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Analytical and statistical methods to evaluate microsatellite allelic imbalance in small amounts of DNA

机译：评估少量DNA中微卫星等位基因失衡的分析和统计方法

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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.
机译：微卫星分析是评估癌细胞中遗传不稳定性和杂合性丧失的强大工具。但是，大多数人类肿瘤都带有大量正常细胞，这可能会导致假阴性结果。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可以对（PCR）产物进行可靠的定量评估，而所需细胞数量却很少。我们报告了高度敏感的协议，用于基于时间释放PCR和毛细管电泳的等位基因不平衡半自动化分析。使用此协议，可以使用最少100个细胞来可靠地评估DNA样品中的等位基因失衡（AI）。使用一组七个微卫星标记，我们确定了一大批杂合淋巴细胞DNA样品中的等位基因变异，并检查了不同统计分析技术的使用。使用这些统计方法，我们描述了一种从微卫星结果评估AI的校准方法。使用一个简单的公式，以预设的置信度水平下的分界点来确定在研究的基因座的给定样本中是否存在等位基因失衡。我们的方法可以用非常少量的DNA可靠地检测AI，并且足够定量以评估非克隆组织标本中的等位基因比率。

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