Yamamoto Miyako, Ahn Ray Hyungjoo, Yamamoto Fumiichiro
Cancer Genetics and Epigenetics Program, Burnham Institute for Medical Research, La Jolla, CA 92037, USA.
Electrophoresis. 2006 Jul;27(13):2529-40. doi: 10.1002/elps.200500875.
We developed the systematic multiplex reverse transcription-PCR (SM RT-PCR) method that is distinguishable from other multiplex RT-PCR methods by optimized PCR conditions allowing amplification of sequences that fall within a single exon of genes of similar band intensity using genomic DNA template as a calibration standard. Using an SM RT-PCR system of proto-oncogenes and tumor suppressor genes, we previously showed that the SM RT-PCR system, which was developed for cDNA expression analysis, could also be used for a more exquisite analysis of copy number changes in genomic DNA. Here we report that the SM PCR method semiquantitatively detected less than a two-fold difference in copy number. Furthermore, we also report the results of subchromosomal scanning of copy number and expression using the SM PCR and SM RT-PCR methods. We identified and characterized the novel homozygous deletion that spans over 12-plus genes on 16p13.3-13.2 in the MDA-MB-468 breast cancer cell line.
我们开发了一种系统多重逆转录聚合酶链反应（SM RT-PCR）方法，该方法与其他多重RT-PCR方法不同，它通过优化的PCR条件，以基因组DNA模板作为校准标准，能够扩增位于相似条带强度基因的单个外显子内的序列。使用原癌基因和肿瘤抑制基因的SM RT-PCR系统，我们之前表明，为cDNA表达分析而开发的SM RT-PCR系统也可用于对基因组DNA中拷贝数变化进行更精确的分析。在此我们报告，SM PCR方法能够半定量检测出拷贝数小于两倍的差异。此外，我们还报告了使用SM PCR和SM RT-PCR方法进行的亚染色体拷贝数和表达扫描结果。我们在MDA-MB-468乳腺癌细胞系中鉴定并表征了一个跨越16p13.3 - 13.2上12个以上基因的新型纯合缺失。
