Competitive amplification and unspecific amplification in polymerase chain reaction with confronting two-pair primers
Competitive amplification and unspecific amplification in polymerase chain reaction with confronting two-pair primers
复制标题
DOI：
10.1016/s1525-1578(10)60688-5
复制
发表时间：
2002-05-01
影响因子：
4.1
通讯作者：
Tajima, K
中科院分区：
文献类型：
--
作者：
Hamajima, N;Saito, T;Tajima, K
聚合酶链反应与对抗双对引物（PCR-CTPP）是一种廉价，节省时间的基因分型方法，适用于大多数单核苷酸多态性。迄今为止，我们已经成功地应用PCR-CTPP对30多个多态性进行了基因分型。本文论证了四种引物在给定熔融温度下，pcr - cctpp在不同退火温度下DNA扩增的差异。以NQO1 C609T （Pro187Ser）多态性基因为例。PCR-CTPP采用两组引物；第一组有不同的熔化温度（罐头），第二组有相似的罐头。与单对引物PCR（等位基因特异性PCR）的比较表明，PCR- ctpp比等位基因特异性PCR更能特异性地扩增DNA。不同Tms的引物对杂合基因型产生竞争性DNA扩增。具有相似Tins的4条引物在较低的退火温度下对两个等位基因进行了非特异性扩增，而在最佳退火温度下对相同DNA样品进行了正确的基因分型。这些发现在PCR-CTPP中是独一无二的，也是设计PCR-CTPP引物和退火温度时的重要特征。这些特征的知识将扩展PCR-CTPP多态性基因分型的适用性。
Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is an inexpensive, time-saving genotyping method that is applicable for most single nucleotide polymorphisms. To date, we have applied PCR-CTPP successfully for the genotyping of more than 30 polymorphisms. This paper demonstrates the differences in DNA amplification among different annealing temperatures of PCR-CTPP with given melting temperatures for four primers. The NQO1 C609T (Pro187Ser) polymorphisin was used as an example. Two sets of four primers were applied for PCR-CTPP; the first set with different melting temperatures (Tins), and the second with similar Tins. The comparisons with one-pair primer PCR (allele-specific PCR) revealed that PCR-CTPP amplified DNA more specifically than allele-specific PCR. The primers with different Tms caused competitive DNA amplification for heterozygous genotype. Four primers with similar Tins amplified both alleles unspecifically at a lower annealing temperature, while the same DNA samples were correctly genotyped under an optimal annealing temperature. These findings are unique for PCR-CTPP, and important characteristics when the primers and annealing temperatures in PCR-CTPP are designed. The knowledge of these characteristics will extend the applicability of PCR-CTPP for polymorphism genotyping.