Format

Send to

Choose Destination
Bioinformatics. 2005 Dec 15;21(24):4363-70. Epub 2005 Oct 18.

A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3'-end subsequences.

Author information

1
Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo Kashiwa, Japan.

Abstract

MOTIVATION:

In contrast with conventional PCR using a pair of specific primers, some applications utilize a single unique primer in combination with a common primer, thereby relying solely on the former for specificity. These applications include rapid amplification of cDNA ends (RACE), adaptor-tagged competitive PCR (ATAC-PCR), PCR-mediated genome walking and so forth. Since the primers designed by conventional methods often fail to work in these applications, an improved strategy is required, particularly, for a large-scale analysis.

RESULTS:

Based on the structure of 'off-target' products in the ATAC-PCR, we reasoned that the practical determinant of the specificity of primers may not be the uniqueness of entire sequence but that of the shortest 3'-end subsequence that exceeds a threshold of duplex stability. We termed such a subsequence as a 'specificity-determining subsequence' (SDSS) and developed a simple algorithm to predict the performance of the primer: the algorithm identifies the SDSS of each primer and examines its uniqueness in the target genome. The primers designed using this algorithm worked much better than those designed using a conventional method in both ATAC-PCR and 5'-RACE experiments. Thus, the algorithm will be generally useful for improving various PCR-based applications.

PMID:
16234322
DOI:
10.1093/bioinformatics/bti716
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Silverchair Information Systems
Loading ...
Support Center