Tanabe Chikako, Aoyagi Kazuhiko, Sakiyama Tokuki, Kohno Takashi, Yanagitani Noriko, Akimoto Shingo, Sakamoto Michiie, Sakamoto Hiromi, Yokota Jun, Ohki Misao, Terada Masaaki, Yoshida Teruhiko, Sasaki Hiroki
Genetics Division, National Cancer Center Research Institute, Tokyo, Japan.
Genes Chromosomes Cancer. 2003 Oct;38(2):168-76. doi: 10.1002/gcc.10269.
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.
高通量基因研究通常需要大量DNA用于各种分析。因此，开发和评估全基因组扩增方法很重要，特别是鉴于当前希望对先前收集的样本进行大规模基因分型，而这些样本中可用的DNA有限。我们开发的方法称为PRSG，它基于随机剪切的基因组DNA的衔接子连接介导的PCR。通过对数百个个体的PRSG产物上随机分布在整个基因组中的367个基因的2607个外显子进行PCR，评估了无偏代表性。在PRSG产物上发现外显子序列的罕见丢失（<1%）。在各种染色体上的307个微卫星中，258个（84%）在PRSG产物和原始DNA中均被扩增，而49个（16%）微卫星仅在PRSG产物中丢失。对287个用于测量相对基因拷贝数的位点进行的阵列比较基因组杂交分析表明检测到低偏差。此外，该方法在来自石蜡包埋组织的100 - 1000个激光捕获细胞上得到了验证。这些数据表明，PRSG可以为各种基因分析以及长期储存以供未来研究提供足够量的基因组序列。
