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<!DOCTYPE html>
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<title>Primerize: Primer Design for PCR Assembly</title>
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<a class="navbar-brand" href="/"> <span>Primerize</span> </a>
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<li class="li-danger"><a href="/protocol/#pipe"><span class="glyphicon glyphicon-bookmark"></span> Pipeline Overview</a></li>
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<li class="li-magenta"><a href="/protocol/#temp_design"><span class="glyphicon glyphicon-edit"></span> Section #1: DNA Template Design</a></li>
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<h2 class="panel-title"><span class="glyphicon glyphicon-indent-left"></span> Primerize</h2>
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<img src="/assets/images/logo_primerize.png" style="clear:both;float:right;padding-left:20px;" height="128"/>
<p class="cap">
Primerize (previously named <code>NA_thermo</code>) is a <a href="https://www.python.org/" target="_blank" rel="noopener noreferrer external"><b>Python</b></a> package for primer design and nucleic acid thermodynamics, developed by the <a href="https://daslab.stanford.edu/" target="_blank" rel="noopener noreferrer external">Das Lab</a> at Stanford University for high-throughput RNA synthesis and design.
</p>
<p>
Given a desired DNA template sequence, Primerize uses a dynamic programming-based algorithm to design primers optimized for PCR assembly into templates for high-throughput RNA synthesis. The algorithm minimizes mispriming during PCR by avoiding primer boundaries that might anneal to incorrect sequences.
</p>
<p>
The algorithm has been tested in the synthesis and rapid purification of numerous RNA sequences from our lab with lengths up to 300 nucleotides. Although developed independently, Primerize is a special case of the general <b>'Gapped Oligo Design'</b> algorithm described by <a href="http://ieeexplore.ieee.org/xpls/abs_all.jsp?arnumber=4375554" target="_blank" rel="noopener noreferrer external">Thachuk and Condon</a>.
</p>
<p>
<a href="https://github.com/ribokit/Primerize" target="_blank" rel="noopener noreferrer external" class="btn btn-success">
<span class="glyphicon glyphicon-cloud-download"></span> Code on GitHub
</a>
<a href="https://ribokit.github.io/Primerize/" target="_blank" rel="noopener noreferrer external" class="btn btn-info">
<span class="glyphicon glyphicon-book"></span> Documentation
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<strong><span class="glyphicon glyphicon-exclamation-sign"></span> The Primerize web server was decommissioned in May 2026.</strong>
To design primers, use the <a href="https://github.com/ribokit/Primerize" class="alert-link">Primerize Python package</a> directly — or use the prompt below with <a href="https://claude.ai/code" class="alert-link" target="_blank" rel="noopener noreferrer external">Claude Code</a> or another AI coding assistant.
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<p>Paste this prompt into <a href="https://claude.ai/code" target="_blank" rel="noopener noreferrer external">Claude Code</a> or another AI coding assistant to design primers for your sequence:</p>
<pre style="white-space: pre-wrap; word-break: break-word; font-size: 12px;">Help me design PCR assembly primers for my RNA construct using the Primerize Python package (https://github.com/ribokit/Primerize), and produce the IDT ordering block.
1. Install: pip install git+https://github.com/ribokit/Primerize.git
2. Run:
import primerize
sequence = "PASTE_YOUR_SEQUENCE_HERE" # RNA or DNA, any case
result = primerize.Primerize_1D.design(sequence, prefix="my_rna")
if result.is_success:
print(result)
result.save() # writes my_rna.txt
3. Open my_rna.txt — scroll to the bottom for the IDT block (between START/END markers),
formatted for direct paste into IDT Bulk Input:
https://www.idtdna.com/site/order/oligoentry
Use "Lab Ready" for Normalization when ordering.
My sequence is: PASTE_YOUR_SEQUENCE_HERE</pre>
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<div class="panel-heading">
<h2 class="panel-title"><span class="glyphicon glyphicon-education"></span> Citation</h2>
</div>
<div class="panel-body">
<b>Please cite use of Primerize with:</b>
<br/>
<blockquote class="h5 alert alert-orange" style="margin-bottom: 0px">
<p>
Tian, S., and Das, R. (<b>2017</b>)
<br/>
<a href="https://academic.oup.com/bioinformatics/article-abstract/33/9/1405/2801460/Primerize-2D-automated-primer-design-for-RNA" target="_blank" rel="noopener noreferrer external"><u>Primerize-2D: automated primer design for RNA multidimensional chemical mapping.</u> <span class="glyphicon glyphicon-new-window"></span></a>
<br/>
<i>Bioinformatics</i> <b>33(9)</b>: 1405-1406.
</p>
</blockquote>
<blockquote class="h5 alert alert-orange">
<p>
Tian, S., Yesselman, J.D., Cordero, P., and Das, R. (<b>2015</b>)
<br/>
<a href="http://nar.oxfordjournals.org/content/43/W1/W522" target="_blank" rel="noopener noreferrer external"><u>Primerize: automated primer assembly for transcribing interesting RNAs.</u> <span class="glyphicon glyphicon-new-window"></span></a>
<br/>
<i>Nucleic Acid Research</i> <b>43(W1)</b>: W522-W526.
</p>
</blockquote>
<b>Publications of <u>mutate-map-rescue</u> pipeline:</b>
<br/>
<blockquote class="h5" style="padding-right: 10px">
<p>
Tian, S., and Das, R. (<b>2016</b>)
<br/>
<a href="http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=10242118&fulltextType=RV&fileId=S0033583516000020" target="_blank" rel="noopener noreferrer external"><u>RNA structure through multidimensional chemical mapping.</u> <span class="glyphicon glyphicon-new-window"></span></a>
<br/>
<i>Quarterly Review of Biophysics</i> <b>49(e7)</b>: 1-30.
</p>
<p>
Tian, S., Cordero, P., Kladwang, W., and Das, R. (<b>2014</b>)
<br/>
<a href="http://rnajournal.cshlp.org/content/20/11/1815" target="_blank" rel="noopener noreferrer external"><u>High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states.</u> <span class="glyphicon glyphicon-new-window"></span></a>
<br/>
<i>RNA</i> <b>20(11)</b>: 1815-1826.
</p>
<p>
Kladwang, W., VanLang, C.C., Cordero P., and Das, R. (<b>2011</b>)
<br/>
<a href="http://www.nature.com/nchem/journal/v3/n12/full/nchem.1176.html" target="_blank" rel="noopener noreferrer external"><u>A two-dimensional mutate-and-map strategy for non-coding RNA structure.</u> <span class="glyphicon glyphicon-new-window"></span></a>
<br/>
<i>Nature Chemistry</i> <b>3</b>: 954-962.
</p>
</blockquote>
</div>
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<h4 style="line-height: 140%;">
<span class="label label-primary">Principal Investigator</span>
</h4>
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<h4 style="line-height: 140%;">
<span class="label label-inverse">
<a href="https://daslab.stanford.edu/" target="_blank" rel="noopener noreferrer external" style="color:#fff;">Rhiju Das</a>
</span>
<a href="mailto:rhiju@stanford.edu" target="_blank" rel="noopener noreferrer external"><span class="glyphicon glyphicon-envelope"></span></a>
</h4>
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<p>Please feel free to contact the <a href="https://daslab.stanford.edu/contact/" target="_blank" rel="noopener noreferrer external">Das Lab</a> for questions, suggestions, or commercial use authorization.</p>
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Designed, built and managed by members of the
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Copyright © 2008-<span id="cp_year"></span>: The Board of Trustees of the Leland Stanford Junior University. All Rights Reserved.
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