Evaluation of a whole-genome amplification method based on adaptor-ligation PCR of randomly sheared genomic DNA
Evaluation of a whole-genome amplification method based on adaptor-ligation PCR of randomly sheared genomic DNA
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DOI：
10.1002/gcc.10269
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发表时间：
2003-10-01
影响因子：
3.7
通讯作者：
Sasaki, H
中科院分区：
文献类型：
--
作者：
Tanabe, C;Aoyagi, K;Sasaki, H
高通量的遗传研究通常需要大量的DNA来进行各种分析。因此，开发和评估全基因组扩增方法是重要的，特别是在目前希望在以前收集的样本中进行大规模基因分型的情况下，可获得的DNA有限。我们开发的方法，称为PRSG，是基于接头连接介导的随机剪切基因组DNA的聚合酶链式反应。通过对随机分布在整个基因组中的367个基因的2,607个外显子进行聚合酶链式反应，对数百个个体的PRSG产品进行了无偏见表示的评估。在PRSG产物上发现罕见的外显子序列丢失(1%)。在不同染色体上的307个微卫星中，258个(84%)在PRSG产物和原始DNA中都被扩增，而49个微卫星(16%)仅在PRSG产物中丢失。对287个基因座的相对基因拷贝数的阵列CGH分析表明，该方法具有较低的偏倚。此外，该方法在100-1000个石蜡包埋组织的激光捕获细胞上得到了验证。这些数据表明，PRSG可以为各种遗传分析提供足够的基因组序列，也可以为未来的工作提供长期存储。(C)2003年Wiley-Liss，Inc.
High-throughput genetic studies often require large quantities of DNA for a variety of analyses. Developing and assessing a whole-genome amplification method is thus important, especially with the current desire for large-scale genotyping in previously collected samples for which limited DNA is available. The method we have developed, called PRSG, is based on an adaptor-ligation-mediated PCR of randomly sheared genomic DNA. An unbiased representation was evaluated by performing PCR on 2,607 exons of 367 genes, which are randomly distributed throughout the genome, on PRSG products of hundreds of individuals. An infrequent loss (< 1%) of the exon sequence on the PRSG products was found. Out of 307 microsatellites on various chromosomes, 258 (84%) were amplified in both the PRSG product and an original DNA, whereas 49 (16%) microsatellites were lost only in the PRSG product. Array CGH analysis of 287 loci for measuring the relative gene copy number demonstrated that a low bias was detected. Moreover, this method was validated on 100-1,000 laser-captured cells from paraffin-embedded tissues. These data show that PRSG can provide a sufficient amount of genomic sequence for a variety of genetic analyses as well as for long-term storage for future work. (C) 2003 Wiley-Liss, Inc.