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<!doctype html>
<!--
Material Design Lite
Copyright 2015 Google Inc. All rights reserved.
Licensed under the Apache License, Version 2.0 (the "License");
you may not use this file except in compliance with the License.
You may obtain a copy of the License at
https://www.apache.org/licenses/LICENSE-2.0
Unless required by applicable law or agreed to in writing, software
distributed under the License is distributed on an "AS IS" BASIS,
WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied.
See the License for the specific language governing permissions and
limitations under the License
-->
<html lang="en">
<head>
<link rel="shortcut icon" href="./favicon.ico" />
<meta charset="utf-8">
<meta http-equiv="X-UA-Compatible" content="IE=edge">
<meta name="description" content="HTP-OligoDesigner: an online primer design tool for high-throughput gene cloning and site-directed mutagenesis">
<meta name="viewport" content="width=device-width, initial-scale=1">
<meta name="theme-color" content="#1976D2">
<meta name="Keywords" content="htp, primer, oligo, designer, amplification, cloning, DNA, RNA, protein, create, primers, tm, calculator, USP, melting temperature, breslauer, sugimoto">
<title>HTP Oligo Designer</title>
<link href="https://fonts.googleapis.com/css?family=Roboto+Mono:regular,bold,italic,thin,light,bolditalic,black,medium&lang=en" rel="stylesheet">
<link href="https://fonts.googleapis.com/icon?family=Material+Icons"
rel="stylesheet">
<link rel="stylesheet" href="./material.min.css">
<link rel="stylesheet" href="./styles.css">
<style>
#view-source {
position: fixed;
display: block;
right: 0;
bottom: 0;
margin-right: 40px;
margin-bottom: 40px;
z-index: 900;
}
</style>
</head>
<body class="mdl-demo mdl-color--grey-100 mdl-color-text--grey-700 mdl-base" >
<div class="mdl-layout mdl-js-layout mdl-layout--fixed-header">
<header class="mdl-layout__header mdl-layout__header--scroll mdl-color--primary">
<div class="mdl-layout--large-screen-only mdl-layout__header-row">
</div>
<div class="mdl-layout__header-row" style="display: inline-flex;
justify-items: flex-center;
align-items: center;
justify-content: center;">
<h2><b>HTP Oligo Designer</b></h2>
</div>
<div class="mdl-layout--large-screen-only mdl-layout__header-row" >
</div>
<div class="mdl-layout--large-screen-only mdl-layout__tab-bar mdl-js-ripple-effect mdl-color--primary-dark" style= "display: inline-flex;
justify-items: flex-center;
align-items: center;
justify-content: center;">
<a href="#amplification" class="mdl-layout__tab is-active"><h1>Gene amplification</h1></a>
<a href="#mutagenesis" class="mdl-layout__tab"><h1>Mutagenesis</h1></a>
<a href="#tmcalculator" class="mdl-layout__tab"><h1>TM Calculator</h1></a>
<a href="#tutorial" class="mdl-layout__tab"><h1>Tutorial</h1></a>
<a href="#about" class="mdl-layout__tab" hidden ><h1>About</h1></a>
<button class="mdl-button mdl-js-button mdl-button--fab mdl-js-ripple-effect mdl-button--colored mdl-shadow--4dp mdl-color--accent" id="add" onClick="window.open('https://gustalima.github.io/htpoligo/')">
<i class="material-icons">add</i>
<a href="http://www.w3schools.com/html/"></a>
</button>
</div>
</header>
<div class="mdl-layout__drawer">
<span class="mdl-layout-title">HTP Oligo Designer</span>
<nav class="mdl-navigation">
<a href="#amplification" class="mdl-navigation__link">Gene amplification</a>
<a href="#mutagenesis" class="mdl-navigation__link">Mutagenesis</a>
<a href="#tmcalculator" class="mdl-navigation__link">TM Calculator</a>
<a href="#tutorial" class="mdl-navigation__link">Tutorial</a>
<a href="#about" class="mdl-navigation__link">About</a>
</nav>
</div>
<main class="mdl-layout__content">
<div class="mdl-layout__tab-panel is-active mdl-color--grey-100" id="amplification">
<section class="section--center mdl-grid mdl-grid--no-spacing">
<!-- Event card -->
<style>
.demo-card-event.mdl-card {
width: 860px;
}
.demo-card-event > .mdl-card__title,
.demo-card-event > .mdl-card__actions > .mdl-button {
color: #0000;
}
</style>
<div class="mdl-card mdl-shadow--2dp demo-card-event">
<div class="mdl-card__title mdl-card--expand">
<!-- Floating Multiline Textfield -->
<form action="#">
<div class="mdl-textfield mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<textarea class="mdl-textfield__input" style="color:black;overflow:auto;resize:none;font-family:'Roboto Mono'" type="text" rows= "15" id="SeqFASTA"></textarea>
<label class="mdl-textfield__label" for="SeqFASTA">Input data sequences in FASTA format:</label>
</div>
</form>
</div>
<div class="mdl-card__actions mdl-card--border">
<input type="file" id="fileinput" />
</div>
</div>
<table style="width:100%">
<tr>
<style>
.demo-card-square.mdl-card {
width: 280px;
height: 220px;
}
.demo-card-square > .mdl-card__title {
color: #fff;
background: #1976D2;
}
</style>
<td><!-- Square card -->
<div class="mdl-card mdl-shadow--2dp demo-card-square">
<div class="mdl-card__title mdl-card--expand" >
<h2 class="mdl-card__title-text"> Optimal TM</h2>
</div><br><br><br>
<!-- Numeric Textfield -->
<form action="#">
<div class="mdl-textfield mdl-js-textfield mdl-textfield--floating-label textfield-demo" style="width: 280px;">
<input class="mdl-textfield__input" type="text" pattern="-?[0-9]*(\.[0-9]+)?" id="Tm" style="width: 280px;"/>
<label class="mdl-textfield__label" for="Tm"> In ºC...</label>
<span class="mdl-textfield__error">Input is not a number!</span>
</div>
</form>
</div>
</td>
<td><!-- Square card -->
<div class="mdl-card mdl-shadow--2dp demo-card-square">
<div class="mdl-card__title mdl-card--expand" >
<h2 class="mdl-card__title-text"> Primer concentration</h2>
</div><br><br><br>
<!-- Numeric Textfield -->
<form action="#">
<div class="mdl-textfield mdl-js-textfield mdl-textfield--floating-label textfield-demo" style="width: 280px;">
<input class="mdl-textfield__input" type="text" pattern="-?[0-9]*(\.[0-9]+)?" id="pConc" value="500" style="width: 280px;"/>
<label class="mdl-textfield__label" for="pConc"> In nM...</label>
<span class="mdl-textfield__error">Input is not a number!</span>
</div>
</form>
</div>
</td>
<td><!-- Square card -->
<div class="mdl-card mdl-shadow--2dp demo-card-square">
<div class="mdl-card__title mdl-card--expand" >
<h2 class="mdl-card__title-text">Monovalent Cation Concentration</h2>
</div><br><br><br>
<!-- Numeric Textfield -->
<form action="#">
<div class="mdl-textfield mdl-js-textfield mdl-textfield--floating-label textfield-demo" style="width: 280px;">
<input class="mdl-textfield__input" type="text" pattern="-?[0-9]*(\.[0-9]+)?" id="salt" value="0.050" style="width: 280px;"/>
<label class="mdl-textfield__label" for="salt"> In M...</label>
<span class="mdl-textfield__error">Input is not a number!</span>
</div>
</form>
</div>
</td>
</tr>
<tr>
<td><!-- Square card -->
<div class="mdl-card mdl-shadow--2dp demo-card-square">
<div class="mdl-card__title mdl-card--expand" >
<h2 class="mdl-card__title-text">Thermodynamic data for Tm calculation</h2>
</div><br><br><br>
<!-- Numeric Textfield -->
<div class="container">
<form>
<div class="form-group">
<select id="combo">
<option value='Breslauer'>Breslauer et al. (1986)</option>
<option value='Sugimoto'>Sugimoto et al. (1996)</option>
</select>
<i class="bar"></i>
</div>
</form>
</div>
</div>
</td>
<td><!-- Square card -->
<div class="mdl-card mdl-shadow--2dp demo-card-square">
<div class="mdl-card__title mdl-card--expand" >
<h2 class="mdl-card__title-text">Append tag sequence to Forward primer</h2>
</div><br><br><br>
<!-- Numeric Textfield -->
<form action="#">
<div class="mdl-textfield mdl-js-textfield mdl-textfield--floating-label textfield-demo" style="width: 280px;">
<input class="mdl-textfield__input" type="text" pattern="-?[A,T,C,G,a,t,c,g]*(\.[A,T,C,G,a,t,c,g]+)?" id="appendFw" style="width: 280px;" />
<label class="mdl-textfield__label" for="appendFw"> Forward TAG...</label>
<span class="mdl-textfield__error">Use only A, T, C or G</span>
</div>
</form>
</div>
</td>
<td><!-- Square card -->
<div class="mdl-card mdl-shadow--2dp demo-card-square">
<div class="mdl-card__title mdl-card--expand" >
<h2 class="mdl-card__title-text">Append tag sequence to Reverse primer</h2>
</div><br><br><br>
<!-- Numeric Textfield -->
<form action="#">
<div class="mdl-textfield mdl-js-textfield mdl-textfield--floating-label textfield-demo" style="width: 280px;">
<input class="mdl-textfield__input" type="text" pattern="-?[A,T,C,G,a,t,c,g]*(\.[A,T,C,G,a,t,c,g]+)?" id="appendRv" style="width: 280px;" />
<label class="mdl-textfield__label" for="appendRv"> Reverse TAG...</label>
<span class="mdl-textfield__error">Input is not a number!</span>
</div>
</form>
</div>
</td>
</tr>
</table>
<div class="mdl-card__title mdl-card--expand">
<button class="mdl-button mdl-js-button mdl-button--raised mdl-js-ripple-effect" onclick='amplification()' id='ampButton'>
Obtain primers
</button>
<button class="mdl-button mdl-js-button mdl-button--raised mdl-js-ripple-effect" onclick='javascript:eraseText();' id='clearAllAmp'>
Clear all
</button>
<div class="mdl-tooltip" for="clearAllAmp">Clear amplification fields</div>
<div class="mdl-tooltip" for="ampButton">Create primers for your sequences</div>
</div>
<!-- Event card -->
<style>
.demo-card-event.mdl-card {
width: 860px;
}
.demo-card-event > .mdl-card__title,
.demo-card-event > .mdl-card__actions > .mdl-button {
color: #0000;
}
</style>
<div class="mdl-card mdl-shadow--2dp demo-card-event">
<div class="mdl-card__title mdl-card--expand">
<!-- Floating Multiline Textfield -->
<form action="#">
<div class="mdl-textfield mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<textarea class="mdl-textfield__input" style="color:black;overflow:auto;resize:none;font-family:'Roboto Mono'" type="text" rows= "10" id="resultForward" ></textarea>
<label class="mdl-textfield__label" for="resultForward">Forward Primers...</label>
</div>
</form>
</div>
</div>
<div class="mdl-card mdl-shadow--2dp demo-card-event">
<div class="mdl-card__title mdl-card--expand">
<!-- Floating Multiline Textfield -->
<form action="#">
<div class="mdl-textfield mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<textarea class="mdl-textfield__input" style="color:black;overflow:auto;resize:none;font-family:'Roboto Mono'" type="text" rows= "10" id="resultReverse" ></textarea>
<label class="mdl-textfield__label" for="resultReverse">Reverse Primers...</label>
</div>
</form>
</div>
</div>
<div class="mdl-card__title mdl-card--expand">
<button id='dl_amp' class="mdl-button mdl-js-button mdl-button--raised mdl-js-ripple-effect mdl-button--accent" onclick='saveTextAsFile_rv();saveTextAsFile_fw()'>
Download as CSV
</button>
<div class="mdl-tooltip" for="dl_amp">Download as CSV using tabs as delimiter</div>
</div>
</section>
</div>
<div class="mdl-layout__tab-panel mdl-color--grey-100" id="mutagenesis">
<section class="section--center mdl-grid mdl-grid--no-spacing">
<!-- Event card -->
<style>
.demo-card-event.mdl-card {
width: 860px;
}
.demo-card-event > .mdl-card__title,
.demo-card-event > .mdl-card__actions > .mdl-button {
color: #0000;
}
</style>
<div class="mdl-card mdl-shadow--2dp demo-card-event">
<div class="mdl-card__title mdl-card--expand">
<!-- Floating Multiline Textfield -->
<form action="#">
<div class="mdl-textfield mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<textarea class="mdl-textfield__input" style="color:black;overflow:auto;resize:none;font-family:'Roboto Mono'" type="text" rows= "15" id="SeqFASTA_mut" ></textarea>
<label class="mdl-textfield__label" for="SeqFASTA_mut">Input data sequences in FASTA format:</label>
</div>
</form>
</div>
<div class="mdl-card__actions mdl-card--border">
<input type="file" id="fileinput2" />
<button id='helperbutton' class="mdl-button mdl-js-button mdl-button--raised mdl-js-ripple-effect mdl-button--accent" style="float: right; color: white;" onClick="window.open('http://www.ifsc.usp.br/htpoligo/helper.html')">Format your entry with this helper</button>
<div class="mdl-tooltip" for="helperbutton">Format your entry using aminoacids references</div>
</div>
</div>
<table style="width:100%" >
<tr >
<style>
.demo-card-square.mdl-card_wide {
width: 420px;
height: 220px;
}
.demo-card-square > .mdl-card__title {
color: #fff;
background: #1976D2;
}
</style>
<td align='center'><!-- Square card -->
<div class="mdl-card_wide mdl-shadow--2dp demo-card-square">
<div class="mdl-card__title mdl-card--expand" >
<h2 class="mdl-card__title-text" style="height: 40px;margin-top:20px;"> Monovalent Cation Concentration</h2>
</div><br><br><br>
<!-- Numeric Textfield -->
<form action="#">
<div class="mdl-textfield_table mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<input class="mdl-textfield__input" type="text" pattern="-?[0-9]*(\.[0-9]+)?" id="salt_mut" value="0.050"/>
<label class="mdl-textfield__label" for="salt_mut"> In M...</label>
<span class="mdl-textfield__error">Input is not a number!</span>
</div>
</form>
</div>
</td>
<td align='center'><!-- Square card -->
<div class="mdl-card_wide mdl-shadow--2dp demo-card-square">
<div class="mdl-card__title mdl-card--expand" >
<h2 class="mdl-card__title-text" style="height: 40px;margin-top:20px;"> Primer Concentration</h2>
</div><br><br><br>
<!-- Numeric Textfield -->
<form action="#">
<div class="mdl-textfield_table mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<input class="mdl-textfield__input" type="text" pattern="-?[0-9]*(\.[0-9]+)?" id="pConc_mut" value="500"/>
<label class="mdl-textfield__label" for="pConc_mut"> In nM...</label>
<span class="mdl-textfield__error">Input is not a number!</span>
</div>
</form>
</div>
</td>
</tr>
<tr >
<td ><!-- Square card -->
<div class="mdl-card_wide mdl-shadow--2dp demo-card-square">
<div class="mdl-card__title mdl-card--expand" >
<h2 class="mdl-card__title-text" style="height: 60px;">Thermodynamic data for Tm calculation</h2>
</div><br><br><br>
<!-- Numeric Textfield -->
<div class="container">
<form>
<div class="form-group">
<select id="combo_mut">
<option value='Breslauer'>Breslauer et al. (1986)</option>
<option value='Sugimoto'>Sugimoto et al. (1996)</option>
</select>
<i class="bar"></i>
</div>
</form>
</div>
</div>
</td>
<td><!-- Square card -->
<div class="mdl-card_wide mdl-shadow--2dp demo-card-square">
<div class="mdl-card__title mdl-card--expand" >
<h2 class="mdl-card__title-text" style="height: 40px;margin-top:20px;"> Choose design method</h2>
</div><br><br><br>
<!-- Numeric Textfield -->
<table align='center'><tr><td>
<div class="container">
<form>
<div class="form-group">
<select id="mut_type">
<option value='size'>By size</option>
<option value='tm'>By TM</option>
</select>
<i class="bar"></i>
</div>
</form></td><td>
<form action="#">
<div class="mdl-textfield_short mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<input class="mdl-textfield__input" type="text" pattern="-?[0-9]*(\.[0-9]+)?" id="Size_temp_mut" style="width: 70px;" />
<label class="mdl-textfield__label" for="Size_temp_mut">Value</label>
<span class="mdl-textfield__error">Input is not a number!</span>
</div>
</div>
</form></td></tr></table>
</div>
</td>
</tr>
</table>
<div class="mdl-card__title mdl-card--expand">
<button id='mutButton' class="mdl-button mdl-js-button mdl-button--raised mdl-js-ripple-effect" onclick="mutatePrimer()" >
Obtain primers
</button>
<button id='clearAllMut' class="mdl-button mdl-js-button mdl-button--raised mdl-js-ripple-effect" onclick="javascript:eraseText_mut();">
Clear all
</button>
<div class="mdl-tooltip" for="clearAllMut">Clear amplification fields</div>
<div class="mdl-tooltip" for="mutButton">Create primers for your sequences</div>
</div>
<!-- Event card -->
<style>
.demo-card-event.mdl-card {
width: 860px;
}
.demo-card-event > .mdl-card__title,
.demo-card-event > .mdl-card__actions > .mdl-button {
color: #0000;
}
</style>
<div class="mdl-card mdl-shadow--2dp demo-card-event">
<div class="mdl-card__title mdl-card--expand">
<!-- Floating Multiline Textfield -->
<form action="#">
<div class="mdl-textfield mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<textarea class="mdl-textfield__input" style="color:black;overflow:auto;resize:none;font-family:'Roboto Mono'" type="text" rows= "10" id="resultForward_mut" ></textarea>
<label class="mdl-textfield__label" for="resultForward_mut">Forward Primers...</label>
</div>
</form>
</div>
</div>
<div class="mdl-card mdl-shadow--2dp demo-card-event">
<div class="mdl-card__title mdl-card--expand">
<!-- Floating Multiline Textfield -->
<form action="#">
<div class="mdl-textfield mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<textarea class="mdl-textfield__input" style="color:black;overflow:auto;resize:none;font-family:'Roboto Mono'" type="text" rows= "10" id="resultReverse_mut" ></textarea>
<label class="mdl-textfield__label" for="resultReverse_mut">Reverse Primers...</label>
</div>
</form>
</div>
</div>
<div class="mdl-card__title mdl-card--expand">
<button id='dl_mut' class="mdl-button mdl-js-button mdl-button--raised mdl-js-ripple-effect mdl-button--accent" onclick='saveTextAsFile_fw_mut();saveTextAsFile_rv_mut()'>
Download as CSV
</button>
<div class="mdl-tooltip" for="dl_mut">Download as CSV using tabs as delimiter</div>
</div>
</section>
</div>
<div class="mdl-layout__tab-panel mdl-color--grey-100" id="tmcalculator">
<section class="section--center mdl-grid mdl-grid--no-spacing">
<table style="width:100%">
<tr>
<style>
.demo-card-square.mdl-card {
width: 280px;
height: 220px;
}
.demo-card-square > .mdl-card__title {
color: #fff;
background: #1976D2;
}
</style>
<td><!-- Square card -->
<div class="mdl-card mdl-shadow--2dp demo-card-square">
<div class="mdl-card__title mdl-card--expand" >
<h2 class="mdl-card__title-text">Thermodynamic data for Tm calculation</h2>
</div><br><br><br>
<!-- Numeric Textfield -->
<div class="container">
<form>
<div class="form-group">
<select id="combotmcalc">
<option value='Breslauer'>Breslauer et al. (1986)</option>
<option value='Sugimoto'>Sugimoto et al. (1996)</option>
</select>
<i class="bar"></i>
</div>
</form>
</div>
</div>
</td>
<td><!-- Square card -->
<div class="mdl-card mdl-shadow--2dp demo-card-square">
<div class="mdl-card__title mdl-card--expand">
<h2 class="mdl-card__title-text"> Primer Concentration</h2>
</div><br><br><br>
<!-- Numeric Textfield -->
<form action="#">
<div class="mdl-textfield mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<input class="mdl-textfield__input" type="text" pattern="-?[0-9]*(\.[0-9]+)?" id="pConc_simple" value="500" />
<label class="mdl-textfield__label" for="pConc_simple"> In nM...</label>
<span class="mdl-textfield__error">Input is not a number!</span>
</div>
</form>
</div>
</td>
<td><!-- Square card -->
<div class="mdl-card mdl-shadow--2dp demo-card-square">
<div class="mdl-card__title mdl-card--expand">
<h2 class="mdl-card__title-text">Monovalent Cation Concentration</h2>
</div><br><br><br>
<!-- Numeric Textfield -->
<form action="#">
<div class="mdl-textfield mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<input class="mdl-textfield__input" type="text" pattern="-?[0-9]*(\.[0-9]+)?" id="salt_simple" value="0.050"/>
<label class="mdl-textfield__label" for="salt_simple"> In M...</label>
<span class="mdl-textfield__error">Input is not a number!</span>
</div>
</form>
</div>
</td>
</tr>
</table>
<div class="mdl-card mdl-shadow--2dp demo-card-event">
<div class="mdl-card__title mdl-card--expand">
<!-- Textfield with Floating Label -->
<form action="#">
<div class="mdl-textfield mdl-js-textfield mdl-textfield--floating-label textfield-demo" style="color:black;width: 515px;">
<input class="mdl-textfield__input" type="text" type="text" pattern="[A-Za-z]{10,}" id="seqCalcFw" style="color:black;width: 515px;" />
<label class="mdl-textfield__label" for="seqCalcFw">Forward primer...</label>
<span class="mdl-textfield__error" id='span_tmCalc_fw'>Sequence too short</span>
</div>
</form>
<!-- Numeric Textfield -->
<form action="#">
<div class="mdl-textfield_short mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<input class="mdl-textfield__input" type="text" pattern="-?[0-9]*(\.[0-9]+)?" id="tmCalcFw" style="width: 70px;color: black;" readonly="readonly"/>
<label class="mdl-textfield__label" for="tmCalcFw">TM...</label>
</div>
</form>
<form action="#">
<div class="mdl-textfield_short mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<input class="mdl-textfield__input" type="text" pattern="-?[0-9]*(\.[0-9]+)?" id="sizeCalcFw" style="width: 70px;color: black;"readonly="readonly"/>
<label class="mdl-textfield__label" for="sizeCalcFw">Size...</label>
</div>
</form>
<form action="#">
<div class="mdl-textfield_short mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<input class="mdl-textfield__input" type="text" pattern="-?[0-9]*(\.[0-9]+)?" id="gcCalcFw" style="width: 70px;color: black;"readonly="readonly"/>
<label class="mdl-textfield__label" for="gcCalcFw">GC %...</label>
</div>
</form>
</div>
<div class="mdl-card__title mdl-card--expand">
<!-- Textfield with Floating Label -->
<form action="#">
<div class="mdl-textfield mdl-js-textfield mdl-textfield--floating-label textfield-demo" style="color:black;width: 515px;">
<input class="mdl-textfield__input" type="text" type="text" pattern="[A-Za-z]{10,}" id="seqCalcRv" style="color:black;width: 515px;"/>
<label class="mdl-textfield__label" for="seqCalcRv">Reverse primer...</label>
<span class="mdl-textfield__error" id='span_tmCalc_rv'>Sequence too short</span>
</div>
</form>
<!-- Numeric Textfield -->
<form action="#">
<div class="mdl-textfield_short mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<input class="mdl-textfield__input" type="text" pattern="-?[0-9]*(\.[0-9]+)?" id="tmCalcRv" style="width: 70px;color: black;" readonly="readonly"/>
<label class="mdl-textfield__label" for="tmCalcRv">TM...</label>
</div>
</form>
<form action="#">
<div class="mdl-textfield_short mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<input class="mdl-textfield__input" type="text" pattern="-?[0-9]*(\.[0-9]+)?" id="sizeCalcRv" style="width: 70px;color: black;"readonly="readonly"/>
<label class="mdl-textfield__label" for="sizeCalcRv">Size...</label>
</div>
</form>
<form action="#">
<div class="mdl-textfield_short mdl-js-textfield mdl-textfield--floating-label textfield-demo">
<input class="mdl-textfield__input" type="text" pattern="-?[0-9]*(\.[0-9]+)?" id="gcCalcRv" style="width: 70px;color: black;"readonly="readonly"/>
<label class="mdl-textfield__label" for="gcCalcRv">GC %...</label>
</div>
</form>
</div>
<script src="https://ajax.googleapis.com/ajax/libs/jquery/1.11.3/jquery.min.js"></script>
<script type="text/javascript">
var jq=jQuery.noConflict();
jq(seqCalcFw).ready( function(){
jq(seqCalcFw).keyup(function(event){
tmcalc()
});
});
</script>
<script type="text/javascript">
var jq=jQuery.noConflict();
jq(seqCalcRv).ready( function(){
jq(seqCalcRv).keyup(function(event){
tmcalc()
});
});
</script>
</div>
<div class="mdl-card__title mdl-card--expand">
<button class="mdl-button mdl-js-button mdl-button--raised mdl-js-ripple-effect" onclick='tmcalc()'>
Calculate TM
</button>
<button class="mdl-button mdl-js-button mdl-button--raised mdl-js-ripple-effect" onclick="cleartmcalc()">
Clear
</button>
</div>
</section>
</div>
<div class="mdl-layout__tab-panel mdl-color--grey-100" id="tutorial">
<section class="section--center mdl-grid mdl-grid--no-spacing mdl-shadow--2dp">
<div class="mdl-tabs mdl-js-tabs mdl-js-ripple-effect">
<div class="mdl-tabs__tab-bar">
<a href="#amp-panel" class="mdl-tabs__tab is-active">Gene Amplification</a>
<a href="#mut-panel" class="mdl-tabs__tab">Mutagenesis</a>
</div>
<div class="mdl-tabs__panel is-active" id="amp-panel">
<div class="mdl-card mdl-cell mdl-cell--12-col">
<div class="mdl-card__supporting-text mdl-grid mdl-grid--no-spacing">
<h3 class="mdl-cell mdl-cell--12-col">HT Gene Amplification</h3>
<div class="section__circle-container mdl-cell mdl-cell--2-col mdl-cell--1-col-phone">
<div class="section__circle-container__circle mdl-color--primary"></div>
</div>
<div class="section__text mdl-cell mdl-cell--10-col-desktop mdl-cell--6-col-tablet mdl-cell--3-col-phone">
<h4>Melting Temperature Calculation</h4>
The melting temperature (Tm) calculations are based on nearest neighbor method as described by Breslauer et al., (1986) (1) and two options of thermodynamic data are available
for calculating cloning primers: from <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC323600/">Breslauer et al., (1986)</a> and from
<a href="http://nar.oxfordjournals.org/content/24/22/4501.long">Sugimoto et al., (1996)</a> <br>
</div>
<div class="section__circle-container mdl-cell mdl-cell--2-col mdl-cell--1-col-phone">
<div class="section__circle-container__circle mdl-color--primary"></div>
</div>
<div class="section__text mdl-cell mdl-cell--10-col-desktop mdl-cell--6-col-tablet mdl-cell--3-col-phone">
<h4>Data Input</h4>
Input data must be a flat text containing coding region sequences of desired target protein genes in the FASTA format. For better usage, use descriptors (header of FASTA sequence)
with a maximum of 20 characters. More than 20 characters will make hard to understand output file, although this is irrelevant if you open it with a CSV reader (like MS Excel or
GNumeric).
</div>
<div class="section__circle-container mdl-cell mdl-cell--2-col mdl-cell--1-col-phone">
<div class="section__circle-container__circle mdl-color--primary"></div>
</div>
<div class="section__text mdl-cell mdl-cell--10-col-desktop mdl-cell--6-col-tablet mdl-cell--3-col-phone">
<h4>Optimal Tm</h4>
The primers will be designed according to the optimal Tm selected by the user. The program uses a maximum Tm variation of 5 degrees or 4 bases from the optimal value and the maximum
Tm difference of 5 degrees or 4 bases between forward and reverse primers. </div>
<div class="section__circle-container mdl-cell mdl-cell--2-col mdl-cell--1-col-phone">
<div class="section__circle-container__circle mdl-color--primary"></div>
</div>
<div class="section__text mdl-cell mdl-cell--10-col-desktop mdl-cell--6-col-tablet mdl-cell--3-col-phone">
<h4>Primer Concentration</h4>
This feature specifies the concentration of primers to be used in Tm calculations. The concentration must be the same as used in PCR reactions. The default value for this field is
500nM, which is the recommended primer concentration for most PCR reactions.
</div>
<div class="section__circle-container mdl-cell mdl-cell--2-col mdl-cell--1-col-phone">
<div class="section__circle-container__circle mdl-color--primary"></div>
</div>
<div class="section__text mdl-cell mdl-cell--10-col-desktop mdl-cell--6-col-tablet mdl-cell--3-col-phone">
<h4>Monovalent Cation Concentration</h4>
In this field the user can define the monovalent cation concentration for Tm calculations according to the polymerase buffer composition. As compositions of most buffers are proprietary,
the standard value of 50mM is set as default.
</div>
<div class="section__circle-container mdl-cell mdl-cell--2-col mdl-cell--1-col-phone">
<div class="section__circle-container__circle mdl-color--primary"></div>
</div>
<div class="section__text mdl-cell mdl-cell--10-col-desktop mdl-cell--6-col-tablet mdl-cell--3-col-phone">
<h4>Append Extra Sequence to Forward and Reverse Primers</h4>
In these fields the user can append extra sequences at the 5' ends of generated primers containing LIC sequences, recombination sites or restriction sites for downstream cloning. Note
that the application does not check for the existence restriction sites within the gene sequence that would interfere in restriction enzyme/ligation methodologies. Thus, we recommend
the use of a sequence-independent cloning methodology like LIC, PIPE, Gateway™ or In-Fusion™. The appended sequences are NOT taken into account for Tm calculation.
</div>
<div class="section__circle-container mdl-cell mdl-cell--2-col mdl-cell--1-col-phone">
<div class="section__circle-container__circle mdl-color--primary"></div>
</div>
<div class="section__text mdl-cell mdl-cell--10-col-desktop mdl-cell--6-col-tablet mdl-cell--3-col-phone">
<h4>Output Data</h4>
Output data from both mutagenesis and cloning tools are given as CSV text containing the GeneID (first uninterrupted characters from FASTA header), primers sequences, lengths and Tm.
</div>
</div>
</div>
</div>
<div class="mdl-tabs__panel" id="mut-panel">
<div class="mdl-card mdl-cell mdl-cell--12-col">
<!-- Wide card with share menu button -->
<div class="mdl-card__supporting-text mdl-grid mdl-grid--no-spacing">
<h3 class="mdl-cell mdl-cell--12-col">HT Mutagenesis</h3>
<style>
.demo-card-wide.mdl-card {
width: 860px;
}
.demo-card-wide > .mdl-card__title {
color: #fff;
height:560px;
background: url('http://i.imgur.com/Ug91qLY.png') center / cover;
}
.demo-card-wide > .mdl-card__menu {
color: #fff;
}
</style>
<div class="mdl-card demo-card-wide">
<div class="mdl-card__title">
</div>
<div class="mdl-card__supporting-text">
<b>Fig 1:</b> Types of mutation by inverse PCR method<br> <br>
<p align='justify'>Mutagenic primers designed by HTP-OligoDesigner are based on inverse PCR method (3). The cloning plasmid or expression vector containing the gene of interest is used as template for a PCR
reaction. Forward (F) and reverse (R) primers anneal back to back to the plasmid, which is entirely amplified by a proofreading polymerase that produces consistently blunt-ended DNA, like
Pfu DNA polymerase or Phusion® High-Fidelity DNA Polymerase. After amplification, PCR product is circularized by blunt-end ligation with T4 DNA ligase and, for this reason, primers must be
synthesized with 5´end phosphorylation or PCR product must be phosphorylated by T4 Polynucleotide Kinase. Circularized PCR product is then transformed into E. coli competent cells. This
method generates up to 75% of mutated DNA copies in the third PCR cycle resulting in nearly 100% of molecules being mutated at a specific site after 30 cycles (3). HTP-OligoDesigner is
capable of designing primers to introduce different types of mutation by inverse PCR method (Fig. 2).</p>
</div>
<div class="mdl-card__actions mdl-card--border">
</div>
</div>
<div class="section__circle-container mdl-cell mdl-cell--2-col mdl-cell--1-col-phone">
<div class="section__circle-container__circle mdl-color--primary"></div>
</div>
<div class="section__text mdl-cell mdl-cell--10-col-desktop mdl-cell--6-col-tablet mdl-cell--3-col-phone">
<h4>Melting Temperature Calculation</h4>
The melting temperature (Tm) calculations are based on nearest neighbor method as described by Breslauer et al., (1986) (1) and two options of thermodynamic data are available
for calculating cloning primers: from <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC323600/">Breslauer et al., (1986)</a> and from
<a href="http://nar.oxfordjournals.org/content/24/22/4501.long">Sugimoto et al., (1996)</a> <br>
</div>
<div class="section__circle-container mdl-cell mdl-cell--2-col mdl-cell--1-col-phone">
<div class="section__circle-container__circle mdl-color--primary"></div>
</div>
<div class="section__text mdl-cell mdl-cell--10-col-desktop mdl-cell--6-col-tablet mdl-cell--3-col-phone">
<h4>Data Input</h4>
Input data must be a flat text containing coding region sequences of desired target protein genes in the FASTA format. For better usage, use descriptors (header of FASTA sequence)
with a maximum of 20 characters. More than 20 characters will make hard to understand output file, although this is irrelevant if you open it with a CSV reader (like MS Excel or
GNumeric). The desired mutations, deletions or insertions for each sequence must be indicated into the sequences by the following code: The modification must be enclosed in parenthesis.
The base(s) to be changed and the new base(s) must be separated by a point
<br>
<table class="mdl-data-table mdl-js-data-table">
<tbody>
<tr>
<td class="mdl-data-table__cell--non-numeric">Point Mutation: </td>
<td class="mdl-data-table__cell--non-numeric">TG(C.A)TTA</td>
<td class="mdl-data-table__cell--non-numeric">replaces C for A in TGCTTA, that becomes TGATTA</td>
</tr>
<tr>
<td class="mdl-data-table__cell--non-numeric">Deletion:</td>
<td class="mdl-data-table__cell--non-numeric">TG(C.)TTA</td>
<td class="mdl-data-table__cell--non-numeric">deletes C in TGCTTA, that becomes TGTTA</td>
</tr>
<tr>
<td class="mdl-data-table__cell--non-numeric">Insertion:</td>
<td class="mdl-data-table__cell--non-numeric">TGC(.GAT)TTA </td>
<td class="mdl-data-table__cell--non-numeric">inserts GAT in TGCTTA, that becomes TGCGATTTA</td>
</tr>
</tbody>
</table>
<br>
</div>
<div class="section__circle-container mdl-cell mdl-cell--2-col mdl-cell--1-col-phone">
<div class="section__circle-container__circle mdl-color--primary"></div>
</div>
<div class="section__text mdl-cell mdl-cell--10-col-desktop mdl-cell--6-col-tablet mdl-cell--3-col-phone">
<h4>Choose Design Method</h4>
This feature allows the user to choose if primer design will be “By Size” or “By Tm”.
In “By Size” option, the number of nucleotides of the forward primer (which contains the mutation) will be fixed and the reverse primer will be calculated to have the same Tm as the forward.
In “By Tm” option, both forward and reverse primers will be designed according to the selected Tm. The program uses a maximum Tm variation of 5 degrees or 4 bases from the optimal value and
the maximum Tm difference of 5 degrees or 4 bases between forward and reverse primers.
</div>
<div class="section__circle-container mdl-cell mdl-cell--2-col mdl-cell--1-col-phone">
<div class="section__circle-container__circle mdl-color--primary"></div>
</div>
<div class="section__text mdl-cell mdl-cell--10-col-desktop mdl-cell--6-col-tablet mdl-cell--3-col-phone">
<h4>Primer Concentration</h4>
This feature specifies the concentration of primers to be used in Tm calculations. The concentration must be the same as used in PCR reactions. The default value for this field is
500nM, which is the recommended primer concentration for most PCR reactions.
</div>
<div class="section__circle-container mdl-cell mdl-cell--2-col mdl-cell--1-col-phone">
<div class="section__circle-container__circle mdl-color--primary"></div>
</div>
<div class="section__text mdl-cell mdl-cell--10-col-desktop mdl-cell--6-col-tablet mdl-cell--3-col-phone">
<h4>Monovalent Cation Concentration</h4>
In this field the user can define the monovalent cation concentration for Tm calculations according to the polymerase buffer composition. As compositions of most buffers are proprietary,
the standard value of 50mM is set as default.
</div>
<div class="section__circle-container mdl-cell mdl-cell--2-col mdl-cell--1-col-phone">
<div class="section__circle-container__circle mdl-color--primary"></div>
</div>
<div class="section__text mdl-cell mdl-cell--10-col-desktop mdl-cell--6-col-tablet mdl-cell--3-col-phone">
<h4>Output Data</h4>
Output data from both mutagenesis and cloning tools are given as CSV text containing the GeneID (first uninterrupted characters from FASTA header), primers sequences, lengths and Tm.
</div>
</div>
</div>
</div>
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<h4>Gustavo Machado Alvares de Lima</h4>
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PhD in Biomolecular Physics
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