Slebos Robbert J C, Umbach David M, Sommer Courtney A, Horner Geoffrey A, Choi Jane Y, Taylor Jack A
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Lab Invest. 2004 May;84(5):649-57. doi: 10.1038/labinvest.3700076.
Microsatellite analysis is a powerful tool for the assessment of genetic instability and loss of heterozygosity in cancer cells. However, most human tumors harbor significant numbers of normal cells, which may contribute to false-negative results. Recent techniques based on fluorescently labeled primers and semiautomated capillary electrophoresis of polymerase chain reaction (PCR) products allow a reliable quantitative assessment of (PCR) products while requiring very small numbers of cells. We report a highly sensitive protocol for the semiautomated analysis of allelic imbalance based on time-release PCR and capillary electrophoresis. With this protocol, as few as 100 cells can be used to reliably assess allelic imbalance (AI) in DNA samples. Using a panel of seven microsatellite markers, we determined allelic variation in a large set of heterozygous lymphocyte DNA samples and examined the use of different statistical analysis techniques. Using these statistical approaches, we describe a calibration method to evaluate AI from microsatellite results. Using a simple formula, cutoff points at preset confidence levels are used to decide whether allelic imbalance exists in a given sample at the loci under investigation. Our method allows the reliable detection of AI with very small amounts of DNA, and is sufficiently quantitative to assess allelic ratios in nonclonal tissue specimens.
微卫星分析是评估癌细胞遗传不稳定性和杂合性缺失的有力工具。然而，大多数人类肿瘤含有大量正常细胞，这可能导致假阴性结果。基于荧光标记引物和聚合酶链反应（PCR）产物的半自动毛细管电泳的最新技术，在只需极少量细胞的情况下就能对PCR产物进行可靠的定量评估。我们报告了一种基于定时释放PCR和毛细管电泳的等位基因不平衡半自动分析的高灵敏度方案。采用该方案，仅需100个细胞就能可靠评估DNA样本中的等位基因不平衡（AI）。我们使用一组7个微卫星标记，确定了大量杂合淋巴细胞DNA样本中的等位基因变异，并研究了不同统计分析技术的使用情况。通过这些统计方法，我们描述了一种从微卫星结果评估AI的校准方法。使用一个简单公式，在预设置信水平下的截断点用于判断在给定样本中所研究位点是否存在等位基因不平衡。我们的方法能够使用极少量DNA可靠检测AI，并且具有足够的定量性来评估非克隆组织标本中的等位基因比率。
