
				GC_CLAMP => $gc_clamp,
				POLYX => $max_poly_x,
				SELF_ANY => $max_self_comp,
				SELF_END => $max_3prime_comp,
				MONO_CATIONS => $monovalent_cations,
				DIVA_CATIONS => $divalent_cations,
				CON_DNTPS => $dNTPs,
				SALT_FORMULAR => $salt_formula,
				TM_METHOD => $tm_method
			}
);

die "Submission failed!!\n" unless $mech->success;

$mech->follow_link(tag=>'meta');
print $mech->content(format => 'text');


##</code><code>##

POST /tools/primer-blast/primertool.cgi HTTP/1.1
Host: www.ncbi.nlm.nih.gov
User-Agent: Mozilla/5.0 (Macintosh; Intel Mac OS X 10.6; rv:2.0.1) Gecko/20100101 Firefox/4.0.1
Accept: text/html,application/xhtml+xml,application/xml;q=0.9,*/*;q=0.8
Accept-Language: en-us,en;q=0.5
Accept-Encoding: gzip, deflate
Accept-Charset: ISO-8859-1,utf-8;q=0.7,*;q=0.7
Keep-Alive: 115
Connection: keep-alive
Referer: http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Cookie: ncbi_sid=8A1A2C9BE1302E51_0026SID

##</code><code>##

GET /tools/primer-blast/primertool.cgi?ctg_time=1310519679&job_key=JSID_01_116688_130.14.24.201_9000 HTTP/1.1
Host: www.ncbi.nlm.nih.gov
User-Agent: Mozilla/5.0 (Macintosh; Intel Mac OS X 10.6; rv:2.0.1) Gecko/20100101 Firefox/4.0.1
Accept: text/html,application/xhtml+xml,application/xml;q=0.9,*/*;q=0.8
Accept-Language: en-us,en;q=0.5
Accept-Encoding: gzip, deflate
Accept-Charset: ISO-8859-1,utf-8;q=0.7,*;q=0.7
Keep-Alive: 115
Connection: keep-alive
Cookie: ncbi_sid=8A1A2C9BE1302E51_0026SID