摘要
以人CYP2D6 10等位基因位点为研究对象,建立了一种由5条引物同时PCR扩增在单管中进行等 位基因快速检测的新方法。该方法在等位基因特异性扩增法的基础上,在内引物3′端引入了一个人工错配 碱基并在5′端附加一段“尾巴”作为引物的锚定部分,使扩增反应的特异性得到了提高。本实验所建立的方 法可在单管中同时完成SNP3种可能等位基因类型的快速检测。实测了47份样品的CYP2D6 10等位基因 的类型,并随机对其中20份样品的测定结果用RFLP法进行了验证,结果完全一致。结果表明本法简便、快 速,结果准确。
An accurate and effective method was developed for rapidly genotyping in a single tube, which was based on di-allele-specific amplification with artificially modified primers. With human CYP2D6 * 10 as an example, five primers were designed for polymerase chain reaction ( PCR) amplification, where two outer primers were used for amplifying deoxyribonucleic acid (DNA) fragments containing SNP point, and two inner primers were single nucleotide polymorphism ( SNP) -specific primers. The fifth primer which was composed of base G and C is used to increase allele-specific amplification. The specificity of amplification was enhanced by introducing an artificially mismatched base into the 3' end area of the inner primers as well as adding a tail on its 5' end as an anchor primer. The templates were amplified in one tube by five primers and the genotype was determined by the length of extended products separated by gel electrophoresis. The PCR amplification conditions including the types of Taq polymerase, the position for introducing the artificially mismatched base, as well as the concentration ratio between primer's pair were also optimized. The developed method was successfully applied to type CYP2D6 * 10 in 47 samples. The typing results of 20 randomly selected genome samples were the same as those by PCR-RFLP. The method is simple, accurate and easy-to-operate, by which the three possible SNP types can be rapidly determined in a single tube at a time.
出处
《分析化学》
SCIE
EI
CAS
CSCD
北大核心
2005年第2期155-160,共6页
Chinese Journal of Analytical Chemistry
基金
国家自然科学基金资助项目(No.30270368)
