Oda S, Oki E, Maehara Y, Sugimachi K
Cancer Center, Kyushu University Hospital, Fukuoka 812-82, Japan.
Nucleic Acids Res. 1997 Sep 1;25(17):3415-20. doi: 10.1093/nar/25.17.3415.
The instability of microsatellite sequences dispersed in the genome has been linked to a deficiency in cellular mismatch repair. This phenotype has been frequently observed in various human neoplasms and is regarded as a major factor in tumorigenesis. To demonstrate alterations in microsatellite sequences, polymerase chain reaction (PCR) and electrophoretic analysis are techniques often used. However, the electrophoretic profiles of PCR-amplified microsatellite sequences have not been well characterized. Moreover, the conventional method using autoradiography has critical problems in detection characteristics and migration accuracy. We made use of fluorescence-labeled PCR and laser scanning with linear detection characteristics, so as to detect bands quantitatively. Next, we characterized Taq polymerase-dependent modification of the amplified microsatellite sequences, using artificially synthesized microsatellite alleles and we optimized the electrophoretic profiles by enzymatic modification with T4 DNA polymerase. We developed a dual fluorescence co-electrophoresis system, in which both samples derived from cancer and normal tissues are electrophoresed in the same lane, in order to minimize migration errors. These improvements remarkably facilitate precise and objective assessments of microsatellite instability. Analyzing many positive cases in cell lines and tissue specimens, we classified all the patterns of microsatellite alteration and set up new criteria for assessing microsatellite instability.
分散在基因组中的微卫星序列的不稳定性与细胞错配修复缺陷有关。这种表型在各种人类肿瘤中经常观察到，并被视为肿瘤发生的主要因素。为了证明微卫星序列的改变，聚合酶链反应（PCR）和电泳分析是常用的技术。然而，PCR扩增的微卫星序列的电泳图谱尚未得到很好的表征。此外，使用放射自显影的传统方法在检测特性和迁移准确性方面存在关键问题。我们利用荧光标记的PCR和具有线性检测特性的激光扫描来定量检测条带。接下来，我们使用人工合成的微卫星等位基因来表征扩增的微卫星序列的Taq聚合酶依赖性修饰，并通过用T4 DNA聚合酶进行酶促修饰来优化电泳图谱。我们开发了一种双荧光共电泳系统，其中来自癌症和正常组织的样本都在同一泳道中进行电泳，以尽量减少迁移误差。这些改进显著促进了对微卫星不稳定性的精确和客观评估。通过分析细胞系和组织标本中的许多阳性病例，我们对微卫星改变的所有模式进行了分类，并建立了评估微卫星不稳定性的新标准。
