Molecular haplotype determination using allele-specific PCR and Pyrosequencing technology
Molecular haplotype determination using allele-specific PCR and Pyrosequencing technology
复制标题
DOI：
10.1016/s0888-7543(03)00177-0
复制
发表时间：
2003-09-01
期刊：
影响因子：
4.4
通讯作者：
Alderborn, A
中科院分区：
文献类型：
--
作者：
Pettersson, M;Bylund, M;Alderborn, A
单核苷酸多态性（SNPs）的单倍型分析通常通过计算分析或耗时的克隆技术进行统计。在这里，我们提出了一个简单的分子方法，可靠的单倍型测定个体样本。该方法基于等位基因特异性PCR（AS-PCR）结合焦磷酸测序分析。使用了所研究SNP的每个等位基因变体的AS-PCR引物。在3'端第二个碱基处引入的错配显著提高了等位基因特异性。使用焦磷酸测序技术分析扩增片段上的多个SNP允许确定单倍型。AS-PCR后的杂合子样品的基因分型给出了典型的单等位基因模式在每个SNP，其中本等位基因的身份依赖于等位基因特异性的初始扩增。单倍型测定所描述的程序被证明是高度可靠的。通过焦磷酸测序技术获得的结果具有真正定量的优点，能够检测任何非特异性等位基因扩增。(C)2003 Elsevier Science（美国）。All rights reserved.
Haplotyping of single-nucleotide polymorphisms (SNPs) is usually performed statistically by computational analysis or by time-consuming cloning techniques. Here we present a simple molecular approach for reliable haplotype determination on individual samples. The procedure is based on allele-specific PCR (AS-PCR) in combination with Pyrosequencing analysis. AS-PCR primers for each allelic variant of the investigated SNPs were used. A mismatch introduced at the second base from the 3' end dramatically improved allele specificity. Analysis of multiple SNPs on amplified fragments using Pyrosequencing technology allowed determination of haplotypes. Genotyping of heterozygote samples after AS-PCR gave a typical monoallelic pattern at each SNP, in which the identity of the present allele depended on the allele-specific initial amplification. Haplotype determination by the described procedure proved to be highly reliable. The results obtained by Pyrosequencing technology have the benefit of being truly quantitative, enabling detection of any nonspecific allele amplification. (C) 2003 Elsevier Science (USA). All rights reserved.