Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene.
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.
Chun
Jong-Yoon
JY
Seegene Institute of Life Science, 65-5 Bangyi-dong, Songpa-gu, Seoul 138-050, South Korea. chun@seegene.com
Kim
Kyoung-Joong
KJ
Hwang
In-Taek
IT
Kim
Yun-Jee
YJ
Lee
Dae-Hoon
DH
Lee
In-Kyoung
IK
Kim
Jong-Kee
JK
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D028962:Coronavirus OC43, Human / Q000235:genetics / Q000302:isolation & purification; D065731:Cytochrome P-450 CYP2C19; D017931:DNA Primers / Q000737:chemistry*; D005838:Genotype; D006801:Humans; D051379:Mice; D006899:Mixed Function Oxygenases / Q000235:genetics*; D009975:Orthomyxoviridae / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012328:RNA Viruses / Q000302:isolation & purification*; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19