Hahn M, Matzen S E, Serth J, Pingoud A
Justus-Liebig-Universität, Giessen, Germany.
Biotechniques. 1995 Jun;18(6):1040-7.
The most frequently altered gene in diverse tumor types is the tumor suppressor gene p53. Typically, normal function is inactivated by point mutation of one allele and deletion of the other. Therefore, loss of heterozygosity (LOH) of intragenic polymorphic markers is a strong indication for p53 involvement in a cancerous lesion. This study shows that a highly polymorphic short tandem repeat (STR) within intron 1 of p53 is an excellent marker for quantitative evaluation of LOH in tumor samples, whose multicolor, fluorescently tagged PCR products are analyzed and quantitated on an automated DNA sequencer. The range of error was analyzed in detail. Discrete allelic profiles were obtained following amplification of DNA from microdissected cell samples of patients with urogenital tumors. By calculating qLOH, the relative allele ratio of a tumor compared with healthy tissue, a quantitative expression for the LOH is obtained. PCR-based tumor DNA typing using fluorescent STR primers and automated analysis provides an enhanced level of accuracy and sensitivity required for routine analysis in clinical practice, where large numbers of tumor samples have to be processed.
在多种肿瘤类型中，最常发生改变的基因是肿瘤抑制基因p53。通常，一个等位基因的点突变和另一个等位基因的缺失会使正常功能失活。因此，基因内多态性标记的杂合性缺失（LOH）是p53参与癌性病变的有力指征。本研究表明，p53基因内含子1内的一个高度多态性短串联重复序列（STR）是肿瘤样本中LOH定量评估的优良标记，其多色、荧光标记的PCR产物在自动DNA测序仪上进行分析和定量。详细分析了误差范围。对泌尿生殖系统肿瘤患者显微切割细胞样本的DNA进行扩增后，获得了离散的等位基因图谱。通过计算qLOH（肿瘤与健康组织的相对等位基因比率），可得到LOH的定量表达。使用荧光STR引物和自动分析的基于PCR的肿瘤DNA分型，为临床实践中的常规分析提供了更高水平的准确性和灵敏度，因为临床实践中需要处理大量肿瘤样本。
