Primer-BLAST Input

The Primer-BLAST interface contains elements of both the Primer3 tool and the NCBI BLAST Web service. The submission form is divided into three sections with commonly adjusted settings relevant to the target template, the primers, and the background specificity check. As with other BLAST services, the more specialized options are available under an expandable section at the bottom of the submission form, called “Advanced parameters” in Primer-BLAST.

Example

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 (Figure 1). The UNG products code for enzymes involved in removing misincorpated uracil during DNA replication. The longer UNG1 transcript (NM_003362 ) codes for a mitochondrial enzyme while the shorter UNG2 transcript (NM_080911) 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).

Figure 1

Primer-BLAST results for UNG transcript variant 2. The NCBI Reference sequence NM_080911 was used as a template. Top panel: Primers specific to the single splice variant are reported by default with the mRNA RefSeq database limited to human sequences. (more...)

Summary

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.

Reference

1. 1. Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386 [PubMed: 10547847]

BLAST 2 Sequences

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: blast.ncbi.nlm.nih.gov/docs/align_seqs.pdf

My NCBI

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 NLM Technical Bulletin article at: www.nlm.nih.gov/pubs/techbull/so08/so08_myncbi_redesign.html . My NCBI is located at: www.ncbi.nlm.nih.gov/sites/myncbi/

BarSTool

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: www.ncbi.nlm.nih.gov/Genbank/barcode.html.

Bookshelf

The Bookshelf has added two new books entitled: Hepatitis C Viruses: Genomes and Molecular Biology and NCBI News. Books can be found at: www.ncbi.nlm.nih.gov/sites/entrez?db=Books.

Microbial Genomes

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: ftp://ftp.ncbi.nih.gov/genbank/genomes/Bacteria/. The RefSeq provisional versions of these genomes are available via FTP at: ftp://ftp.ncbi.nih.gov/genomes/Bacteria/.

Discovery PubMed Pages

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.

RefSeq

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.

The RefSeq website is: www.ncbi.nlm.nih.gov/RefSeq/.

Genome Assembly

Genome annotation for Ciona intestinalis build 1.1 and Arabidopsis thaliana build 8.1 were released in September. For more annotation updates and links to Genome Resource pages see: www.ncbi.nlm.nih.gov/Genomes/.