Analysis of minisatellite mutation for performing a highly reliable paternity test without false exclusion

利用聚合酶链式反应(PCR)构建了小卫星变异重复序列(MVR)图谱，用于定位微小变异重复序列沿小卫星串联阵列的分布模式。MVR-PCR在几个座位上发现了巨大的等位基因结构多样性，远远超过了等位基因长度分析所能解决的问题。我们发现，MVR-PCR的应用导致了比其他12个经典DNA标记更高的父子关系概率。然而，MS32和MS31A等高变小卫星会显示出显著的胚系突变率，导致新的长度等位基因发生突变，这可能会在亲子鉴定案例中产生虚假排除，尽管当检查几个MVR基因座时，显示父亲等位基因在一个以上突变的亲子鉴定案例将非常罕见。对突变过程的详细了解，结合等位基因结构的MVR分析，可以帮助区分突变和非亲子关系。我们使用真实数据和模拟数据…展示了突变等位基因与其祖先有多么相似并演示了如何使用MVR-PCR来识别突变的父亲等位基因，在亲子鉴定案例中表现出明显的排斥性。我们分析了日本人群中的小卫星B6.7基因座。等位基因大小分布显示，与北欧人相比，日本人保留了广泛的等位基因长度变异性，但等位基因明显较小。对92个日本等位基因进行了MVR-PCR分析。这些等位基因表现出多种多样的内部MVR结构，大多数等位基因在样本中只观察到一次。真正的杂合度估计为99.95%，在日本人群中存在2000多个不同的等位基因。点阵分析表明，在日本人和北欧人中可以识别出具有相同结构基序的相关等位基因组，并且这些组是群体特有的，没有任何日本和北欧等位基因之间显著相似的例子。因此，小卫星B6.7在日本和北欧人群中表现出巨大的等位基因变异性和快速的重复更新，为探索人类种群历史上最近的事件提供了新的谱系标记。较少

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) was devised to map the interspersion pattern of subtle variant repeats along minisatellite tandem arrays. MVR-PCR has revealed enormous diversity of allele structures at, several loci, far more than can be resolved by allele length analysis. We showed that the application of MVR-PCR resulted in higher paternity probabilities than for a set of 12 other classic DNA markers. Hypervariable minisatellites like MS32 and MS31A can however show significant germline mutation rates to new length alleles which can generate false exclusions in paternity cases although paternity cases showing mutant paternal alleles at more than one locus will be rare when several MVR loci are examined. Detailed knowledge of mutation processes coupled with MVR analysis of allele structure can help distinguish mutation from non-paternity. We show how similar mutant alleles are to their progenitors using both real and simulated data, … More and demonstrate how MVR-PCR can be used to identify mutant paternal allele in paternity cases showing apparent exclusions.We analyse minisatellite B6.7 locus in the Japanese population. Allele size distributions showed that Japanese retain extensive allele length variability but have significantly smaller alleles compared to north Europeans. Ninety-two Japanese alleles were further characterised by MVR-PCR. These alleles showed a wide variety of internal MVR structures with most alleles observed only once in the sample. The true heterozygosity is estimated at 99.95%, with well in excess of 2000 different alleles existing in the Japanese population. Dot matrix analysis showed that groups of related alleles sharing structural motifs could be identified within Japanese and in north Europeans, and that these groups are population-specific with no examples of significant similarity between any Japanese and north European alleles. Minisatellite B6.7 therefore shows huge allele variability and fast repeat turnover in Japanese as well as north European populations, and provides novel lineage markers for exploring very recent events in human population history. Less