"

From 2012.igem.org

Revision as of 00:00, 12 July 2012 (view source) Sander.kromwijk (Talk | contribs) ← Older edit		Revision as of 00:25, 12 July 2012 (view source) Sander.kromwijk (Talk | contribs) Newer edit →
Line 378:		Line 378:
	"AAGCTT": "HindIII",		"AAGCTT": "HindIII",
	"GCGGCCGC": "NotI",		"GCGGCCGC": "NotI",
		+	"TTATAA": "PsiI",
	"CTGCAG": "PstI",		"CTGCAG": "PstI",
	"CCGCGG": "SacII",		"CCGCGG": "SacII",

---

Revision as of 00:25, 12 July 2012

window.tails = [ { name: "Biobrick (gene)", value: "biobrickGene", primer1Tail: "GTT TCT TCG AAT TCG CGG CCG CTT CTA G", primer2Tail: "GTT TCT TCC TGC AGC GGC CGC TAC TAG TA TTA TTA", messages: [ "Don't forget to include the first ATG and to exclude the stop codon!", "The recommended minimum length of a BioBrick primer is 20bp", "The recommended Tm of a BioBrick primer is between 55 and 65 °C", "Check that the following restriction sites are absent: EcoRI, SpeI, XbaI, PstI, NotI", "Check out <a href='http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication'>OpenWetWare's reference page</a>" ], validate: function(primer1, primer2){ var start = primer1.substr(0, 3).toUpperCase();

if(start != "ATG"){ return "The starting ATG seems to be absent." } var end = primer2.substr(0, 3).toUpperCase(); // TAA TGA TAG if(end == "TTA" || end == "TCA" || end == "CTA"){ return "Remove the stop codons from the sequence!" } } }, { name: "Biobrick (other)", value: "biobrickOther", primer1Tail: "GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG", primer2Tail: "GTT TCT TCC TGC AGC GGC CGC TAC TAG TA", messages: [ "The recommended minimum length of a BioBrick primer is 20bp", "The recommended Tm of a BioBrick primer is between 55 and 65 °C", "Check that the following restriction sites are absent: EcoRI, SpeI, XbaI, PstI, NotI", "Check out <a href='http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication'>OpenWetWare's reference page</a>" ] } ]; window.calc = function(){ var data = document.getElementById("data").value; var isNormal = document.getElementById("primerTypeNormal").checked; var isCustom = document.getElementById("primerTypeCustom").checked; var isRS = document.getElementById("primerTypeRestrictionSites").checked; var isKnown = document.getElementById("primerTypeKnownTail").checked; var isNumbered = document.getElementById("toggleLines").checked; var goalTm = document.getElementById("goalTm").value;

try{ var messages = []; var toAdd = {}; var p = new Primer(data); var validate = function(primer1, primer2){};

if(isCustom){ p.setTails(Sequence.fromString(document.getElementById("primer1Tail").value), Sequence.fromString(document.getElementById("primer2Tail").value)); }

if(isRS){ var rs1 = Sequence.fromString(document.getElementById("primer1RS").value); var rs2 = Sequence.fromString(document.getElementById("primer2RS").value);

var match1 = rs1.findRS(); var match2 = rs2.findRS(); toAdd.primer1RSMatch = match1.length > 0 ? "- "+match1 : ""; toAdd.primer2RSMatch = match2.length > 0 ? "- "+match2 : "";

p.setTails(rs1, rs2.reverseComplement()); p.addRandomEdges(4);

messages.push("Click the 'Calculate' button to regenerate the random bases added on the end of the primers."); messages.push( "In the case of a plasmid where the piece between A and B is cut out, and A is used for the primer 1 RS (and B for the second). " + "The generated primers will put the RS such that the given DNA starts at the A side and ends at the B side (in short, " + "it will probably work as you expected, but still check what happens with some other tool)." ); }

if(isKnown){ var selected = $("#knownTailSelect").val();

var tail1 = ""; var tail2 = "";

for(var i = 0; i < window.tails.length; i++){ if(window.tails[i].value == selected){ tail1 = window.tails[i].primer1Tail; tail2 = window.tails[i].primer2Tail; if(window.tails[i].messages) messages = [].concat(window.tails[i].messages); if(window.tails[i].validate) validate = window.tails[i].validate; } }

p.setTails(Sequence.fromString(tail1), Sequence.fromString(tail2)); }

if(!p.isSameAsLast(data, goalTm) || window.isCurrentlyNumbered != isNumbered){ window.isCurrentlyNumbered = isNumbered; var primers = p.generate(goalTm);

for(var key in toAdd){ primers[key] = toAdd[key]; }

var result = validate(primers.primer1NoTail, primers.primer2NoTail);

if(result) messages.push(result);

if(messages.length > 0){ window.output({ primers: primers, messages: messages, numbered: isNumbered }); }else{ window.output({ primers: primers, numbered: isNumbered }); } } }catch(e){ window.output({ error: e.message, numbered: isNumbered }); } }; window.isCurrentlyNumbered = -1; window.output = function(options){ var output = { message: [], primer1: "No data", primer1Temp: "NaN", primer1bp: "NaN", primer1Tailbp: "NaN", primer1RSMatch: "", primer2: "No data", primer2Temp: "NaN", primer2bp: "NaN", primer2Tailbp: "NaN", primer2RSMatch: "", full: "", numbered: false }; if(options.numbered){ output.numbered = options.numbered; }

if(options.error){ output.message.push(options.error); } if(options.messages){ output.message = output.message.concat(options.messages); } if(options.primers){ for(var key in options.primers){ if(key == "message"){ output[key].push(options.primers[key]); }else{ output[key] = options.primers[key]; } } }

if(Math.abs(output.primer1Temp - output.primer2Temp) > 5){ output.message.push("The difference is more than 5 °C"); }

if(Math.min(output.primer1Temp, output.primer2Temp) > 72){ output.message.push("Consider using the two-step PCR protocol"); }

if(Math.min(output.primer1Temp, output.primer2Temp) < 45){ output.message.push("The annealing temperature should be more than 45 °C"); }

document.getElementById("primer1RSMatch").innerHTML = output.primer1RSMatch; document.getElementById("primer2RSMatch").innerHTML = output.primer2RSMatch;

document.getElementById("messages").innerHTML = output.message.length > 0 ? "

• "+output.message.join("
• ")+"

" : "";

document.getElementById("primer1").innerHTML = output.primer1; document.getElementById("primer1Temp").innerHTML = output.primer1Temp; document.getElementById("primer1bp").innerHTML = output.primer1Tailbp != 0 && output.primer1Tailbp != "NaN" ? output.primer1Tailbp+" + "+output.primer1bp : output.primer1bp; document.getElementById("primer2").innerHTML = output.primer2; document.getElementById("primer2Temp").innerHTML = output.primer2Temp; document.getElementById("primer2bp").innerHTML = output.primer2Tailbp != 0 && output.primer2Tailbp != "NaN" ? output.primer2Tailbp+" + "+output.primer2bp : output.primer2bp; document.getElementById("annealingTemp").innerHTML = Math.min(output.primer1Temp, output.primer2Temp);

var width = 80; var lines = window.wrap(output.full, width); var outputLines = []; if(output.numbered){ for(var i = 0; i < lines.length; i++){ outputLines[i] = window.pad(i*width+1, 6, true)+" - "+window.pad((i+1)*width, 6)+" "+lines[i]; } }else{ outputLines = lines; } document.getElementById("finalResult").innerHTML = outputLines.join("\n"); };

window.pad = function(str, width, right){ str += ""; while(str.length < width){ if(right){ str = " "+str; }else{ str += " "; } } return str; }

window.wrap = function(str, width){ var chunks = []; var i = 0, l = str.length; while(i < l){ if(i + width >= l){ chunks.push(str.substr(i, l-i)); }else{ chunks.push(str.substr(i, width)); } i += width; } return chunks; };

window.updateRadioElements = function(){ if(document.getElementById('primerTypeRestrictionSites').checked){ $('#restrictionSiteRow').show(); }else{ $('#restrictionSiteRow').hide(); } if(document.getElementById('primerTypeCustom').checked){ $('#customRow').show(); }else{ $('#customRow').hide(); } if(document.getElementById('primerTypeKnownTail').checked){ $('#knownTailRow').show(); }else{ $('#knownTailRow').hide(); } };var Primer;

Primer = (function() {

 Primer.minLength = 8;

 Primer.maxLength = 50;

 Primer.maxDiff = 8;

 Primer.lastData = "";

 Primer.lastTail1 = "";

 Primer.lastTail2 = "";

 Primer.lastTm = 0;

 Primer.lastRandEdgeSize = -1;

 Primer.reset = function() {
   this.lastData = "";
   this.lastTm = 0;
   this.lastTail1 = "";
   this.lastTail2 = "";
   return this.lastRandEdgeSize = -1;
 };

 function Primer(data) {
   this.seq = Sequence.fromString(data);
   this.tail1 = new Sequence([]);
   this.tail2 = new Sequence([]);
   this.randEdgeSize = 0;
 }

 Primer.prototype.setTails = function(tail1, tail2) {
   this.tail1 = tail1;
   this.tail2 = tail2;
 };

 Primer.prototype.addRandomEdges = function(randEdgeSize) {
   this.randEdgeSize = randEdgeSize;
 };

 Primer.prototype.isSameAsLast = function(data, tm) {
   return Primer.lastTail1.toString() === this.tail1.toString() && Primer.lastTail2.toString() === this.tail2.toString() && Primer.lastData === Sequence.fromString(data).toString() && Primer.lastTm === tm && Primer.lastRandEdgeSize === this.randEdgeSize;
 };

 Primer.prototype.generate = function(goal) {
   var diff, l1, l2, maxLength, primer1Length, primer1Temp, primer2Length, primer2Temp, rand1, rand2, revCompl, temp, _i, _j, _ref, _ref1;
   if (2 * Primer.minLength > this.seq.length()) {
     throw new Error("The given sequence is too short");
   }
   Primer.lastData = this.seq.toString();
   Primer.lastTm = goal;
   Primer.lastTail1 = this.tail1;
   Primer.lastTail2 = this.tail2;
   Primer.lastRandEdgeSize = this.randEdgeSize;
   maxLength = Math.min(Math.floor(this.seq.length() / 2), Primer.maxLength);
   revCompl = this.seq.reverseComplement();
   primer1Length = -1;
   primer1Temp = 0;
   primer2Length = -1;
   primer2Temp = 0;
   for (l1 = _i = _ref = Primer.minLength; _ref <= maxLength ? _i <= maxLength : _i >= maxLength; l1 = _ref <= maxLength ? ++_i : --_i) {
     temp = this.seq.snip(l1).tm();
     diff = Math.abs(temp - goal);
     if (diff <= Primer.maxDiff && (primer1Length === -1 || Math.abs(primer1Temp - goal) > diff)) {
       primer1Length = l1;
       primer1Temp = temp;
     }
     if (temp >= goal) {
       break;
     }
   }
   for (l2 = _j = _ref1 = Primer.minLength; _ref1 <= maxLength ? _j <= maxLength : _j >= maxLength; l2 = _ref1 <= maxLength ? ++_j : --_j) {
     temp = revCompl.snip(l2).tm();
     diff = Math.abs(temp - goal);
     if (diff <= Primer.maxDiff && (primer2Length === -1 || Math.abs(primer2Temp - goal) > diff)) {
       primer2Length = l2;
       primer2Temp = temp;
     }
     if (temp >= goal) {
       break;
     }
   }
   if (primer1Length < 0 || primer2Length < 0) {
     throw new Error("Couldn't find a primer of correct length that satisfies the conditions");
   }
   rand1 = Sequence.fromRandom(this.randEdgeSize);
   rand2 = Sequence.fromRandom(this.randEdgeSize);
   return {
     primer1: rand1.toString() + " " + this.tail1.toString() + " " + this.seq.snip(primer1Length).toString(),
     primer1NoTail: this.seq.snip(primer1Length).toString(),
     primer1Temp: primer1Temp,
     primer1bp: primer1Length,
     primer1Tailbp: rand1.length() + this.tail1.length(),
     primer2: rand2.toString() + " " + this.tail2.toString() + " " + revCompl.snip(primer2Length).toString(),
     primer2NoTail: revCompl.snip(primer2Length).toString(),
     primer2Temp: primer2Temp,
     primer2bp: primer2Length,
     primer2Tailbp: rand2.length() + this.tail2.length(),
     full: rand1.toString() + this.tail1.toString() + this.seq.toString() + this.tail2.reverseComplement().toString() + rand2.reverseComplement().toString()
   };
 };

 return Primer;

})(); /* Immutable class that makes it possible to work with (very simplified) DNA sequences

• /

var Sequence,

 __indexOf = [].indexOf || function(item) { for (var i = 0, l = this.length; i < l; i++) { if (i in this && this[i] === item) return i; } return -1; };

Sequence = (function() {

 Sequence.allowed = ["A", "C", "G", "T"];

 Sequence.RS = {
   "CCTAGG": "AvrII",
   "ATCGAT": "ClaI",
   "GGATCC": "BamHI",
   "TGATCA": "BclI",
   "AGATCT": "BglII",
   "TTTAAA": "DraI",
   "GAATTC": "EcoRI",
   "GATATC": "EcoRV",
   "AAGCTT": "HindIII",
   "GCGGCCGC": "NotI",
   "TTATAA": "PsiI",
   "CTGCAG": "PstI",
   "CCGCGG": "SacII",
   "GTCCAG": "SalI",
   "CCTGCAGG": "SbfI",
   "GGCCNNNNNGGCC": "SfiI",
   "CCCGGG": "SmaI/XmaI",
   "ACTAGT": "SpeI",
   "TCTAGA": "XbaI"
 };

 Sequence.fromString = function(data) {
   var seq;
   seq = new Sequence(data.toUpperCase().replace(/\s/ig, "").split());
   seq.validateData();
   return seq;
 };

 Sequence.fromRandom = function(length) {
   var data, i;
   if (length === 0) {
     return new Sequence([]);
   } else {
     data = (function() {
       var _i, _results;
       _results = [];
       for (i = _i = 1; 1 <= length ? _i <= length : _i >= length; i = 1 <= length ? ++_i : --_i) {
         _results.push(Sequence.allowed[Math.floor(Math.random() * Sequence.allowed.length)]);
       }
       return _results;
     })();
     return new Sequence(data);
   }
 };

 /*
   The constructor assumes the data has been validated (for performance reasons -> premature and non-consequent
   optimization ^^). Anyway, the Sequence.fromString function is probably what you were looking for!
 */

 function Sequence(data) {
   this.data = data;
 }

 Sequence.prototype.validateData = function() {
   if (this.data.some(function(x) {
     return __indexOf.call(Sequence.allowed, x) < 0;
   })) {
     throw new Error("Invalid character encountered");
   }
 };

 Sequence.prototype.findRS = function() {
   var i, key, match, name, seq, _i, _ref, _ref1;
   key = this.data.join("");
   if (Sequence.RS[key]) {
     return Sequence.RS[key];
   } else {
     _ref = Sequence.RS;
     for (seq in _ref) {
       name = _ref[seq];
       if (seq.indexOf("N") >= 0 && seq.length === key.length) {
         match = true;
         for (i = _i = 0, _ref1 = seq.length - 1; 0 <= _ref1 ? _i <= _ref1 : _i >= _ref1; i = 0 <= _ref1 ? ++_i : --_i) {
           if (seq[i] !== "N" && seq[i] !== key[i]) {
             match = false;
           }
         }
         if (match) {
           return name;
         }
       }
     }
     return "";
   }
 };

 Sequence.prototype.length = function() {
   return this.data.length;
 };

 Sequence.prototype.snip = function(length) {
   if (length >= 0 && length <= this.data.length) {
     return new Sequence(this.data.slice(0, length));
   } else {
     throw new Error("Invalid snip length (tried to snip a piece of length " + length + " in a sequence of " + this.length() + "bp)");
   }
 };

 Sequence.prototype.reverseComplement = function() {
   var complement, k, l, revCompl, v, _i, _len, _ref;
   revCompl = [];
   complement = function(x) {
     switch (x) {
       case "A":
         return "T";
       case "T":
         return "A";
       case "C":
         return "G";
       case "G":
         return "C";
       default:
         throw new Error("Invalid character encountered (" + x + ")");
     }
   };
   l = this.data.length - 1;
   _ref = this.data;
   for (k = _i = 0, _len = _ref.length; _i < _len; k = ++_i) {
     v = _ref[k];
     revCompl[l - k] = complement(v);
   }
   return new Sequence(revCompl);
 };

 Sequence.prototype.append = function(seqToAdd) {
   return new Sequence(this.data.concat(seqToAdd.data));
 };

 Sequence.prototype.tm = function() {
   return TmCalc.calc(this.data);
 };

 Sequence.prototype.toString = function() {
   return this.data.join("");
 };

 return Sequence;

})(); /* Container class that helps with Tm calculations. It has been seperated to enable easy improvements to the algorithm.

• /

var TmCalc;

TmCalc = (function() {

 function TmCalc() {}

 TmCalc.maxCacheSize = 500;

 TmCalc.cache = {};

 TmCalc.calc = function(data) {
   var key, keys, result;
   if (!this.cache[data]) {
     result = Math.round(this.calcFull(data) * 10) / 10;
     if (this.cache.length >= this.maxCacheSize) {
       keys = Object.keys(TmCalc.cache);
       key = keys[Math.floor(Math.random() * keys.length)];
       delete TmCalc.cache[key];
     }
     this.cache[data] = result;
   }
   return this.cache[data];
 };

 TmCalc.complement = function(n) {
   switch (n) {
     case "A":
       return "T";
     case "T":
       return "A";
     case "C":
       return "G";
     case "G":
       return "C";
     default:
       throw new Error("Invalid character encountered (" + n + ")");
   }
 };

 TmCalc.neighborH = {
   AA: 9.1,
   AT: 8.6,
   TA: 6.0,
   CA: 5.8,
   GT: 6.5,
   CT: 7.8,
   GA: 5.6,
   CG: 11.9,
   GC: 11.1,
   GG: 11.0
 };

 TmCalc.getH = function(n1, n2) {
   return this.get(this.neighborH, n1, n2);
 };

 TmCalc.neighborG = {
   AA: 1.9,
   AT: 1.5,
   TA: 0.9,
   CA: 1.9,
   GT: 1.3,
   CT: 1.6,
   GA: 1.6,
   CG: 3.6,
   GC: 3.1,
   GG: 3.1
 };

 TmCalc.getG = function(n1, n2) {
   return this.get(this.neighborG, n1, n2);
 };

 TmCalc.neighborS = {
   AA: 24.0,
   AT: 23.9,
   TA: 16.9,
   CA: 12.9,
   GT: 17.3,
   CT: 20.8,
   GA: 13.5,
   CG: 27.8,
   GC: 26.7,
   GG: 26.6
 };

 TmCalc.getS = function(n1, n2) {
   return this.get(this.neighborS, n1, n2);
 };

 TmCalc.get = function(searchVar, n1, n2) {
   switch (n1 + n2) {
     case "AA":
       return searchVar["AA"];
     case "TT":
       return searchVar["AA"];
     case "AT":
       return searchVar["AT"];
     case "TA":
       return searchVar["TA"];
     case "TT":
       return searchVar["TA"];
     case "CA":
       return searchVar["CA"];
     case "TG":
       return searchVar["CA"];
     case "GT":
       return searchVar["GT"];
     case "AC":
       return searchVar["GT"];
     case "CT":
       return searchVar["CT"];
     case "AG":
       return searchVar["CT"];
     case "GA":
       return searchVar["GA"];
     case "TC":
       return searchVar["GA"];
     case "CG":
       return searchVar["CG"];
     case "TC":
       return searchVar["CG"];
     case "GC":
       return searchVar["GC"];
     case "TC":
       return searchVar["GC"];
     case "GG":
       return searchVar["GG"];
     case "CC":
       return searchVar["GG"];
     default:
       throw new Error("No idea what happened... (tried getting " + n1 + n2 + ")");
   }
 };

 TmCalc.calcFull = function(data) {
   /* See (Breslauer et al. 1986): http://www.pnas.org/content/83/11/3746.full.pdf
   */

   var R, i, n1, n2, naConc, predG, predH, predS, primerConc, _i, _ref;
   predH = 0;
   predG = -(5 + 0.4);
   predS = 0;
   primerConc = 500 * 10e-9;
   naConc = 0.03;
   R = 1.987;
   n1 = data[0];
   for (i = _i = 1, _ref = data.length - 1; 1 <= _ref ? _i <= _ref : _i >= _ref; i = 1 <= _ref ? ++_i : --_i) {
     n2 = data[i];
     predH += this.getH(n1, n2);
     predG += this.getG(n1, n2);
     predS += this.getS(n1, n2);
     n1 = n2;
   }
   return (1000 * (predH - 3.4) / (predS + R * Math.log(1 / primerConc))) - 272.9 + 7.21 * Math.log(naConc / 1000) / Math.log(10);
 };

 /* Again, old
 @calcFull: (data, options) ->
   ##
   For more details see: http://www.basic.northwestern.edu/biotools/oligocalc.html
   ##
   freq = @calcFrequencies(data)
   
   if not options
     options = {}
   
   if not options.Na
     options.Na = 0.05
   
   GC = freq["G"] + freq["C"]
   AT = freq["A"] + freq["T"]
   N = data.length
   
   if N < 14
     AT*2 + GC*4 - 16.6*Math.log(0.050)/Math.log(10) + 16.6*Math.log(options.Na)/Math.log(10)
   else if N < 50 #18
     100.5 + (41 * GC/N) - (820/N) + 16.6*Math.log(options.Na)/Math.log(10)
   #else if N < 50
   #  81.5 + (41 * GC/N) - (500/N) + 16.6*Math.log(options.Na) - 0.62*options.F
   else
     79.8 + 18.5*Math.log(options.Na)/Math.log(10) + (58.4 * GC/N) + (11.8 * (GC/N) * (GC/N)) - (820/N) 
   
   
 
 @calcFrequencies: (data) ->
   freq = {A: 0, C: 0, G: 0, T: 0}
   data.forEach (x) ->
     freq[x]++
   freq
 */

 return TmCalc;

})();

/* Old version (not very accurate):

 @calc: (data) ->
   Math.round(@calcFull(data)*100)/100
 
 @calcFull: (data) ->
   freq = @calcFrequencies(data)
   if data.length < 14
     4 * (freq["G"] + freq["C"]) + 2 * (freq["A"] + freq["T"])
   else
     64.9 + 41 * (freq["G"] + freq["C"] - 16.4)/data.length
 
 @calcFrequencies: (data) ->
   freq = {A: 0, C: 0, G: 0, T: 0}
   data.forEach (x) ->
     freq[x]++
   freq

• /