Detection of DNA sequence polymorphisms by enzymatic amplification and direct genomic sequencing.
Detection of DNA sequence polymorphisms by enzymatic amplification and direct genomic sequencing.
复制标题
DOI：
--
复制
发表时间：
1989-10
影响因子：
9.8
通讯作者：
D. Yandell;T. Dryja
中科院分区：
文献类型：
--
作者：
D. Yandell;T. Dryja
RFLPs的发现及其作为遗传标记的应用已经彻底改变了人类分子遗传学的研究。然而，人类基因组中只有一小部分DNA序列多态性会影响限制性片段的长度，从而导致RFLP。未被检测为RFLPs的多态性通常在筛选过程中被忽略，尽管它们代表了信息遗传标记的潜在重要来源。我们已经使用了一种快速的方法来检测自然发生的DNA序列变异，这是基于酶扩增和基因组DNA的直接测序。这种方法基本上可以检测到筛选区域内所有有用的序列变化。我们通过将其应用于人类视网膜母细胞瘤易感位点来证明该技术的可行性。我们从9个个体中筛选了3712 bp的基因组DNA，发现了4个DNA序列多态性。在三个遗传性视网膜母细胞瘤家族中，至少有一个DNA序列多态性是有信息的，而这些DNA序列多态性与该位点的任何已知RFLPs都没有信息。我们认为，直接测序是筛选DNA序列多态性的一种合理的替代方法，它代表了在过去信息最少的特征位点上获得信息标记的一步。
The discovery of RFLPs and their utilization as genetic markers has revolutionized research in human molecular genetics. However, only a fraction of the DNA sequence polymorphisms in the human genome affect the length of a restriction fragment and hence result in an RFLP. Polymorphisms that are not detected as RFLPs are typically passed over in the screening process though they represent a potentially important source of informative genetic markers. We have used a rapid method for the detection of naturally occurring DNA sequence variations that is based on enzymatic amplification and direct sequencing of genomic DNA. This approach can detect essentially all useful sequence variations within the region screened. We demonstrate the feasibility of the technique by applying it to the human retinoblastoma susceptibility locus. We screened 3,712 bp of genomic DNA from each of nine individuals and found four DNA sequence polymorphisms. At least one of these DNA sequence polymorphisms was informative in each of three families with hereditary retinoblastoma that were not informative with any of the known RFLPs at this locus. We believe that direct sequencing is a reasonable alternative to other methods of screening for DNA sequence polymorphisms and that it represents a step forward for obtaining informative markers at well-characterized loci that have been minimally informative in the past.