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Nucleic Acids Research Advance Access originally published online on February 7, 2007
Nucleic Acids Research 2007 35(6):e40; doi:10.1093/nar/gkm051
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Nucleic Acids Research, 2007, Vol. 35, No. 6 e40
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene

Jong-Yoon Chun*, Kyoung-Joong Kim, In-Taek Hwang, Yun-Jee Kim, Dae-Hoon Lee, In-Kyoung Lee and Jong-Kee Kim

Seegene Institute of Life Science, 65-5 Bangyi-dong, Songpa-gu, Seoul 138-050, South Korea

*To whom correspondence should be addressed. Tel: 82 2 2240 4100; Fax: 82 2 2240 4042; Email: chun{at}seegene.com

Received October 13, 2006. Revised January 15, 2007. Accepted January 16, 2007.

Successful PCR starts with proper priming between an oligonucleotide primer and the template DNA. However, the inevitable risk of mismatched priming cannot be avoided in the currently used primer system, even though considerable time and effort are devoted to primer design and optimization of reaction conditions. Here, we report a novel dual priming oligonucleotide (DPO) which contains two separate priming regions joined by a polydeoxyinosine linker. The linker assumes a bubble-like structure which itself is not involved in priming, but rather delineates the boundary between the two parts of the primer. This structure results in two primer segments with distinct annealing properties: a longer 5'-segment that initiates stable priming, and a short 3'-segment that determines target-specific extension. This DPO-based system is a fundamental tool for blocking extension of non-specifically primed templates, and thereby generates consistently high PCR specificity even under less than optimal PCR conditions. The strength and utility of the DPO system are demonstrated here using multiplex PCR and SNP genotyping PCR.


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