Detection of deletion mutations extending beyond the HPRT gene by multiplex PCR analysis.
Detection of deletion mutations extending beyond the HPRT gene by multiplex PCR analysis.
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通过多重 PCR 分析检测超出 HPRT 基因的缺失突变。
DOI：
10.1007/bf02257484
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发表时间：
1994
期刊：
影响因子：
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通讯作者：
Pilia,G
中科院分区：
文献类型：
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作者：
Fuscoe,JC;Nelsen,AJ;Pilia,G
建立了一种快速检测次黄嘌呤磷酸核糖转移酶(HPRT)基因缺失大小的多重聚合酶链式反应方法。测定了hprt基因两侧作图DNA片段的DNA序列。这些克隆的DNA来自一组重叠的酵母人工染色体(YAC)的末端，在Xq26上定义了8Mb的重叠群，包括hprt。我们用“气泡”聚合酶链式反应分离了一个额外的YAC末端克隆。从克隆的DNA序列分析中获得了7对引物，并将其纳入多重PCR分析。这些引物对定义了位于hprt上游约750kb和350kb以及hprt下游约300kb、540kb、900kb、1260kb和1400kb的基因座。在多重反应中还包括一对未连接和未选择的基因序列(K-ras)的引物，以作为内部阳性对照。使用这种新的检测方法，可以筛选hprt突变DNA以确定缺失的程度。大于2Mb的缺失已经被发现，并表明在这个半合子基因座上可以容忍大的缺失。
A multiplex PCR assay was developed for the rapid analysis of deletion size at the hypoxanthine phosphoribosyltransferase (hprt) locus. The DNA sequence of mapped DNA segments flanking thehprtgene was determined. These cloned DNAs were derived from the ends of a set of overlapping yeast artificial chromosomes (YAC) defining a contig of 8 Mb at Xq26 and includinghprt. We used “bubble” PCR to isolate an additional YAC end-clone. Seven primer pairs were derived from DNA sequence analysis of the clones and incorporated into a multiplex PCR assay. These primer pairs define loci located approximately 750 kb and 350 kb upstream ofhprtand 300 kb, 540 kb, 900 kb, 1260 kb, and 1400 kb downstream ofhprt. A primer pair for an unlinked and unselected gene sequence (K-ras) was also included in the multiplex reaction to serve as an internal positive control. Using this new assay,hprtmutant DNAs can be screened to determine the extent of deletion. Deletions larger than 2 Mb have been identified and show that large deletions can be tolerated at this hemizygous locus.