Problem With Microsatellite Polymerphism Analysis and its Practical Solution Detection of Complex STR Alleles by Single-Strand Conformation Polymorphism (SSCP) Analysis and Denaturing Gradient Gel Electrophoresis (DGGE)

我们扩增了复杂的短串联重复（STR）位点D11S488和D11S554，在变性聚丙烯酰胺凝胶上电泳扩增，并用Southern blotting进行检测。对262 bp长的D11S488等位基因进行测序，发现5个不同的等位基因存在结构变异。D115554位点的序列分析显示，237 bp、225 bp和217 bp等位基因分别有3个、2个和2个不同的序列。D115488扩增子的单链构象多态性（SSCP）在6%的天然聚丙烯酰胺凝胶上进行分析，使我们能够区分出4个相同长度的等位基因。同样地，我们可以在5%聚丙烯酰胺凝胶上电泳，鉴定出相同长度的不同D11S554等位基因。通过20-50%的变性梯度凝胶电泳（DGGE）成功分离出4个不同的D115488等位基因（262 bp）， 25-35%的DGGE成功分离出上述所有不同的D115554等位基因。然而，不同长度的等位基因表现出相同的移动性。我们将SSCP技术应用于两例正常的三人争议亲子关系，检测了包括D115488在内的四个STR位点。这三个人在变性凝胶上具有明显相同的262 bp D115488等位基因。在一个案例中，SSCP分析增加了亲子关系的综合可能性，因为孩子和所谓的父亲共享一个罕见的等位基因。在另一种情况下，孩子与母亲共享等位基因，但不与所谓的父亲共享，这就排除了亲子关系。结果，四个基因座中有三个被排除了亲子关系。允许简单和快速的检测序列多态性，我们的方法适合于实际应用。

We amplified the complex short tandem repeat (STR) loci D11S488 and D11S554, electrophoresed amplicons on denaturing polyacrylamide gels, and detected them by Southern blotting. Sequencing of 262 bp-long D11S488 alleles revealed five different alleles with structural variations. Sequence analysis of the D115554 locus showed that 237 bp, 225 bp and 217 bp alleles had three, two, and two different sequences, respectively.Single-strand conformation polymorphism (SSCP) analysis of D115488 amplicons on a 6% native polyacrylamide gel at 4ﾟC enabled us to distinguish four alleles of the same length. Similarly, we were able to identify different D11S554 alleles of the same length by electrophoresis on a 5% polyacrylamide gel at 15ﾟC.We successfully distinguished four different D115488 alleles (262 bp) by 20-50% denaturing gradient gel electrophoresis (DGGE), and all of the different D115554 alleles mentioned above by 25-35% DGGE.However, some alleles of different lengths showed the same mobility.We applied the SSCP technique to two normal trio cases of disputed parentage, in which four STR loci including D115488 were tested for. Both trios had an apparently identical 262 bp D115488 allele on a denaturing gel. In one case, SSCP analysis increased the combined probability of paternity, ' because the child and the alleged father shared a rare allele. In the other case, the child shared the allele with the mother, but not with the alleged father, which excluded paternity. As a result, paternity was excluded by three of the four loci tested.Allowing simple and rapid detection of sequence polymorphisms, our method is suited for practical use.