LENGTH                   ".strlen($primer)."\n";
        $cg=round(100*CountCG($primer)/strlen($primer),1);
        print "C+G%                     $cg\n";
        Mol_wt($primer);
        print "

";
        if (strlen($primer)!=CountATCG($primer)){
                print "Minimun        ".Tm_min($primer)." °C\n";
                print "Maximum        ".Tm_max($primer)." °C";
        }else{
                print "Tm:                 ".Tm_min($primer)." °C";
        }
        print "

";
        tm_Base_Stacking($primer,$_GET["cp"],$_GET["cs"],$_GET["cmg"]);
        print "

Basic Melting Temperature (Tm) Calculations

For sequences less than 14 nucleotides the formula is:

     Tm= (wA+xT) * 2 + (yG+zC) * 4

     where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively.

For sequences longer than 13 nucleotides, the equation used is

     Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC)

When degenerated nucleotides are included in the primer sequence (Y,R,W,S,K,M,D,V,H,B or N), those nucleotides will be internally substituted prior to minimum and maximum Tm calculation.

    Example:
     Primer sequence:                            CTCTRYCTWSCTCTCT
     Sequence for minimum Tm calculation:        CTCTATCTAGCTCTCT
     Sequence for maximum Tm calculation:        CTCTGCCTAGCTCTCT

ASSUMPTIONS:

Both equations assume that the annealing occurs under the standard conditions of 50 nM primer, 50 mM Na⁺, and pH 7.0.

Base-Stacking Melting Temperature (Tm) Calculations

SantaLucia J. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1460-5. NCBI

von Ahsen N, Oellerich M, Armstrong VW, Schütz E. Application of a thermodynamic nearest-neighbor model to estimate nucleic acid stability and optimize probe design: prediction of melting points of multiple mutations of apolipoprotein B-3500 and factor V with a hybridization probe genotyping assay on the LightCycler. Clin Chem. 1999 Dec;45(12):2094-101. NCBI ".round($tm,1)." °C"; print "\n Enthalpy: ".round($h,2)."\n Entropy: ".round($s,2).""; } function Mol_wt($primer){ $upper_mwt=molwt($primer,"DNA","upperlimit"); $lower_mwt=molwt($primer,"DNA","lowerlimit"); if ($upper_mwt==$lower_mwt){ print "Molecular weight: $upper_mwt"; }else{ print "Upper Molecular weight: $upper_mwt\nLower Molecular weight: $lower_mwt"; } } function CountCG($c){ $cg=substr_count($c,"G")+substr_count($c,"C"); return $cg; } function CountATCG($c){ $cg=substr_count($c,"A")+substr_count($c,"T")+substr_count($c,"G")+substr_count($c,"C"); return $cg; } function Tm_min($primer){ $primer_len=strlen($primer); $primer2=preg_replace("/A|T|Y|R|W|K|M|D|V|H|B|N/","A",$primer); $n_AT=substr_count($primer2,"A"); $primer2=preg_replace("/C|G|S/","G",$primer); $n_CG=substr_count($primer2,"G"); if ($primer_len > 0) { if ($primer_len < 14) { return round(2 * ($n_AT) + 4 * ($n_CG)); }else{ return round(64.9 + 41*(($n_CG-16.4)/$primer_len),1); } } } function Tm_max($primer){ $primer_len=strlen($primer); $primer=primer_max($primer); $n_AT=substr_count($primer,"A"); $n_CG=substr_count($primer,"G"); if ($primer_len > 0) { if ($primer_len < 14) { return round(2 * ($n_AT) + 4 * ($n_CG)); }else{ return round(64.9 + 41*(($n_CG-16.4)/$primer_len),1); } } } function primer_min($primer){ $primer=preg_replace("/A|T|Y|R|W|K|M|D|V|H|B|N/","A",$primer); $primer=preg_replace("/C|G|S/","G",$primer); return $primer; } function primer_max($primer){ $primer=preg_replace("/A|T|W/","A",$primer); $primer=preg_replace("/C|G|Y|R|S|K|M|D|V|H|B|N/","G",$primer); return $primer; } function molwt($sequence,$moltype,$limit) { // the following are single strand molecular weights / base $rna_A_wt = 329.245; $rna_C_wt = 305.215; $rna_G_wt = 345.245; $rna_U_wt = 306.195; $dna_A_wt = 313.245; $dna_C_wt = 289.215; $dna_G_wt = 329.245; $dna_T_wt = 304.225; $water = 18.015; $dna_wts = array('A' => array($dna_A_wt, $dna_A_wt), // Adenine 'C' => array($dna_C_wt, $dna_C_wt), // Cytosine 'G' => array($dna_G_wt, $dna_G_wt), // Guanine 'T' => array($dna_T_wt, $dna_T_wt), // Thymine 'M' => array($dna_C_wt, $dna_A_wt), // A or C 'R' => array($dna_A_wt, $dna_G_wt), // A or G 'W' => array($dna_T_wt, $dna_A_wt), // A or T 'S' => array($dna_C_wt, $dna_G_wt), // C or G 'Y' => array($dna_C_wt, $dna_T_wt), // C or T 'K' => array($dna_T_wt, $dna_G_wt), // G or T 'V' => array($dna_C_wt, $dna_G_wt), // A or C or G 'H' => array($dna_C_wt, $dna_A_wt), // A or C or T 'D' => array($dna_T_wt, $dna_G_wt), // A or G or T 'B' => array($dna_C_wt, $dna_G_wt), // C or G or T 'X' => array($dna_C_wt, $dna_G_wt), // G, A, T or C 'N' => array($dna_C_wt, $dna_G_wt) // G, A, T or C ); $rna_wts = array('A' => array($rna_A_wt, $rna_A_wt), // Adenine 'C' => array($rna_C_wt, $rna_C_wt), // Cytosine 'G' => array($rna_G_wt, $rna_G_wt), // Guanine 'U' => array($rna_U_wt, $rna_U_wt), // Uracil 'M' => array($rna_C_wt, $rna_A_wt), // A or C 'R' => array($rna_A_wt, $rna_G_wt), // A or G 'W' => array($rna_U_wt, $rna_A_wt), // A or U 'S' => array($rna_C_wt, $rna_G_wt), // C or G 'Y' => array($rna_C_wt, $rna_U_wt), // C or U 'K' => array($rna_U_wt, $rna_G_wt), // G or U 'V' => array($rna_C_wt, $rna_G_wt), // A or C or G 'H' => array($rna_C_wt, $rna_A_wt), // A or C or U 'D' => array($rna_U_wt, $rna_G_wt), // A or G or U 'B' => array($rna_C_wt, $rna_G_wt), // C or G or U 'X' => array($rna_C_wt, $rna_G_wt), // G, A, U or C 'N' => array($rna_C_wt, $rna_G_wt) // G, A, U or C ); $all_na_wts = array('DNA' => $dna_wts, 'RNA' => $rna_wts); //print_r($all_na_wts); $na_wts = $all_na_wts[$moltype]; $mwt = 0; $NA_len = strlen($sequence); if($limit=="lowerlimit"){$wlimit=1;} if($limit=="upperlimit"){$wlimit=0;} for ($i = 0; $i < $NA_len; $i++) { $NA_base = substr($sequence, $i, 1); $mwt += $na_wts[$NA_base][$wlimit]; } $mwt += $water; return $mwt; } ?>