Rapid determination of clonality by detection of two closely-linked X chromosome exonic polymorphisms using allele-specific PCR.
Rapid determination of clonality by detection of two closely-linked X chromosome exonic polymorphisms using allele-specific PCR.
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使用等位基因特异性 PCR 检测两个紧密连锁的 X 染色体外显子多态性，快速确定克隆性。
DOI：
10.1172/jci119366
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Prchal,JT
中科院分区：
文献类型：
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作者：
Liu,Y;Phelan,J;Go,RC;Prchal,JF;Prchal,JT
我们报告了两个具体的，可重复的，和定量的克隆性检测的基础上检测的X染色体基因p55和G6 PD外显子多态性使用rtPCR-LDR。这些测定不便于筛选目的。本研究旨在开发一种简单、重复性好、实用的p55/G6 PD基因型筛选方法，并快速确定克隆性，以确定这些密切相关的基因座之间的连锁关系。ASPCR的突出特点是两轮PCR的性能。第一步生成模板;第二种方法是在两个反应管中使用第一轮产物的一个等分试样，每个反应管含有一个等位基因特异性引物，检测每个等位基因。ASPCR和rtPCR-LDR在91个正常女性基因组DNA和12个克隆性造血疾病cDNA中产生相同的p55/G6 PD结果，证实了测定的有效性。通过ASPCR分析了209名女性和207名男性基因组DNA样本的p55/G6 PD基因型; 60%的女性在一个或两个位点上杂合。G6 PD和p55等位基因频率在非洲裔美国人男性和女性之间存在显著差异，但在白人男性和女性之间没有显著差异。这些基因座在非裔美国人中处于连锁平衡，但在高加索人中不处于连锁平衡。ASPCR是一种快速、灵敏、特异的方法，用于筛选大量基因组DNA，并用于快速克隆性测定。
We reported two specific, reproducible, and quantitative clonality assays based on detection of exonic polymorphisms of the X chromosome genes p55 and G6PD using rtPCR-LDR. These assays are inconvenient for screening purposes. This study sought to develop a simple, reproducible assay, practical for screening genomic DNA samples for p55/G6PD genotypes, rapid clonality determination, and to determine the linkage relationship between these closely related loci. The salient feature of ASPCR is the performance of two PCR rounds. The first generates template; the second, using one aliquot of first-round products in two reaction tubes, each containing one allele-specific primer, detects each allele. ASPCR and rtPCR-LDR produced identical p55/G6PD results in 91 normal female genomic DNAs, and in 12 clonal hematopoietic disorder cDNAs, confirming assay validity. 209 female and 207 male genomic DNA samples were analyzed for p55/G6PD genotype by ASPCR; 60% of females were heterozygous at one or both loci. G6PD and p55 allelic frequencies were significantly different among African-American men and women, but were not significantly different among Caucasian men and women. These loci were in linkage equilibrium among African Americans, but not among Caucasians. ASPCR is a rapid, sensitive, and specific method for screening large numbers of genomic DNAs, and for rapid clonality determination.