Cheung V G, Nelson S F
Department of Pediatrics, University of California, Los Angeles 90095, USA.
Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14676-9. doi: 10.1073/pnas.93.25.14676.
Genetic analysis of limiting quantities of genomic DNA play an important role in DNA forensics, paleoarcheology, genetic disease diagnosis, genetic linkage analysis, and genetic diversity studies. We have tested the ability of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) to amplify picogram quantities of human genomic DNA for the purpose of increasing the amount of template for genotyping with microsatellite repeat markers. DNA was uniformly amplified at a large number of typable loci throughout the human genome with starting template DNAs from as little as 15 pg to as much as 400 ng. A much greater-fold enrichment was seen for the smaller genomic DOP-PCRs. All markers tested were amplified from starting genomic DNAs in the range of 0.6-40 ng with amplifications of 200- to 600-fold. The DOP-PCR-amplified genomic DNA was an excellent and reliable template for genotyping with microsatellites, which give distinct bands with no increase in stutter artifact on di-, tri-, and tetranucleotide repeats. There appears to be equal amplification of genomic DNA from 55 of 55 tested discrete microsatellites implying near complete coverage of the human genome. Thus, DOP-PCR appears to allow unbiased, hundreds-fold whole genome amplification of human genomic DNA for genotypic analysis.
对极少量基因组DNA进行遗传分析在DNA法医鉴定、古考古学、遗传疾病诊断、遗传连锁分析和遗传多样性研究中发挥着重要作用。我们测试了简并寡核苷酸引物聚合酶链反应（DOP-PCR）扩增皮克级人类基因组DNA的能力，目的是增加用于微卫星重复标记基因分型的模板量。使用低至15 pg至高达400 ng的起始模板DNA，在整个人类基因组的大量可分型位点上对DNA进行了均匀扩增。对于较小的基因组DOP-PCR，观察到了更大倍数的富集。所有测试的标记物均从0.6 - 40 ng范围内的起始基因组DNA中扩增出来，扩增倍数为200至600倍。DOP-PCR扩增的基因组DNA是用于微卫星基因分型的优良且可靠的模板，在二核苷酸、三核苷酸和四核苷酸重复序列上产生清晰的条带，且口吃伪影没有增加。在测试的55个离散微卫星中的55个中，基因组DNA似乎得到了同等程度的扩增，这意味着几乎完全覆盖了人类基因组。因此，DOP-PCR似乎能够对人类基因组DNA进行无偏差的、数百倍的全基因组扩增以进行基因分型分析。
