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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>NCBI News, November 2008 - NCBI News - NCBI Bookshelf</title>
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<meta name="citation_author" content="Peter Cooper">
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yet</button><a id="jr-fip-next" class="wsprkl btn" title="Jump to next match">▶</a></nav></nav></div><div id="jr-epub-interstitial" class="hidden"></div><div id="jr-content"><article data-type="main"><div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><div class="fm-sec"><h1 id="_NBK2593_"><span class="title" itemprop="name">NCBI News, November 2008</span></h1><p class="contribs">Lipshultz D, Cooper P.</p><p class="fm-aai"><a href="#_NBK2593_pubdet_">Publication Details</a></p><p><em>Estimated reading time: 6 minutes</em></p></div></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="nov08.Featured_Resource_Pr"><h2 id="_nov08_Featured_Resource_Pr_">Featured Resource: Primer-BLAST—NCBI’s Primer Designer and
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Specificity Checker</h2><p>NCBI now offers Primer-BLAST, a Web service for designing target specific oligonucleotide
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primers for use in polymerase chain reaction (PCR) protocols. Primer-BLAST may be accessed
|
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from the NCBI BLAST Homepage or through the direct URL:</p><p>
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<a href="/tools/primer-blast" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://www.ncbi.nlm.nih.gov/tools/primer-blast/</a>
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</p><p>This service integrates primer designing code from the popular Primer3<a href="#nov08.Reference">
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<sup>1</sup>
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</a> software with a specificity check that uses a custom BLAST search. Primer-BLAST
|
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eliminates the need to design primers at another site and then perform a search with those
|
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primers using the main NCBI BLAST service to check specificity. Moreover, Primer-BLAST can
|
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design primers that amplify only a particular splice variant of a gene – an
|
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important feature for PCR protocols measuring tissue specific expression. Primer-BLAST has a
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number of additional enhancements that make it better at picking specific primers than
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Primer3 and NCBI BLAST used separately.</p><div id="nov08.PrimerBLAST_Input"><h3>Primer-BLAST Input</h3><p>The Primer-BLAST interface contains elements of both the Primer3 tool and the NCBI BLAST
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Web service. The submission form is divided into three sections with commonly adjusted
|
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settings relevant to the <b>target template</b>, <b>the primers,</b> and the
|
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background <b>specificity check</b>. As with other BLAST services, the more
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specialized options are available under an expandable section at the bottom of the
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submission form, called “Advanced parameters” in Primer-BLAST. </p><div id="nov08.Template"><h4>Template</h4><p>The target template sequence or accession number to be analyzed is placed in the large
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text area in the <b>PCR Template</b> section of the form. Specific binding regions
|
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on the template may be specified using the range boxes. With just the target template,
|
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Primer-BLAST will design primer pairs that are specific to the target template and
|
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unique in the target database. </p></div><div id="nov08.Primers"><h4>Primers</h4><p>One or both primers may be supplied in the <b>Primer Parameters</b> section. The
|
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most important and commonly modified primer-specific parameters such as desired product
|
|
size, Tm ranges and Tm difference may be modified here as well. Primers may accompany a
|
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template, but may also be used without a template. With only a primer pair, Primer-BLAST
|
|
identifies potential templates in the target database. In this case, the results are
|
|
more precise than a specificity check with ordinary BLAST and have additional PCR
|
|
condition parameters such as melting temperature (Tm) provided in the results. </p></div><div id="nov08.Specificity"><h4>Specificity</h4><p>Parameters that may be adjusted in the <b>Specificity Checking </b>section of the
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form affect the nature of the background database and the level of stringency for
|
|
avoiding nonspecific matches. The database specificity check does not simply find BLAST
|
|
matches but includes adjustable mismatch cutoffs that focus on the 3′ end of
|
|
the binding site to more effectively avoid mispriming. The most informative results are
|
|
obtained by selecting the target database and organism that best match the template
|
|
sequence source. The organism selection is managed by typing the name of the organism of
|
|
interest in the “Organism” box. An auto-complete feature will
|
|
find the closest match in the NCBI taxonomy database. </p><p>Four BLAST nucleotide databases are available for searching: RefSeq mRNA, Genome
|
|
(selected reference assemblies), Genome (all chromosomes), and nr (the standard
|
|
non-redundant database). The first two databases will give the most precise results as
|
|
these are the best annotated and analyzed sets of sequences. In particular, if an NCBI
|
|
mRNA Reference Sequence is used as a template with the RefSeq mRNA database,
|
|
Primer-BLAST will design primers specific to the single splice variant represented by
|
|
the reference sequence. The well characterized genome sequences of human, chimpanzee,
|
|
mouse, rat, cow, dog, chicken, zebrafish, fruit fly, honeybee,
|
|
<i>Arabidopsis</i>, and rice are available as the selected reference
|
|
assemblies. These selected reference assemblies will produce results that are less
|
|
redundant and easier to interpret when using genomic templates from these organisms. The
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remaining Genome database (all chromosomes) contains all chromosome Reference Sequences
|
|
(NC_ accessions) including the selected reference assemblies, as well as their
|
|
alternative assemblies, and chromosome records for other organisms, most notably
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microbial genome sequences. Finally, the nr database is available for the widest
|
|
coverage of organisms.</p></div><div id="nov08.Advanced_Parameters"><h4>Advanced Parameters</h4><p>The <b>Advanced Parameters</b> section contains numerous adjustable settings
|
|
affecting primer binding and efficacy from the Primer3 program. Two additional features
|
|
that are only available in Primer-BLAST are options to avoid making primers that match
|
|
repetitive or regions with biased base composition (low complexity regions) and an
|
|
option to avoid regions that contain reported nucleotide polymorphisms (SNPs) from
|
|
NCBI’s SNP database. As with Primer3, Primer-BLAST also has an option to
|
|
design an internal hybridization probe for each product that can be used in various
|
|
product detection assays.</p></div></div><div id="nov08.Example"><h3>Example</h3><p>Using Primer-BLAST to design primers to distinguish the two transcripts of the human
|
|
uracil-DNA glycosylase genes (UNG, GeneID: 7374) demonstrates the utility and precision of
|
|
Primer BLAST (<a class="figpopup" href="/books/NBK2593/figure/nov08.nov08_fig1/?report=objectonly" target="object" rid-figpopup="fignov08nov08fig1" rid-ob="figobnov08nov08fig1">Figure 1</a>). The UNG products code for enzymes
|
|
involved in removing misincorpated uracil during DNA replication. The longer UNG1
|
|
transcript (<a href="/nuccore/1677530039" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">NM_003362</a> ) codes for a mitochondrial enzyme while the shorter UNG2 transcript
|
|
(<a href="/nuccore/1677498868" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">NM_080911</a>) that lacks the portion coding for the mitochondrial targeting sequence
|
|
apparently functions in the nuclear genome of replicating cells. The top panel of Figure 1
|
|
shows the Primer-BLAST results that are specific to the UNG2 splice variant. In this case
|
|
the NCBI reference sequence was used as a template with RefSeq mRNA database limited to
|
|
human. By default the service designs only UNG2 specific primers with the reference
|
|
sequence as the template. These conditions may be relaxed by checking the option for
|
|
“Allow primer to amplify mRNA splice variants.” The bottom panel
|
|
of Figure 1 shows that under these conditions new primers are found that will amplify both
|
|
transcripts from the UNG gene. Expanding the database coverage provides a means for
|
|
checking primers in additional species. The results using the nr database predict that
|
|
many of the primer pairs designed against the human UNG transcripts should also amplify
|
|
transcripts from other primate species (not shown). </p><div class="iconblock whole_rhythm clearfix ten_col fig" id="fignov08nov08fig1" co-legend-rid="figlgndnov08nov08fig1"><a href="/books/NBK2593/figure/nov08.nov08_fig1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="fignov08nov08fig1" rid-ob="figobnov08nov08fig1"><img class="small-thumb" src="/books/NBK2593/bin/nov08.G1.gif" src-large="/books/NBK2593/bin/nov08.G1.jpg" alt="Figure 1. Primer-BLAST results for UNG transcript variant 2." /></a><div class="icnblk_cntnt" id="figlgndnov08nov08fig1"><h4 id="nov08.nov08_fig1"><a href="/books/NBK2593/figure/nov08.nov08_fig1/?report=objectonly" target="object" rid-ob="figobnov08nov08fig1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Primer-BLAST results for UNG transcript variant 2. The NCBI
|
|
Reference sequence NM_080911 was used as a template. <i>Top panel:</i> Primers
|
|
specific to the single splice variant are reported by default with the mRNA RefSeq
|
|
database limited to human sequences. <a href="/books/NBK2593/figure/nov08.nov08_fig1/?report=objectonly" target="object" rid-ob="figobnov08nov08fig1">(more...)</a></p></div></div></div><div id="nov08.Summary"><h3>Summary</h3><p>Primer-BLAST provides a new more powerful and more comprehensive primer design tool by
|
|
combining oligonucleotide primer design with specific genome and transcriptome level
|
|
searches, and will save time and money by eliminating some of the trial and error of PCR
|
|
primer design.</p></div><div id="nov08.Reference"><h3>Reference</h3><ol><li><div class="bk_ref" id="nov08.MN.1">1. Steve Rozen and Helen J. Skaletsky (2000)
|
|
Primer3 on the WWW for general users and for biologist programmers. <strong>In:</strong>
|
|
Krawetz S, Misener S (eds) <em>Bioinformatics Methods and Protocols: Methods in
|
|
Molecular Biology.</em> Humana Press, Totowa, NJ, pp 365-386 [<a href="https://pubmed.ncbi.nlm.nih.gov/10547847" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10547847</span></a>]</div></li></ol></div></div><div id="nov08.New_Databases_and_To"><h2 id="_nov08_New_Databases_and_To_">New Databases and Tools</h2><div id="nov08.BLAST_2_Sequences"><h3>BLAST 2 Sequences</h3><p>The NCBI BLAST web pages have a new option to align a query against a set of target
|
|
sequences, rather than a BLAST database. This option allows you to align your query to one
|
|
or more subject sequences and still use the standard BLAST web interface to optimize your
|
|
search and change algorithm parameters. Each search is assigned a "Request ID" (RID) and
|
|
is also listed under the "Recent Results" tab that you can access from the BLAST home
|
|
page. The results are formatted as a standard BLAST report, except a "Dot Matrix view" (a
|
|
"dot-plot" like graphic of the alignments) is available in the new report design if only
|
|
one subject sequence was searched. Links to BLAST web pages can be found at:
|
|
blast.ncbi.nlm.nih.gov/BLAST.cgi Step-by-step instructions can be found at: <a href="http://blast.ncbi.nlm.nih.gov/docs/align_seqs.pdf" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">blast.ncbi.nlm.nih.gov/docs/align_seqs.pdf</a></p></div><div id="nov08.My_NCBI"><h3>My NCBI</h3><p>My NCBI version 2.0 was released in September with new features and updated functions.
|
|
New features include: account and username options; navigation, preferences, and filter
|
|
improvements; My Saved Searches tool; and My Collections tool. Another new feature, My
|
|
Bibliography, allows authors to search and collect citations for their publications. For a
|
|
full description of new and improved features, please see the <i>NLM Technical
|
|
Bulletin</i> article at: <a href="http://www.nlm.nih.gov/pubs/techbull/so08/so08_myncbi_redesign.html" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">www.nlm.nih.gov/pubs/techbull/so08/so08_myncbi_redesign.html</a> . My NCBI is
|
|
located at: <a href="/sites/myncbi" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">www.ncbi.nlm.nih.gov/sites/myncbi/</a></p></div><div id="nov08.BarSTool"><h3>BarSTool</h3><p>The Barcode of Life project is one in which DNA barcode sequences are being collected
|
|
from vouchered specimens of all biological species. The idea is to make it easier to
|
|
identify biological specimens via a short gene sequence from a standardized position in
|
|
the genome. NCBI has created a submission tool, Barcode Submission Tool (BarSTool), to
|
|
submit barcode sequences to GenBank. For more information about BarSTool or the Barcode of
|
|
Life project see: <a href="http:///Local%20Settings/Temporary%20Internet%20Files/OLK80/www.ncbi.nlm.nih.gov/Genbank/barcode.html" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">www.ncbi.nlm.nih.gov/Genbank/barcode.html</a>.</p></div><div id="nov08.Bookshelf"><h3>Bookshelf</h3><p>The Bookshelf has added two new books entitled: <i>Hepatitis C Viruses: Genomes and
|
|
Molecular Biology</i> and <i>NCBI News</i>. Books can be found at:
|
|
<a href="/sites/entrez?db=Books" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">www.ncbi.nlm.nih.gov/sites/entrez?db=Books</a>.</p></div><div id="nov08.Microbial_Genomes"><h3>Microbial Genomes</h3><p>Thirty-two finished microbial genomes were released (August 15-October 10). The original
|
|
sequence data files submitted to GenBank/EMBL/DDBJ can be found at: <a href="ftp://ftp.ncbi.nih.gov/genbank/genomes/Bacteria" ref="pagearea=body&targetsite=external&targetcat=link&targettype=ftp">ftp://ftp.ncbi.nih.gov/genbank/genomes/Bacteria/</a>. The RefSeq provisional
|
|
versions of these genomes are available via FTP at: <a href="ftp://ftp.ncbi.nih.gov/genomes/Bacteria" ref="pagearea=body&targetsite=external&targetcat=link&targettype=ftp">ftp://ftp.ncbi.nih.gov/genomes/Bacteria/</a>.</p></div></div><div id="nov08.GenBank_News"><h2 id="_nov08_GenBank_News_">GenBank News</h2><p>GenBank release 167.0 is available via web and FTP that includes information as of August
|
|
19, 2008.</p></div><div id="nov08.Updates_and_Enhancem"><h2 id="_nov08_Updates_and_Enhancem_">Updates and Enhancements</h2><div id="nov08.Discovery_PubMed_Pag"><h3>Discovery PubMed Pages</h3><p>PubMed Summary results pages have begun showing related information from other resources
|
|
on the right side of the page. Drug Sensor, an NCBI tool that detects drug names in a
|
|
search, was released in August. As of September, additional resources will be shown to a
|
|
percentage of users to aid in the discovery process.</p></div><div id="nov08.RefSeq"><h3>RefSeq</h3><p>RefSeq Release 31 is available via web and FTP. This full release incorporates genomic,
|
|
transcript, and protein data available as of August 30, 2008 and includes 9,145,702
|
|
records, 5,859,648  proteins, and sequences from 5,513 different organisms. </p><p>The RefSeq website is: <a href="/RefSeq" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">www.ncbi.nlm.nih.gov/RefSeq/</a>.</p></div><div id="nov08.Genome_Assembly"><h3>Genome Assembly</h3><p>Genome annotation for <i>Ciona intestinalis</i> build 1.1 and
|
|
<i>Arabidopsis thaliana</i> build 8.1 were released in September. For more
|
|
annotation updates and links to Genome Resource pages see: <a href="/Genomes" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">www.ncbi.nlm.nih.gov/Genomes/</a>.</p></div></div><div id="nov08.Announce_Lists_and_R"><h2 id="_nov08_Announce_Lists_and_R_">Announce Lists and RSS Feeds</h2><p>Fifteen topic-specific mailing lists are described on the Announcement List summary page.
|
|
Announce lists provide email announcements about changes and updates to NCBI resources.
|
|
<a href="/Sitemap/Summary/email_lists.html" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://www.ncbi.nlm.nih.gov/Sitemap/Summary/email_lists.html</a></p><p>Seven RSS feeds are now available from NCBI including news on PubMed, PubMed Central, NCBI
|
|
Bookshelf, LinkOut, HomoloGene, UniGene, and NCBI Announce. <a href="/feed" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://www.ncbi.nlm.nih.gov/feed/</a></p><p>Comments and questions about NCBI resources may be sent to NCBI at:
|
|
<a href="mailto:dev@null" data-email="vog.hin.mln.ibcn@ofni" class="oemail">vog.hin.mln.ibcn@ofni</a>, or by calling 301-496-2475 between the hours of 8:30
|
|
a.m. and 5:30 p.m. EST, Monday through Friday.</p></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK2593_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><div class="contrib half_rhythm"><span itemprop="author">Dawn Lipshultz</span>, M.S.<div class="affiliation small">NCBI<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="vog.hin.mln.ibcn@tluhspil" class="oemail">vog.hin.mln.ibcn@tluhspil</a></div></div></div><div class="contrib half_rhythm"><span itemprop="author">Peter Cooper</span>, Ph.D.<div class="affiliation small">NCBI<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="vog.hin.mln.ibcn@repooc" class="oemail">vog.hin.mln.ibcn@repooc</a></div></div></div><h3>Publication History</h3><p class="small">Created: <span itemprop="datePublished">October 15, 2008</span>; Last Update: <span itemprop="dateModified">October 28, 2008</span>.</p><h3>Copyright</h3><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div></div><h3>Publisher</h3><p><a href="http://www.ncbi.nlm.nih.gov/news" ref="pagearea=page-banner&targetsite=external&targetcat=link&targettype=publisher">National Center for Biotechnology Information (US)</a>, Bethesda (MD)</p><h3>NLM Citation</h3><p>Lipshultz D, Cooper P. NCBI News, November 2008. 2008 Oct 15 [Updated 2008 Oct 28]. In: NCBI News [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 1991-2012. <span class="bk_cite_avail"></span></p></div><div class="small-screen-prev"><a href="/books/n/newsncbi/dec08/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M75,30 c-80,60 -80,0 0,60 c-30,-60 -30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Prev</text></svg></a></div><div class="small-screen-next"><a href="/books/n/newsncbi/aug08/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M25,30c80,60 80,0 0,60 c30,-60 30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Next</text></svg></a></div></article><article data-type="fig" id="figobnov08nov08fig1"><div id="nov08.nov08_fig1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK2593/bin/nov08.G1.jpg" alt="Figure 1. Primer-BLAST results for UNG transcript variant 2." /></div><h3><span class="label">Figure 1</span><span class="title">Primer-BLAST results for UNG transcript variant 2</span></h3><div class="caption"><p>The NCBI
|
|
Reference sequence <a href="/nuccore/1677498868" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">NM_080911</a> was used as a template. <b>Top panel:</b> Primers
|
|
specific to the single splice variant are reported by default with the mRNA RefSeq
|
|
database limited to human sequences. <b>Bottom panel:</b> Primers that amplify
|
|
both splice variants are found with the option to allow splice variants.</p></div></div></article></div><div id="jr-scripts"><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/libs.min.js"> </script><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.min.js"> </script></div></div>
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