Rapid PCR analysis of the St14 (DXS52) VNTR.
Rapid PCR analysis of the St14 (DXS52) VNTR.
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St14 (DXS52) VNTR 的快速 PCR 分析。
DOI：
10.1093/nar/19.8.1944
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发表时间：
1991
影响因子：
14.9
通讯作者：
Horn,GT
中科院分区：
文献类型：
--
作者：
Richards,B;Heilig,R;Oberlé,I;Storjohann,L;Horn,GT
前言：由于可变数目的串联重复序列(VNTR)，人类基因组的某些片段呈现出多态。用酶法扩增VNTR基因座可以快速分析和区分大小相近的等位基因。我们报道了一种基于聚合酶链式反应的方法来分析高度多态的St14VNTR。来源和描述：Stl4-1探针是从人类DNA中克隆的一个3kb的EcoRI片段，它检测到DXS52基因座的几个多态，包括Taqi发现的高度多态的VNTRRFLP。Southern Transfer分析表明，有10个等位基因的大小在3.4-6.6kb之间，其中黑人个体(1，3)的等位基因长达15kb。负责Taqi多态的区域是从一个克隆的等位基因测序的，并且被发现是一个几乎完全重复的60bp重复(2)。为了用聚合酶链式反应(PCR)检测VNTR，在重复序列的两侧设计了扩增引物。然而，由于高变区两侧有额外的重复序列，所以聚合酶链式反应引物没有直接放置在VNTR附近。结果表明，扩增产物包括约650个碱基的侧翼序列。PCR扩增：在5‘-GGCATGTCATCACTTCTCATGTT-3’和5‘-CACCACTGCCCTCACGTCACTT-3’，‘标准’PCR缓冲液和dNTP浓度(4)，1/%g基因组DNA，以及24个循环的PCR中使用1/AM，包括94℃20秒，55℃30秒，74℃20秒，最后在74℃孵育5分钟，结果不太理想。扩增产物之间的大小差异与Southern blotts上看到的一致；等位基因产物的孟德尔遗传也得到了验证(数据未显示)。频率：对50例无血缘关系的高加索男性进行分析。扩增出的等位基因大小和频率分别为：3000bp(2%)、2900(8%)、2400(12%)、1690(36%)、1630(2%)、1570(14%)、1390(10%)、1300(2%)、1220(2%)和700(12%)。我们还看到了这50个样本中没有的其他稀有等位基因产物(880、1750、1810和2100碱基)。所有这些产品(除了880个基点的产品)如下所示。染色体定位：DXS52基因座定位于Xq26-28，与血友病A、B、脆性X综合征、肾上腺脑白质营养不良等疾病基因连锁。St14 VNTR基因定位于Xq28，距离血友病A-凝血因子VHI基因座约2 cM，但其大量的等位基因使其在血友病A的诊断中非常有用。
Introduction: Some segments of the human genome exhibit polymorphism due to a variable number of tandem repeats (VNTR). The enzymatic amplification of VNTR loci can allow for rapid analysis and discrimination of closely sized alleles. We report a PCR-based method foranalysis of the highly polymorphic Stl4 VNTR. Source and Description: The Stl4-1 probe is a 3 kb EcoRI fragment cloned from human DNA which detects several polymorphisms at the DXS52 loci, including a highly polymorphic VNTR RFLP revealedby TaqI. Analysis of this VNTR by Southern transfer has revealed ten alleles ranging in size from 3.4-6.6 kb, with alleles as large as 15 kb in Black individuals (1, 3). The region responsible for the TaqI polymorphism was sequenced from a cloned allele, and was found to be an almost perfectly duplicated 60 bp repeat (2). To assay the VNTR by PCR, amplification primers were designed on either side of the repeat. However, due to additional repetitive sequence flanking the hypervariable region, the PCR primers were not placed directly adjacent to the VNTR. As a result, amplified products include about 650 bp of flanking sequence. PCR Amplification: We used 1/AM in each of the primers 5'GGCATGTCATCACTTCTCTCATGTT-3'and 5'-CACCACTGCCCTCACGTCACTT-3','standard'PCR buffer and dNTP concentrations (4), 1/% g genomic DNA, and 24 cycles of PCR consisting of 20 sec at 94 C, 30 sec at 55 C, and 20 sec at 74 C, with a final incubation for 5 min at 74 C. Less favorable results were obtained with additional cycles. Size differences between the amplified products were found to correspond with those seen on Southern blots; Mendelian inheritance of the allelic products was alsoverified (data not shown). Frequency: 50 unrelated Caucasian maleswere analyzedusing the above primers. The amplified allele sizes and frequencies observed are: 3000 bp (2%), 2900 (8%), 2400 (12%), 1690 (36%), 1630 (2%), 1570 (14%), 1390 (10%), 1300 (2%), 1220 (2%), and 700 (12%). We have also seen other rare allelic products (880, 1750, 1810 and 2100 bp) that were not present in these 50 samples. All of these products (except for the 880 bp product) are shown below. The size of the smallest product (700 bp) suggests that it represents an allele with just one 60 bp repeat; and the most frequent product of 1690 bp appears to contain 17 tandem repeats.Chromosomal Location: The DXS52 loci map to Xq26-28, and are linked to several disease genes including hemophilias A and B, fragile X syndrome, and adrenoleukodystrophy (2). The St14 VNTR specifically maps to Xq28 and is about 2 cM from the hemophilia A-coagulation factor VHI locus, yet its large number of alleles makes it very useful in the diagnosis of hemophilia A.