Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA
Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA
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DOI：
10.1073/pnas.93.25.14676
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发表时间：
1996-12-10
影响因子：
11.1
通讯作者：
Nelson, SF
中科院分区：
文献类型：
--
作者：
Cheung, VG;Nelson, SF
有限量基因组DNA的遗传分析在DNA法医学、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起着重要作用。为了提高微卫星重复序列标记基因分型的模板量，我们测试了简并寡核苷酸引物聚合酶链反应（DOP-PCR）扩增皮克量人类基因组DNA的能力，DNA在整个人类基因组中的大量模板基因座处均匀扩增，起始模板DNA从少至15 pg到多至400 ng。所有测试的标记都是从0.6-40 ng范围内的起始基因组DNA扩增的，扩增倍数为200- 600倍。DOP-PCR扩增的基因组DNA是用于微卫星基因分型的优异且可靠的模板，其给出了不同的条带，而在二-、三-、和四核苷酸重复。从55个测试的离散微卫星中的55个似乎有相等的基因组DNA扩增，这意味着几乎完全覆盖人类基因组，因此，DOP-PCR似乎允许无偏的。用于基因型分析人基因组DNA的数百倍全基因组扩增。
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 template 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.