596 lines
37 KiB
HTML
596 lines
37 KiB
HTML
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
<!doctype html>
|
||
<html lang="en">
|
||
<head>
|
||
<meta name="viewport" content="width=device-width, initial-scale=1, shrink-to-fit=no">
|
||
<meta http-equiv="X-UA-Compatible" content="IE=edge" />
|
||
<meta http-equiv="Content-Type" content="text/html; charset=utf-8" />
|
||
<meta http-equiv="X-UA-Compatible" content="IE=edge,chrome=1"/>
|
||
<link rel="preconnect" href="https://fonts.googleapis.com">
|
||
<link rel="preconnect" href="https://fonts.gstatic.com" crossorigin>
|
||
<link href="https://fonts.googleapis.com/css2?family=Roboto:wght@100;300;400;500;700&display=swap" rel="stylesheet">
|
||
<link rel="stylesheet" href="https://use.fontawesome.com/releases/v5.0.10/css/all.css" integrity="sha384-+d0P83n9kaQMCwj8F4RJB66tzIwOKmrdb46+porD/OvrJ+37WqIM7UoBtwHO6Nlg" crossorigin="anonymous">
|
||
<link rel="schema.DC" href="http://purl.org/dc/elements/1.1/" title="The Dublin Core metadata Element Set" />
|
||
|
||
<script src="https://assets.nlm.nih.gov/uswds/js/uswds-init.min.js"></script>
|
||
<link rel="stylesheet" href="/home_assets/uswds/css/styles.css">
|
||
<link rel="stylesheet" href="/home_assets/uswds/css/uswds_accordion_nav.css">
|
||
<link rel="stylesheet" href="/home_assets/css/mwm_sidebar.css">
|
||
|
||
|
||
<title>Practice Primer-BLAST Examples</title>
|
||
<link rel="schema.DC" href="http://purl.org/dc/elements/1.1/" title="The Dublin Core metadata Element Set" />
|
||
<meta name="DC.Title" content="Practice Primer-BLAST Examples" />
|
||
|
||
<meta name="DC.Publisher" content="U.S. National Library of Medicine" />
|
||
<meta name="DC.Date.Issued" content="2023-09-22" />
|
||
<meta name="DC.Date.Modified" content="2023-09-22" />
|
||
<meta name="NLMDC.Date.LastReviewed" content="2023-09-22" />
|
||
<meta name="NLM.Contact.Email" content="workshops@ncbi.nlm.nih.gov" />
|
||
<meta name="DC.Type" content="Training Material and Manuals" />
|
||
<meta name="DC.Rights" content="Public Domain" />
|
||
<meta name="DC.Language" content="eng" />
|
||
|
||
|
||
<!-- Google Tag Manager --><script>(function(w,d,s,l,i){w[l]=w[l]||[];w[l].push(
|
||
{'gtm.start': new Date().getTime(),event:'gtm.js'}
|
||
);var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-MT6MLL');</script>
|
||
<!-- End Google Tag Manager -->
|
||
</head>
|
||
<body>
|
||
<!-- Google Tag Manager -->
|
||
<noscript><iframe src="//www.googletagmanager.com/ns.html?id=GTM-MT6MLL" height="0" width="0" style="display:none;visibility:hidden" title="googletagmanager"></iframe></noscript>
|
||
<!-- End Google Tag Manager -->
|
||
<!-- TOP NAV -->
|
||
<a class="usa-skipnav" href="#main">Skip to main content</a>
|
||
<div class="usa-banner site-banner" aria-label="Official government website">
|
||
<div class="usa-accordion">
|
||
<header class="usa-banner__header">
|
||
<div class="usa-banner__inner">
|
||
<div class="grid-col-auto"> <img class="usa-banner__header-flag" src="https://assets.nlm.nih.gov/uswds/img/us_flag_small.png" alt="U.S. flag"/> </div>
|
||
<div class="grid-col-fill tablet:grid-col-auto">
|
||
<p class="usa-banner__header-text"> An official website of the United States government </p>
|
||
<p class="usa-banner__header-action" aria-hidden="true"> Here’s how you know </p>
|
||
</div>
|
||
<button class="usa-accordion__button usa-banner__button" aria-expanded="false" aria-controls="gov-banner"> <span class="usa-banner__button-text">Here’s how you know</span> </button>
|
||
</div>
|
||
</header>
|
||
<div class="usa-banner__content usa-accordion__content" id="gov-banner">
|
||
<div class="grid-row grid-gap-lg">
|
||
<div class="usa-banner__guidance tablet:grid-col-6"> <img class="usa-banner__icon usa-media-block__img" src="https://assets.nlm.nih.gov/uswds/img/icon-dot-gov.svg" role="img" alt="" aria-hidden="true"/>
|
||
<div class="usa-media-block__body">
|
||
<p> <strong> Official websites use .gov </strong> <br />
|
||
A <strong>.gov</strong> website belongs to an official government
|
||
organization in the United States. </p>
|
||
</div>
|
||
</div>
|
||
<div class="usa-banner__guidance tablet:grid-col-6"> <img class="usa-banner__icon usa-media-block__img" src="https://assets.nlm.nih.gov/uswds/img/icon-https.svg" role="img" alt="" aria-hidden="true"/>
|
||
<div class="usa-media-block__body">
|
||
<p> <strong> Secure .gov websites use HTTPS </strong> <br />
|
||
A <strong>lock</strong> ( <span class="icon-lock">
|
||
<svg xmlns="http://www.w3.org/2000/svg" width="52" height="64" viewBox="0 0 52 64" class="usa-banner__lock-image" role="img" aria-labelledby="banner-lock-title-default banner-lock-description-default" focusable="false">
|
||
<title id="banner-lock-title-default">Lock</title>
|
||
<desc id="banner-lock-description-default">A locked padlock</desc>
|
||
<path fill="#000000" fill-rule="evenodd" d="M26 0c10.493 0 19 8.507 19 19v9h3a4 4 0 0 1 4 4v28a4 4 0 0 1-4 4H4a4 4 0 0 1-4-4V32a4 4 0 0 1 4-4h3v-9C7 8.507 15.507 0 26 0zm0 8c-5.979 0-10.843 4.77-10.996 10.712L15 19v9h22v-9c0-6.075-4.925-11-11-11z"/>
|
||
</svg>
|
||
</span> ) or <strong>https://</strong> means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites. </p>
|
||
</div>
|
||
</div>
|
||
</div>
|
||
</div>
|
||
</div>
|
||
</div>
|
||
|
||
<!-- HEADER -->
|
||
<header id="siteheader" class="usa-header usa-header--basic">
|
||
<div class="usa-nav-container tablet:padding-x-4 mobile-lg:padding-x-2 padding-y-1">
|
||
<div class="grid-row padding-y-105">
|
||
<div class="grid-col-8 desktop:grid-col-4 tablet-lg:grid-col-4 tablet:grid-col-6"> <a href="https://www.nlm.nih.gov/"> <img src="https://assets.nlm.nih.gov/uswds/img/NLM_White.png" alt="NLM logo" class="logo margin-top-1"> </a> </div>
|
||
<div class="desktop:grid-col-4 desktop:grid-offset-4 tablet-lg:grid-col-6 tablet-lg:grid-offset-2 tablet:grid-col-6 grid-col-12">
|
||
<form class="usa-search desktop:margin-top-2 tablet:margin-top-2 mobile:margin-top-1" role="search" data-gtm-form-interact-id="0" method="get" action="//vsearch.nlm.nih.gov/vivisimo/cgi-bin/query-meta" target="_self" name="searchForm" id="searchForm2">
|
||
<input class="usa-input ui-autocomplete-input" aria-label="Search" type="search" name="query" data-gtm-form-interact-field-id="0" id="search2" placeholder="Search NLM" autocomplete="off">
|
||
<input type="hidden" name="v:project" value="nlm-main-website">
|
||
<button class="usa-button border border-top border-bottom border-right border-white" role="button" aria-label="Search" type="submit"> <span class="usa-search__submit-text"> <i class="fas fa-search"></i> </span> </button>
|
||
</form>
|
||
</div>
|
||
</div>
|
||
</div>
|
||
</header>
|
||
<div class="bg-secondary insertCOOP">
|
||
<div class="usa-nav-container">
|
||
<div class="usa-navbar ">
|
||
<button class="usa-menu-btn">Menu</button>
|
||
</div>
|
||
<nav aria-label="Primary navigation" class="usa-nav">
|
||
<button class="usa-nav__close"><img src="https://assets.nlm.nih.gov/uswds/img/close.svg" alt="close"></button>
|
||
<ul class="usa-nav__primary usa-accordion insertNav">
|
||
|
||
<li class="usa-nav__primary-item desktop-lg:margin-x-5 desktop:margin-x-3 tablet:margin-x-0">
|
||
<button type="button" class="usa-accordion__button usa-nav__link usa-current" aria-expanded="false" aria-controls="basic-nav-section-one"> <span>Products and Services <i class="fas fa-caret-down margin-left-05"></i> </span> </button>
|
||
<ul id="basic-nav-section-one" class="usa-nav__submenu bg-secondary" hidden="">
|
||
<li class="usa-nav__submenu-item"> <a href="//eresources.nlm.nih.gov/nlm_eresources/"><span>All Products and Services</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="//clinicaltrials.gov/"><span>ClinicalTrials.gov</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="//collections.nlm.nih.gov/"><span>Digital Collections</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="//catalog.nlm.nih.gov"><span>LocatorPlus Catalog</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="//meshb.nlm.nih.gov/search"><span>Medical Subject Headings (MeSH)</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="//medlineplus.gov/"><span>MedlinePlus</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="//pubmed.ncbi.nlm.nih.gov/"><span>PubMed/MEDLINE</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="//uts.nlm.nih.gov/uts/"><span>Unified Medical Language System (UMLS)</span></a> </li>
|
||
</ul>
|
||
</li>
|
||
<li class="usa-nav__primary-item desktop-lg:margin-x-5 desktop:margin-x-3 tablet:margin-x-0">
|
||
<button type="button" class="usa-accordion__button usa-nav__link usa-current" aria-expanded="false" aria-controls="basic-nav-section-two"> <span> Resources for You <i class="fas fa-caret-down margin-left-05"></i></span> </button>
|
||
<ul id="basic-nav-section-two" class="usa-nav__submenu bg-secondary" hidden="">
|
||
<li class="usa-nav__submenu-item"> <a href="/portals/researchers.html"><span>For Researchers</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="/portals/publishers.html "><span>For Publishers</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="/portals/librarians.html"><span>For Librarians</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="/training.html "><span>For Educators/Trainers </span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="/portals/healthcare.html"><span>For Health care Professionals</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="/portals/public.html "><span>For the Public</span></a> </li>
|
||
</ul>
|
||
</li>
|
||
<li class="usa-nav__primary-item desktop-lg:margin-x-5 desktop:margin-x-3 tablet:margin-x-0">
|
||
<button type="button" class="usa-accordion__button usa-nav__link usa-current" aria-expanded="false" aria-controls="basic-nav-section-three"> <span>Explore NLM <i class="fas fa-caret-down margin-left-05"></i> </span> </button>
|
||
<ul id="basic-nav-section-three" class="usa-nav__submenu bg-secondary" hidden="">
|
||
<li class="usa-nav__submenu-item"> <a href="/about/index.html"><span>About the Library</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="/about/visitor.html"><span>Visit the Library</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="/hmd/index.html"><span>History of Medicine</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="/about/org.html"><span>NLM by Organization</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="/news/newsandevents.html"><span>News, Events, and Updates</span></a> </li>
|
||
</ul>
|
||
</li>
|
||
<li class="usa-nav__primary-item desktop-lg:margin-x-5 desktop:margin-x-3 tablet:margin-x-0">
|
||
<button type="button" class="usa-accordion__button usa-nav__link usa-current" aria-expanded="false" aria-controls="basic-nav-section-four"> <span> Grants and Research <i class="fas fa-caret-down margin-left-05"></i> </span> </button>
|
||
<ul id="basic-nav-section-four" class="usa-nav__submenu bg-secondary" hidden="">
|
||
<li class="usa-nav__submenu-item"> <a href="/ep/index.html"><span>NLM Extramural Programs</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="/research/index.html"><span>NLM Intramural Research Program</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="https://www.ncbi.nlm.nih.gov/"><span>National Center for Biotechnology Information</span></a> </li>
|
||
<li class="usa-nav__submenu-item"> <a href="//lhncbc.nlm.nih.gov/"><span>Lister Hill National Center for Biomedical Communications</span></a> </li>
|
||
</ul>
|
||
</li>
|
||
|
||
</ul>
|
||
</nav>
|
||
</div>
|
||
</div>
|
||
|
||
<!-- End of TOP NAV -->
|
||
|
||
|
||
|
||
|
||
<!-- Breadcrumbs -->
|
||
<div class="grid-container">
|
||
<nav class="usa-breadcrumb usa-breadcrumb--wrap padding-top-1" aria-label="Breadcrumbs">
|
||
<ol class="usa-breadcrumb__list">
|
||
<li class="usa-breadcrumb__list-item"> <a href="/" class="usa-breadcrumb__link"><span>Home</span></a> </li>
|
||
</ol>
|
||
</nav>
|
||
</div>
|
||
<!-- End Breadcrumbs -->
|
||
|
||
|
||
|
||
|
||
|
||
<main class="padding-bottom-5" id="main">
|
||
<div class="grid-container">
|
||
<div class="grid-row grid-gap">
|
||
<div class="tablet:grid-col-3">
|
||
<section class="nav-wrap">
|
||
|
||
<!-- start accordion nav block -->
|
||
<nav class="acnav" role="navigation">
|
||
<!-- start level 1 -->
|
||
<ul class="acnav__list acnav__list--level1 padding-left-0" style="list-style-type:none">
|
||
|
||
<li><a class="notactive" href="/ncbi/workshops/2023-09_Primer-BLAST/workshop-details.html"> Workshop Details </a></li><li><a class="notactive" href="/ncbi/workshops/2023-09_Primer-BLAST/workshop-video.html"> Workshop Video </a></li><li class="has-children"><div class="acnav__label"> <i class="arrow down"></i><a class="notactive" href="/ncbi/workshops/2023-09_Primer-BLAST/PCR.html"> The Polymerase Chain Reaction </a></div><ul class="acnav__list acnav__list--level2" style="list-style-type: none; display: none;"><li class="li nav-item"><a class="notactive nav-link link10314" href="/ncbi/workshops/2023-09_Primer-BLAST/PCR.html#PCR">Advent of PCR</a></li><li class="li nav-item"><a class="notactive nav-link link10325" href="/ncbi/workshops/2023-09_Primer-BLAST/PCR.html#Primers">Designing Primers</a></li></ul></li><li><a class="notactive" href="/ncbi/workshops/2023-09_Primer-BLAST/PB_description.html"> What is Primer-BLAST? </a></li><li><a class="notactive" href="/ncbi/workshops/2023-09_Primer-BLAST/PB_access.html"> Accessing Primer-BLAST </a></li><li class="has-children"><div class="acnav__label"> <i class="arrow down"></i><a class="notactive" href="/ncbi/workshops/2023-09_Primer-BLAST/interface.html"> The Interface </a></div><ul class="acnav__list acnav__list--level2" style="list-style-type: none; display: none;"><li class="li nav-item"><a class="notactive nav-link link10639" href="/ncbi/workshops/2023-09_Primer-BLAST/interface.html#Template">Input Sequences</a></li><li class="li nav-item"><a class="notactive nav-link link106410" href="/ncbi/workshops/2023-09_Primer-BLAST/interface.html#Exon">Exon/Intron Selection</a></li><li class="li nav-item"><a class="notactive nav-link link106511" href="/ncbi/workshops/2023-09_Primer-BLAST/interface.html#Specificity">Specificity Check</a></li><li class="li nav-item"><a class="notactive nav-link link106612" href="/ncbi/workshops/2023-09_Primer-BLAST/interface.html#Advanced">Advanced Parameters</a></li></ul></li><li class="has-children"><div class="acnav__label"> <i class="arrow down"></i><a class="notactive" href="/ncbi/workshops/2023-09_Primer-BLAST/results.html"> Results Page </a></div><ul class="acnav__list acnav__list--level2" style="list-style-type: none; display: none;"><li class="li nav-item"><a class="notactive nav-link link107714" href="/ncbi/workshops/2023-09_Primer-BLAST/results.html#Results">Results</a></li><li class="li nav-item"><a class="notactive nav-link link107815" href="/ncbi/workshops/2023-09_Primer-BLAST/results.html#Recent">Recent Results</a></li></ul></li><li class="has-children is-open"><div class="acnav__label"> <i class="arrow up"></i><a class="active" href="/ncbi/workshops/2023-09_Primer-BLAST/examples.html"> Practice Examples </a></div><ul class="acnav__list acnav__list--level2" style="list-style-type: none; display: block;"><li class="li nav-item"><a class="notactive nav-link link108917" href="/ncbi/workshops/2023-09_Primer-BLAST/examples.html#Ex1">Example 1: MPO mRNA</a></li><li class="li nav-item"><a class="notactive nav-link link1081018" href="/ncbi/workshops/2023-09_Primer-BLAST/examples.html#Ex2">Example 2: MPO Exon</a></li><li class="li nav-item"><a class="notactive nav-link link1081119" href="/ncbi/workshops/2023-09_Primer-BLAST/examples.html#Ex3">Example 3: Whale myoglobins</a></li></ul></li><li><a class="notactive" href="/ncbi/workshops/2023-09_Primer-BLAST/job_ids.html"> Today's Job IDs </a></li>
|
||
|
||
</ul>
|
||
<!-- end level 1 -->
|
||
</nav>
|
||
<!-- end accordion nav block -->
|
||
|
||
</section>
|
||
</div>
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
<div class="tablet:grid-col-9">
|
||
<div class="grid-row">
|
||
<div class="desktop:grid-col-8 tablet:grid-col-12">
|
||
<h1>Practice Primer-BLAST Examples</h1>
|
||
</div>
|
||
|
||
|
||
|
||
|
||
|
||
|
||
</div>
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
<br />We'll cover three main examples today and explore options and some related resources. We'll use Primer-BLAST to design primers for:<br />
|
||
<ol>
|
||
<li><a href="#Ex1">A human transcript from the myeloperoxidase (MPO) gene and use the exon/intron selection to make them specific for the transcript</a></li>
|
||
<li><a href="#Ex2">A specific exon of the human MPO gene that contains a clinically significant SNP</a>
|
||
<ol>
|
||
<li>Starting from the nucleotide record</li>
|
||
<li>Starting from the sequence viewer</li>
|
||
</ol>
|
||
</li>
|
||
<li><a href="#Ex3">A set of whale myoglobin transcript sequences to generate cross-species primers and use these to identify the myoglobin gene in an unannotated grey whale genome assembly</a></li>
|
||
</ol>
|
||
<p><a href="/ncbi/workshops/2023-09_Primer-BLAST/job_ids.html" target="_blank" rel="noopener"><strong>Today's results job IDs</strong></a></p>
|
||
<h2><a id="Ex1"></a>Example 1: Human MPO mRNA</h2>
|
||
<h3>General primers</h3>
|
||
<ul>
|
||
<li>Use the provided defaults and design primers for human myeloperoxidase mRNA (<a href="https://www.ncbi.nlm.nih.gov/nuccore/NM_000250" target="_blank" rel="noopener">NM_000250</a>). You can use the <a class="brieflinkpopperctrl" role="button" href="https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?ORGANISM=9606&INPUT_SEQUENCE=NM_000250.2">Pick Primers</a> link to send the sequence to Primer-BLAST as a template. Expand the Advanced Parameters to generate an internal hybridization oligo as well. </li>
|
||
</ul>
|
||
<a href="https://go.usa.gov/xejjA" target="_blank" rel="noopener">Pre-configured Primer-BLAST form with MPO mRNA, no exon/intron selection</a>.<br /><br /><a href="https://go.usa.gov/xzRS5" target="_blank" rel="noopener">Pre-configured Primer-BLAST form as above with internal hybridization oligo</a>.<br />
|
||
<ul>
|
||
<li>Check your primer pair 2 against “Genomes for selected organisms” see if they would amplify human genomic DNA.</li>
|
||
</ul>
|
||
<pre style="padding-left: 320px;">Single exon pair 2
|
||
ATCCCCGGGACTTTGTCAAC
|
||
GGGCCCATAAGTCAACCACA
|
||
</pre>
|
||
<a href="https://go.usa.gov/xebFQ" target="_blank" rel="noopener">Pre-configured Primer-BLAST form with single exon primer pair</a>.<br /><br />
|
||
<h3>Expression-specific primers</h3>
|
||
<ul>
|
||
<li>Re-design primers with the “at least one intron” check box selected.</li>
|
||
</ul>
|
||
<p><a href="https://go.usa.gov/xeben" target="_blank" rel="noopener">Pre-configured Primer-BLAST form with skip one intron checkbox checked</a>.</p>
|
||
<ul>
|
||
<li>Check the new primer pair 5 on genomic DNA by searcing “Genomes for selected organisms” limited to human.</li>
|
||
</ul>
|
||
<pre style="padding-left: 320px;">Different exon pair 5
|
||
GCATCAAGAACCAAGCCGAC
|
||
CATGGGCTGGTACCGATTGT</pre>
|
||
<p><a href="https://go.usa.gov/xebMS" target="_blank" rel="noopener">Pre-configured Primer-BLAST form with primer pair 5 against human genomic DNA</a> </p>
|
||
<ul>
|
||
<li>Try primer pair 5 against RefSeq RNA restricted to Primates to see if they would work in other species.</li>
|
||
</ul>
|
||
<p><a href="https://go.usa.gov/xebtX" target="_blank" rel="noopener">Pre-configured Primer-BLAST form with primer pair 5 against primate RefSeq mRNA</a><br /><br /><br /></p>
|
||
<h2><a id="Ex2"></a>Example 2: Primers for MPO exon with pathogenic SNPs</h2>
|
||
<h3>From the standard Entrez display</h3>
|
||
<ul>
|
||
<li>Perform an <a href="https://www.ncbi.nlm.nih.gov/search/all/?term=MPO" target="_blank" rel="noopener">“All databases” search on the NCBI homepage for MPO</a></li>
|
||
<li>Click the button on the results panel to retrieve the human <a href="https://www.ncbi.nlm.nih.gov/nuccore/NG_009629.1" target="_blank" rel="noopener">MPO RefSeq Gene record </a></li>
|
||
<li>Navigate to exon 10 in the FEATURES table of the record</li>
|
||
<li>Click on the linked “exon” feature to highlight it then display it in <a href="https://www.ncbi.nlm.nih.gov/nuccore/NG_009629.1?from=13018&to=13188&report=fasta" target="_blank" rel="noopener">FASTA</a> or <a href="https://www.ncbi.nlm.nih.gov/nuccore/NG_009629.1?from=13018&to=13188&report=gbwithparts" target="_blank" rel="noopener">GenBank</a> format</li>
|
||
<li>Click the <a href="https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?ORGANISM=9606&INPUT_SEQUENCE=NG_009629.1&PRIMER5_START=13018&PRIMER3_END=13188" target="_blank" rel="noopener">“Pick Primers” link</a> on the right-hand side to send the exon 10 sequence to Primer-BLAST</li>
|
||
<li>Adjust the primer ranges so that primers are within 100 base pairs upstream and downstream of the exon (e.g., 12918 To 13018, 13188 To 13288)</li>
|
||
<li>Search against “Genomes for selected organisms”</li>
|
||
<li>Run the search (Get Primers)</li>
|
||
</ul>
|
||
Your search will be interrupted by an intermediate page (screenshot below). This is because the template RefSeq Gene record is not in the genome database. Primer-BLAST will offer the identical region of chromosome 17 as an alternative match. Check the box for NC_000017.11 and click "Submit."<br /><br /><img src="/ncbi/workshops/2022-10_Primer-BLAST/Images/chromosome_17_in_the_database.JPG" alt="Image of the Primer-BLAST intermediate page with a RefSeq Gene record is used as a template with the genome database" width="900" /><br />
|
||
<ul>
|
||
<li>Once the results load, you can use "Tracks -> Configure tracks" menu on the Primer-BLAST graphical view to add the Clinical Variants track to see clinical SNPs that are present in the exon.</li>
|
||
</ul>
|
||
<img src="/ncbi/workshops/2022-10_Primer-BLAST/Images/Adding_clinical_tracks.JPG" alt="Image of the configure tracks menu on the Primer-BLAST graphical display" width="900" /><br /><br /><a href="https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?&INPUT_SEQUENCE=NG_009629.1&PRIMER5_START=12918&PRIMER5_END=13018&PRIMER3_START=13188&PRIMER3_END=13288&OVERLAP_5END=7&OVERLAP_3END=4&PRIMER_PRODUCT_MIN=70&PRIMER_PRODUCT_MAX=1000&PRIMER_NUM_RETURN=10&PRIMER_MIN_TM=57.0&PRIMER_OPT_TM=60.0&PRIMER_MAX_TM=63.0&PRIMER_MAX_DIFF_TM=3&PRIMER_ON_SPLICE_SITE=0&SEARCHMODE=0&SPLICE_SITE_OVERLAP_5END=7&SPLICE_SITE_OVERLAP_3END=4&SPLICE_SITE_OVERLAP_3END_MAX=8&SPAN_INTRON=off&MIN_INTRON_SIZE=1000&MAX_INTRON_SIZE=1000000&SEARCH_SPECIFIC_PRIMER=on&EXCLUDE_ENV=off&EXCLUDE_XM=off&TH_OLOGO_ALIGNMENT=off&TH_TEMPLATE_ALIGNMENT=off&ORGANISM=9606&PRIMER_SPECIFICITY_DATABASE=PRIMERDB/genome_selected_species&TOTAL_PRIMER_SPECIFICITY_MISMATCH=1&PRIMER_3END_SPECIFICITY_MISMATCH=1&MISMATCH_REGION_LENGTH=5&TOTAL_MISMATCH_IGNORE=6&MAX_TARGET_SIZE=4000&ALLOW_TRANSCRIPT_VARIANTS=off&HITSIZE=50000&EVALUE=30000&WORD_SIZE=7&MAX_CANDIDATE_PRIMER=500&PRIMER_MIN_SIZE=15&PRIMER_OPT_SIZE=20&PRIMER_MAX_SIZE=25&PRIMER_MIN_GC=20.0&PRIMER_MAX_GC=80.0&GC_CLAMP=0&NUM_TARGETS_WITH_PRIMERS=1000&NUM_TARGETS=20&MAX_TARGET_PER_TEMPLATE=100&POLYX=5&SELF_ANY=8.00&SELF_END=3.00&PRIMER_MAX_END_STABILITY=9&PRIMER_MAX_END_GC=5&PRIMER_MAX_TEMPLATE_MISPRIMING_TH=40.00&PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH=70.00&PRIMER_MAX_SELF_ANY_TH=45.0&PRIMER_MAX_SELF_END_TH=35.0&PRIMER_PAIR_MAX_COMPL_ANY_TH=45.0&PRIMER_PAIR_MAX_COMPL_END_TH=35.0&PRIMER_MAX_HAIRPIN_TH=24.0&PRIMER_MAX_TEMPLATE_MISPRIMING=12.00&PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING=24.00&PRIMER_PAIR_MAX_COMPL_ANY=8.00&PRIMER_PAIR_MAX_COMPL_END=3.00&PRIMER_MISPRIMING_LIBRARY=AUTO&NO_SNP=off&LOW_COMPLEXITY_FILTER=on&MONO_CATIONS=50.0&DIVA_CATIONS=1.5&CON_ANEAL_OLIGO=50.0&CON_DNTPS=0.6&SALT_FORMULAR=1&TM_METHOD=1&PRIMER_INTERNAL_OLIGO_MIN_SIZE=18&PRIMER_INTERNAL_OLIGO_OPT_SIZE=20&PRIMER_INTERNAL_OLIGO_MAX_SIZE=27&PRIMER_INTERNAL_OLIGO_MIN_TM=57.0&PRIMER_INTERNAL_OLIGO_OPT_TM=60.0&PRIMER_INTERNAL_OLIGO_MAX_TM=63.0&PRIMER_INTERNAL_OLIGO_MAX_GC=80.0&PRIMER_INTERNAL_OLIGO_OPT_GC_PERCENT=50&PRIMER_INTERNAL_OLIGO_MIN_GC=20.0&PICK_HYB_PROBE=off&NEWWIN=off&NEWWIN=off&SHOW_SVIEWER=true" target="_blank" rel="noopener">Pre-configured Primer-BLAST form with MPO exon 10 and primer binding regions</a><br />
|
||
<p> </p>
|
||
<h4 style="padding-left: 40px;">Graphical Primer-BLAST results showing the Clinical SNPs track with variants in exon 10 of MPO</h4>
|
||
<p> <img src="Images/exon10_clinical_snps.JPG" alt="Primer BLAST results with clinical SNP in exon 10 of MPO gene" width="900" /></p>
|
||
<h3>From the graphical sequence viewer display</h3>
|
||
<ul>
|
||
<li>Click the "Graphics link" on either the FASTA or GenBank display of exon 10 from the MPO RefSeq Gene to load the <a href="https://www.ncbi.nlm.nih.gov/nuccore/NG_009629.1?report=graph&from=13018&to=13188" target="_blank" rel="noopener">graphics view</a>.</li>
|
||
<li>Use the zoom feature to zoom out to get flanking intron sequence on either side of exon 10.</li>
|
||
<li>Click in the ruler and drag out a 100 bp selection upstream of exon 10.</li>
|
||
<li>Hold the Ctrl key down and drag out a 100 bp selection downstream of exon 10.</li>
|
||
<li>Use the pop-up menu to “Primer BLAST” the selection.</li>
|
||
</ul>
|
||
<p> </p>
|
||
<h4 style="padding-left: 40px;">Sending flanking sequence of exon 10 of MPO to Primer-BLAST from the graphical sequence view</h4>
|
||
<p><img src="Images/Primer-BLAST_from_graphics.jpg" alt="Image of graphical sequence viewer showing highlighted flanking sequence to send to Primer-BLAST" width="900" /></p>
|
||
<ul>
|
||
<li>The primer BLAST results load into the graphical sequence viewer as shown in the screenshot below</li>
|
||
</ul>
|
||
<p><img src="Images/primers_in_seq_viewer.jpg" alt="Image of exon 10 of the MPO gene in the graphical sequence viewer showing location of primers" width="900" /></p>
|
||
<p> </p>
|
||
<h2><a id="Ex3"></a>Example 3: Making Common Primers for a Set of Related Sequences (whale myoglobin transcripts)</h2>
|
||
<h3>Designing common primers</h3>
|
||
<ul>
|
||
<li>Select the "Primers common for a group of sequences" tab on the Primer-BLAST form.</li>
|
||
<li>Copy and paste the following accession numbers for whale myoglobin transcript sequences into the template box on the Primer-BLAST page.</li>
|
||
</ul>
|
||
XM_007165704.1 <br />XM_022599904.2 <br />XM_036866958.1 <br />XM_004286206.2 <br />XM_030855394.1 <br />XM_027108256.1 <br />NM_001290722.1 <br />XM_032646419.1<br />XM_024743462.1 <br />XM_007456255.1
|
||
<ul>
|
||
<li>Use the RefSeq mRNA database with an organism limit to whales (cetacea).</li>
|
||
<li>Run the search.</li>
|
||
</ul>
|
||
<p><a href="https://go.usa.gov/xejBr" target="_blank" rel="noopener">Pre-configured Primer-BLAST page for whale myoglobin transcripts</a></p>
|
||
<ul>
|
||
<li>Note that the search finds additional whale myoglobin transcripts (unintended targets).</li>
|
||
<li>Re-run the search after accepting the additional transcripts as shown below.</li>
|
||
</ul>
|
||
<p><img src="Images/Re-submit_unintended.jpg" alt="Image of Primer-BLAST results with check boxes to re-submit with additional targets" width="900" /></p>
|
||
<h3>Testing whale myoglobin primers in the grey whale assembly</h3>
|
||
<ul>
|
||
<li>Perform an <a href="https://www.ncbi.nlm.nih.gov/search/all/?term=grey%20whale" target="_blank" rel="noopener">"All Databases" search for grey whale</a> from the NCBI Homepage.</li>
|
||
</ul>
|
||
<p><img src="Images/grey_whale_search.jpg" alt="Screenshot of grey whale search results with link to genomes highlighted" width="800" /></p>
|
||
<ul>
|
||
<li>Follow the link to Genomes to see list of genome assemblies and copy the assembly accession for the reference genome (<a href="https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_028021215.1/" target="_blank" rel="noopener">GCA_028021215.1</a>). You'll use this as a Custom database in Primer-BLAST.</li>
|
||
<li>Open the <a href="https://www.ncbi.nlm.nih.gov/tools/primer-blast/" target="_blank" rel="noopener">Primer-BLAST submission form</a> , select Custom as a Database option and enter the grey whale assembly accession in the box.</li>
|
||
</ul>
|
||
<p><img src="Images/custom_db.jpg" alt="Screenshot of Custom Database Box with the accession GCA_028021215.1 Entered" width="800" /></p>
|
||
<ul>
|
||
<li>Use your primer pair 4 from the previous example to search the assembly. This primer pair should bind within the last exon based on the conserved structure of mammalian myoglobin genes and should work in genomic DNA.</li>
|
||
</ul>
|
||
<pre style="padding-left: 320px;">Primer pair 4, last exon
|
||
ACTGTTCCGGAAGGACATCG
|
||
GAGAGCCAAGCTTCTCCTG
|
||
</pre>
|
||
<p><a href="https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=bookmark&PRIMER_LEFT_INPUT=ACTGTTCCGGAAGGACATCG&PRIMER_RIGHT_INPUT=GAGAGCCAAGCTTCTCCTG&OVERLAP_5END=7&OVERLAP_3END=4&PRIMER_PRODUCT_MIN=70&PRIMER_PRODUCT_MAX=1000&PRIMER_NUM_RETURN=10&PRIMER_MIN_TM=57.0&PRIMER_OPT_TM=60.0&PRIMER_MAX_TM=63.0&PRIMER_MAX_DIFF_TM=3&PRIMER_ON_SPLICE_SITE=0&SEARCHMODE=0&SPLICE_SITE_OVERLAP_5END=7&SPLICE_SITE_OVERLAP_3END=4&SPLICE_SITE_OVERLAP_3END_MAX=8&SPAN_INTRON=off&MIN_INTRON_SIZE=1000&MAX_INTRON_SIZE=1000000&SEARCH_SPECIFIC_PRIMER=on&EXCLUDE_ENV=off&EXCLUDE_XM=off&TH_OLOGO_ALIGNMENT=off&TH_TEMPLATE_ALIGNMENT=off&PRIMER_SPECIFICITY_DATABASE=genomic/9764/GCA_028021215.1&TOTAL_PRIMER_SPECIFICITY_MISMATCH=1&PRIMER_3END_SPECIFICITY_MISMATCH=1&MISMATCH_REGION_LENGTH=5&TOTAL_MISMATCH_IGNORE=6&MAX_TARGET_SIZE=4000&ALLOW_TRANSCRIPT_VARIANTS=off&HITSIZE=50000&EVALUE=30000&WORD_SIZE=7&MAX_CANDIDATE_PRIMER=500&PRIMER_MIN_SIZE=15&PRIMER_OPT_SIZE=20&PRIMER_MAX_SIZE=25&PRIMER_MIN_GC=20.0&PRIMER_MAX_GC=80.0&GC_CLAMP=0&NUM_TARGETS_WITH_PRIMERS=1000&NUM_TARGETS=20&MAX_TARGET_PER_TEMPLATE=100&POLYX=5&SELF_ANY=8.00&SELF_END=3.00&PRIMER_MAX_END_STABILITY=9&PRIMER_MAX_END_GC=5&PRIMER_MAX_TEMPLATE_MISPRIMING_TH=40.00&PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH=70.00&PRIMER_MAX_SELF_ANY_TH=45.0&PRIMER_MAX_SELF_END_TH=35.0&PRIMER_PAIR_MAX_COMPL_ANY_TH=45.0&PRIMER_PAIR_MAX_COMPL_END_TH=35.0&PRIMER_MAX_HAIRPIN_TH=24.0&PRIMER_MAX_TEMPLATE_MISPRIMING=12.00&PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING=24.00&PRIMER_PAIR_MAX_COMPL_ANY=8.00&PRIMER_PAIR_MAX_COMPL_END=3.00&PRIMER_MISPRIMING_LIBRARY=AUTO&NO_SNP=off&LOW_COMPLEXITY_FILTER=on&MONO_CATIONS=50.0&DIVA_CATIONS=1.5&CON_ANEAL_OLIGO=50.0&CON_DNTPS=0.6&SALT_FORMULAR=1&TM_METHOD=1&PRIMER_INTERNAL_OLIGO_MIN_SIZE=18&PRIMER_INTERNAL_OLIGO_OPT_SIZE=20&PRIMER_INTERNAL_OLIGO_MAX_SIZE=27&PRIMER_INTERNAL_OLIGO_MIN_TM=57.0&PRIMER_INTERNAL_OLIGO_OPT_TM=60.0&PRIMER_INTERNAL_OLIGO_MAX_TM=63.0&PRIMER_INTERNAL_OLIGO_MAX_GC=80.0&PRIMER_INTERNAL_OLIGO_OPT_GC_PERCENT=50&PRIMER_INTERNAL_OLIGO_MIN_GC=20.0&PICK_HYB_PROBE=off&NEWWIN=off&NEWWIN=off&SHOW_SVIEWER=true" target="_blank" rel="noopener">Preconfigured Primer-BLAST form with myoglobin primer pair 4</a><br /><strong>Note</strong>: There is no way to set the custom database. You will need to enter the assembly accession, GCA_028021215.1, in the Custom box.</p>
|
||
<h3>Verifying the myoglobin gene in the identified grey whale sequence</h3>
|
||
<ul>
|
||
<li>Retrieve the grey whale contig MB region <a href="https://go.usa.gov/xeDWR" target="_blank" rel="noopener">NIPP01005289.1 : 123350 to135659</a>.</li>
|
||
<li>Click the “Run BLAST” link on the right-hand side of the sequence record</li>
|
||
<li>Choose the Reference RNA sequences (refseq_rna), organism limit Cetacea</li>
|
||
<li>What gene products does the search find?</li>
|
||
</ul>
|
||
<p><a href="https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&USER_FORMAT_DEFAULTS=on&SET_SAVED_SEARCH=true&PAGE=MegaBlast&PROGRAM=blastn&QUERY=NIPP01005289.1&QUERY_TO=135659&QUERY_FROM=123350&GAPCOSTS=0%200&MATCH_SCORES=1,-2&DATABASE=refseq_rna&BLAST_PROGRAMS=megaBlast&MAX_NUM_SEQ=100&SHORT_QUERY_ADJUST=on&EXPECT=0.05&WORD_SIZE=28&REPEATS=repeat_9606&TEMPLATE_TYPE=0&TEMPLATE_LENGTH=0&FILTER=L&FILTER=m&EQ_MENU=Cetacea%20%28taxid%3A9721%29&PROG_DEFAULTS=on&SHOW_OVERVIEW=on&SHOW_LINKOUT=on&ALIGNMENT_VIEW=Pairwise&MASK_CHAR=2&MASK_COLOR=1&GET_SEQUENCE=on&NUM_OVERVIEW=100&DESCRIPTIONS=100&ALIGNMENTS=100&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML" target="_blank" rel="noopener">Pre-configured nucleotide BLAST form with the grey whale contig, RefSeq RNA database, cetacea organism limit</a></p>
|
||
<p><a href="https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Get&RID=GW79TEEF013" target="_blank" rel="noopener">Grey whale MB region nucleotide results, RID=GW79TEEF013</a></p>
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|
||
</div>
|
||
</div>
|
||
|
||
<p class=”margin-top-5”><small>Last Reviewed: September 22, 2023</small></p>
|
||
|
||
</div>
|
||
</main>
|
||
<!-- FOOTER -->
|
||
<footer class="usa-footer__primary-section padding-top-5 padding-bottom-3 insertfooter">
|
||
|
||
<div class="grid-container">
|
||
<div class="grid-row">
|
||
<div class="desktop:grid-col-3 grid-col-6"> <a href="https://www.nlm.nih.gov/socialmedia/index.html">
|
||
<p class="text-white margin-bottom-1">Connect with NLM</p>
|
||
</a>
|
||
<ul class="social_media add-list-reset">
|
||
<li class="margin-right-05"><a href="https://www.facebook.com/nationallibraryofmedicine"><img class="bg-secondary" src="https://www.nlm.nih.gov/images/facebook.svg" alt="Facebook"></a></li>
|
||
<li class="margin-right-05"><a title="External link: please review our privacy policy." href="https://www.linkedin.com/company/national-library-of-medicine-nlm/"><img class="bg-secondary" src="//www.nlm.nih.gov/images/linkedin.svg" alt="LinkedIn"></a></li>
|
||
<li class="margin-right-05"><a title="External link: please review our privacy policy." href="https://twitter.com/NLM_NIH"><img src="https://www.nlm.nih.gov/images/twitter.svg" class="padding-1 bg-secondary" alt="Twitter"></a></li>
|
||
<li class="margin-right-05"><a title="External link: please review our privacy policy." href="https://www.youtube.com/user/NLMNIH"><img src="//www.nlm.nih.gov/images/youtube.svg" class="bg-secondary" alt="You Tube"></a></li>
|
||
<li class="margin-right-05"><a title="External link: please review our privacy policy." href="https://public.govdelivery.com/accounts/USNLMOCPL/subscriber/new?preferences=true"><img src="//www.nlm.nih.gov/images/mail.svg" class=" bg-secondary" alt="Government Delivery"></a></li>
|
||
</ul>
|
||
</div>
|
||
<div class="desktop:grid-col-3 grid-col-6">
|
||
<p class="address_footer text-white"> National Library of Medicine <br>
|
||
<a href="https://www.google.com/maps/place/8600+Rockville+Pike,+Bethesda,+MD+20894/@38.9959508,-77.101021,17z/data=!3m1!4b1!4m5!3m4!1s0x89b7c95e25765ddb:0x19156f88b27635b8!8m2!3d38.9959508!4d-77.0988323" class="text-white"> 8600 Rockville Pike <br>
|
||
Bethesda, MD 20894 </a></p>
|
||
</div>
|
||
<div class="desktop:grid-col-3 grid-col-6">
|
||
<p><a href="/web_policies.html" class="text-white"> Web Policies </a><br>
|
||
<a href="https://www.nih.gov/institutes-nih/nih-office-director/office-communications-public-liaison/freedom-information-act-office" class="text-white"> FOIA </a><br>
|
||
<a href="https://www.hhs.gov/vulnerability-disclosure-policy/index.html" class="text-white">HHS Vulnerability Disclosure</a> </p>
|
||
</div>
|
||
<div class="desktop:grid-col-3 grid-col-6">
|
||
<p><a class="supportLink text-white" href="//support.nlm.nih.gov?from="> NLM Support Center </a> <br>
|
||
<a href="/accessibility.html" class="text-white"> Accessibility </a><br>
|
||
<a href="/careers/careers.html" class="text-white"> Careers </a></p>
|
||
</div>
|
||
</div>
|
||
<div class="grid-row">
|
||
<div class="grid-col-12">
|
||
<p class="text-center text-white"> <a class="text-white" href="//www.nlm.nih.gov/">NLM</a> | <a class="text-white" href="https://www.nih.gov/">NIH</a> | <a class="text-white" href="https://www.hhs.gov/">HHS</a> | <a class="text-white" href="https://www.usa.gov/">USA.gov</a></p>
|
||
</div>
|
||
</div>
|
||
</div>
|
||
|
||
</footer>
|
||
<script src="//assets.nlm.nih.gov/uswds/js/uswds.min.js"></script>
|
||
<script src="//assets.nlm.nih.gov/jquery/jquery-latest.min.js"></script>
|
||
<script src="//assets.nlm.nih.gov/jquery/jquery-migrate-latest.min.js"></script>
|
||
<script src="/scripts/nlm_autocomplete.js"></script>
|
||
<script src="/scripts/nlm_uswds.js"></script>
|
||
<script src="/scripts/uswds_accordion_nav.js"></script>
|
||
|
||
|
||
|
||
|
||
|
||
</body>
|
||
</html>
|
||
|
||
|
||
|
||
|
||
|
||
|
||
|