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Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction.

Publication ,  Journal Article
Ye, J; Coulouris, G; Zaretskaya, I; Cutcutache, I; Rozen, S; Madden, TL
Published in: BMC Bioinformatics
June 18, 2012

BACKGROUND: Choosing appropriate primers is probably the single most important factor affecting the polymerase chain reaction (PCR). Specific amplification of the intended target requires that primers do not have matches to other targets in certain orientations and within certain distances that allow undesired amplification. The process of designing specific primers typically involves two stages. First, the primers flanking regions of interest are generated either manually or using software tools; then they are searched against an appropriate nucleotide sequence database using tools such as BLAST to examine the potential targets. However, the latter is not an easy process as one needs to examine many details between primers and targets, such as the number and the positions of matched bases, the primer orientations and distance between forward and reverse primers. The complexity of such analysis usually makes this a time-consuming and very difficult task for users, especially when the primers have a large number of hits. Furthermore, although the BLAST program has been widely used for primer target detection, it is in fact not an ideal tool for this purpose as BLAST is a local alignment algorithm and does not necessarily return complete match information over the entire primer range. RESULTS: We present a new software tool called Primer-BLAST to alleviate the difficulty in designing target-specific primers. This tool combines BLAST with a global alignment algorithm to ensure a full primer-target alignment and is sensitive enough to detect targets that have a significant number of mismatches to primers. Primer-BLAST allows users to design new target-specific primers in one step as well as to check the specificity of pre-existing primers. Primer-BLAST also supports placing primers based on exon/intron locations and excluding single nucleotide polymorphism (SNP) sites in primers. CONCLUSIONS: We describe a robust and fully implemented general purpose primer design tool that designs target-specific PCR primers. Primer-BLAST offers flexible options to adjust the specificity threshold and other primer properties. This tool is publicly available at http://www.ncbi.nlm.nih.gov/tools/primer-blast.

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Published In

BMC Bioinformatics

DOI

EISSN

1471-2105

Publication Date

June 18, 2012

Volume

13

Start / End Page

134

Location

England

Related Subject Headings

  • Software
  • Polymorphism, Single Nucleotide
  • Polymerase Chain Reaction
  • Molecular Sequence Data
  • Introns
  • Humans
  • DNA Primers
  • Carrier Proteins
  • Bioinformatics
  • Algorithms
 

Citation

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Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., & Madden, T. L. (2012). Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics, 13, 134. https://doi.org/10.1186/1471-2105-13-134
Ye, Jian, George Coulouris, Irena Zaretskaya, Ioana Cutcutache, Steve Rozen, and Thomas L. Madden. “Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction.BMC Bioinformatics 13 (June 18, 2012): 134. https://doi.org/10.1186/1471-2105-13-134.
Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 2012 Jun 18;13:134.
Ye, Jian, et al. “Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction.BMC Bioinformatics, vol. 13, June 2012, p. 134. Pubmed, doi:10.1186/1471-2105-13-134.
Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 2012 Jun 18;13:134.
Journal cover image

Published In

BMC Bioinformatics

DOI

EISSN

1471-2105

Publication Date

June 18, 2012

Volume

13

Start / End Page

134

Location

England

Related Subject Headings

  • Software
  • Polymorphism, Single Nucleotide
  • Polymerase Chain Reaction
  • Molecular Sequence Data
  • Introns
  • Humans
  • DNA Primers
  • Carrier Proteins
  • Bioinformatics
  • Algorithms