BioPHP - Melting Temperature (Tm) calculation (original)
Original code submitted by josebaCode bellow is covered by GNU GPL v2 license.
Description
Last change: 2013/05/05 14:20 | Recent ChangesThis tool will calculate melting temperature for an oligonucleotide. Formulas are explained in the script.
Code
Last change: 2013/05/05 14:20 | Download original | Recent Changes | Original code and
<?php
// author Joseba Bikandi
// license GNU GPL v2
// source code available at biophp.org
?>
<html><head><title>Melting Temperature (Tm) Calculation</title></head><body bgcolor=FFFFFF>
<center><table border=0><tr><td>
<center><h2>Melting Temperature (Tm) Calculation</h2></center>
<?php
error_reporting(0);
$primer="";
$primer=strtoupper($_GET["primer"]);
$primer=preg_replace("/\W|[^ATGCYRWSKMDVHBN]|\d/","",$primer);
?>
<form method=get action=<? print $_SERVER["PHP_SELF"]; ?>>
<b>Primer </b>(6-50 bases):<br>
<input type=text name=primer value="<? print $primer; ?>" size=40>
<input type=submit value="Calculate Tm">
<?
if ($primer!="" and strlen($primer)>=6 and strlen($primer)<=50){
print "<table width=100%><tr><td bgcolor=DDDDFF><pre>LENGTH ".strlen($primer)."\n";
$cg=round(100*CountCG($primer)/strlen($primer),1);
print "C+G% $cg\n";
Mol_wt($primer);
print "</pre></td></tr></table>\n";
}
?>
<table width=100%>
<tr>
<td valign=top>
<input type=checkbox name=basic value=1<? if ($_GET["basic"]==1){print " checked";} ?>>
</td>
<td valign=top>
<a href=?formula=basic>Basic Tm</a>
<br><font size=-1> Degenerated nucleotides are allowed</a></font>
</td>
<?
if($primer!="" and $_GET["basic"]==1){
print "<tr><td> </td><td bgcolor=DDDDFF><pre>";
if (strlen($primer)!=CountATCG($primer)){
print "Minimun <font color=880000><b>".Tm_min($primer)." °C</b></font>\n";
print "Maximum <font color=880000><b>".Tm_max($primer)." °C</b></font>";
}else{
print "Tm: <font color=880000><b>".Tm_min($primer)." °C</b></font>";
}
print "</pre></td>";
}
?>
</tr>
<tr>
<td valign=top>
<input type=checkbox name=NearestNeighbor value=1<? if ($_GET["NearestNeighbor"]==1){print " checked";} ?>>
</td>
<td valign=top>
<a href=?formula=Base-Stacking>Base-Stacking Tm</a>
<br><font size=-1> Degenerated nucleotides are NOT allowed</a></font>
<br>Primer concentration: <input type=text name=cp value=<? if ($_GET["cp"]==""){print "200";}else{print $_GET["cp"];} ?> size=4> nM
<br>Salt concentration: <input type=text name=cs value=<? if ($_GET["cs"]==""){print "50";}else{print $_GET["cs"];} ?> size=4> mM
<br>Mg<font size=-2><sup>2+</sup></font> concentration: <input type=text name=cmg value=<? if ($_GET["cmg"]==""){print "0";}else{print $_GET["cmg"];} ?> size=4> mM
</td>
<?
if($primer!="" and $_GET["NearestNeighbor"]==1){
print "<tr><td> </td><td bgcolor=DDDDFF><pre>";
tm_Base_Stacking($primer,$_GET["cp"],$_GET["cs"],$_GET["cmg"]);
print "</pre></td>";
}
?>
</tr>
</table>
</form>
<hr>
<font size=-1>
Source code is freely downloable at <a href=http://www.biophp.org/minitools/melting_temperature/>biophp.org</a>
</font>
</td></tr></table></center>
<? if ($_GET["formula"]=="basic"){ ?>
<hr>
<h2>Basic Melting Temperature (Tm) Calculations</h2>
Two standard approximation calculations are used.
<p>For sequences less than 14 nucleotides
the formula is:
<p> Tm= (wA+xT) * 2 + (yG+zC) * 4
<p> where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively.
<p>For sequences longer than 13 nucleotides, the equation used is
<p> Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC)
<p>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.
<p><pre> Example:
Primer sequence: CTCT<b>RY</b>CT<b>WS</b>CTCTCT
Sequence for minimum Tm calculation: CTCT<b>AT</b>CT<b>AG</b>CTCTCT
Sequence for maximum Tm calculation: CTCT<b>GC</b>CT<b>AG</b>CTCTCT</pre>
<p><b>ASSUMPTIONS:</b>
<p>Both equations assume that the annealing occurs under the standard conditions of 50 nM primer, 50
mM Na<sup><font size=-2>+</font></sup>, and pH 7.0.
<? } ?>
<? if ($_GET["formula"]=="Base-Stacking"){ ?>
<hr>
<h2>Base-Stacking Melting Temperature (Tm) Calculations</h2>
This aproximation uses Thermodynamical concepts to compute T<sub>m</sub>.
The following references were used to develop the script:
<p>SantaLucia J. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor
thermodynamics. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1460-5.
<a href=http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=9465037>NCBI</a>
<p>von Ahsen N, Oellerich M, Armstrong VW, Schütz E. Application of a thermodynamic nearest-neighbor
model to estimate nucleic acid stability and optimize probe design: prediction of melting points
of multiple mutations of apolipoprotein B-3500 and factor V with a hybridization probe genotyping
assay on the LightCycler. Clin Chem. 1999 Dec;45(12):2094-101.
<a href=http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10585340>NCBI</a>
<? } ?>
</body></html>
<?
function tm_Base_Stacking($c,$conc_primer,$conc_salt,$conc_mg){
if (CountATCG($c)!= strlen($c)){print "The oligonucleotide is not valid";return;}
$h=$s=0;
// from table at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC19045/table/T2/ (SantaLucia, 1998)
// enthalpy values
$array_h["AA"]= -7.9;
$array_h["AC"]= -8.4;
$array_h["AG"]= -7.8;
$array_h["AT"]= -7.2;
$array_h["CA"]= -8.5;
$array_h["CC"]= -8.0;
$array_h["CG"]=-10.6;
$array_h["CT"]= -7.8;
$array_h["GA"]= -8.2;
$array_h["GC"]= -9.8;
$array_h["GG"]= -8.0;
$array_h["GT"]= -8.4;
$array_h["TA"]= -7.2;
$array_h["TC"]= -8.2;
$array_h["TG"]= -8.5;
$array_h["TT"]= -7.9;
// entropy values
$array_s["AA"]=-22.2;
$array_s["AC"]=-22.4;
$array_s["AG"]=-21.0;
$array_s["AT"]=-20.4;
$array_s["CA"]=-22.7;
$array_s["CC"]=-19.9;
$array_s["CG"]=-27.2;
$array_s["CT"]=-21.0;
$array_s["GA"]=-22.2;
$array_s["GC"]=-24.4;
$array_s["GG"]=-19.9;
$array_s["GT"]=-22.4;
$array_s["TA"]=-21.3;
$array_s["TC"]=-22.2;
$array_s["TG"]=-22.7;
$array_s["TT"]=-22.2;
// effect on entropy by salt correction; von Ahsen et al 1999
// Increase of stability due to presence of Mg;
$salt_effect= ($conc_salt/1000)+(($conc_mg/1000) * 140);
// effect on entropy
$s+=0.368 * (strlen($c)-1)* log($salt_effect);
// terminal corrections. Santalucia 1998
$firstnucleotide=substr($c,0,1);
if ($firstnucleotide=="G" or $firstnucleotide=="C"){$h+=0.1; $s+=-2.8;}
if ($firstnucleotide=="A" or $firstnucleotide=="T"){$h+=2.3; $s+=4.1;}
$lastnucleotide=substr($c,strlen($c)-1,1);
if ($lastnucleotide=="G" or $lastnucleotide=="C"){$h+=0.1; $s+=-2.8;}
if ($lastnucleotide=="A" or $lastnucleotide=="T"){$h+=2.3; $s+=4.1;}
// compute new H and s based on sequence. Santalucia 1998
for($i=0; $i<strlen($c)-1; $i++){
$subc=substr($c,$i,2);
$h+=$array_h[$subc];
$s+=$array_s[$subc];
}
$tm=((1000*$h)/($s+(1.987*log($conc_primer/2000000000))))-273.15;
print "Tm: <font color=880000><b>".round($tm,1)." °C</b></font>";
print "\n<font color=008800> Enthalpy: ".round($h,2)."\n Entropy: ".round($s,2)."</font>";
}
function Mol_wt($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;
}
?>