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目的探讨多重置换扩增(MDA)与激光捕获显微切割(LCM)技术相结合,并应用到胃癌细胞杂合性丢失(LOH)分析等研究的可行性。方法利用LCM技术从胃癌组织冰冻切片中分别获取正常胃黏膜细胞和胃癌细胞,提取基因组DNA后,进行全基因组多重置换扩增。进而,将MDA产物用于聚合酶链反应(PCR)扩增ACE2、TP53和ACTB基因片段,以及微卫星位点的LOH分析。结果位于不同染色体的3个基因片段均得到较好的扩增,而且MDA产物的扩增效率明显高于未经MDA的基因组DNA。另外,MDA产物的LOH分析结果与未经MDA的基因组DNA结果一致。结论MDA与LCM技术相结合,是一种可行的全基因组扩增技术路线,可用于后续的基因组学研究。
Objective To investigate the feasibility of combining multiple displacement amplification (MDA) with laser capture microdissection (LCM) and to investigate the loss of heterozygosity (LOH) in gastric cancer cells. Methods The normal gastric mucosal cells and gastric cancer cells were obtained from frozen sections of gastric cancer tissue by LCM technique. Genomic DNA was extracted and amplified by whole genome multiple replacement. Furthermore, MDA products were used for polymerase chain reaction (PCR) amplification of ACE2, TP53 and ACTB gene fragments, and LOH analysis of microsatellite loci. Results All three genes located on different chromosomes were amplified well, and the amplification efficiency of MDA product was significantly higher than that of MDA-free genomic DNA. In addition, the results of LOH analysis of MDA products are consistent with the results of genomic DNA without MDA. Conclusion The combination of MDA and LCM technology is a viable approach to genome-wide amplification and can be used in subsequent genomics research.