Amplification of a highly polymorphic VNTR segment by the polymerase chain reaction.
Amplification of a highly polymorphic VNTR segment by the polymerase chain reaction.
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通过聚合酶链式反应扩增高度多态性 VNTR 片段。
DOI：
10.1093/nar/17.5.2140
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发表时间：
1989
影响因子：
14.9
通讯作者：
Katherine W. Klinger
中科院分区：
文献类型：
--
作者：
G. Horn;B. Richards;Katherine W. Klinger
由于串联重复序列 (VNTR) 数量可变，人类基因组的某些片段表现出多态性。对这些 VNTR 区域的分析在遗传连锁研究、亲子鉴定和法医鉴定中极其有用 (1)。由探针 pYNZ22（HGM 位点 D1 7S30）检测到的 VNTR 片段是迄今为止已鉴定的最高多态性区域之一 (2)。 Southern blotting已观察到10多个等位基因，杂合度达86%。除了作为多态性标记物的用途外，该探针还与 Miller-Dieker 综合征密切相关 (3)，并且在超过 60% 的乳腺癌肿瘤中被删除 (4)。最近对 pYNZ22 侧翼 DNA 序列的测定 (5) 表明可以通过聚合酶链式反应 (PCR) 来分析该基因座。通过重复应用合成 DNA 引物和热稳定 DNA 聚合酶来扩增基因组的特定区域 (6)。然而，令人担忧的是在扩增其他 VNTR 基因座时遇到的困难 (7)。为了避免与重复单元部分同源的片段，我们设计了两个寡核苷酸引物来指导跨 pYNZ22 区域的扩增。经过 28 个循环的引物在 550°C 退火后，从 1 pg 基因组 DNA 中观察到具有该基因座预期大小的 PCR 产物（图 1）。产物的范围为 170 bp 至 870 bp，正如引物之间 1 至 11 个重复单元所预期的那样。这些等位基因显示出孟德尔遗传模式（泳道 2 至 6）。 1250 bp（泳道 10）的 PCR 产物显然与 pYNZ22 多态性无关，可以通过在扩增中使用更少的引物和更多的总 dNTP 来抑制。
Some segments of the human genome exhibit polymorphism due to a variable number of tandem repeats (VNTR). Analysis of these VNTR regions is extremely useful in genetic linkage studies, paternity testing, and forensic identification (1). The VNTR segment detected by probe pYNZ22 (HGM locus Dl 7S30) is one of the most highly polymorphic regions yet identified (2). Over ten alleles have been observed by Southern blotting, with a heterozygosity of 86%. Besides its usefulness as a polymorphic marker, this probe is also closely associated with the Miller-Dieker syndrome (3), and is deleted in over 60% of breast cancer tumors (4). The recent determination of DNA sequence flanking pYNZ22 (5) suggested that this locus might be analyzed by the polymerase chain reaction (PCR). where specific regions of the genome are amplified by repeated application of synthetic DNA primers and a heat-stable DNA polymerase (6). Of concern, however, were the difficulties encountered when amplifying other VNTR loci (7). Avoiding segments partially homologous to the repeat unit, we designed two oligonucleotide primers to direct amplification across the pYNZ22 region. PCR products with sizes expected for this locus were observed from I pg of genomic DNA after 28 cycles of amplification with primer annealing at 550C (Fig. 1). The products ranged from 170 bp to 870 bp, as expected for I to 11 repeat units between the primers. These alleles showed a Mendelian pattern of inheritance (lanes 2 to 6). A PCR product of 1250 bp (lane 10), apparently unrelated to the pYNZ22 polymorphism, can be suppressed by using less primer and more total dNTP in the amplification.