Hampton G M, Larson A A, Baergen R N, Sommers R L, Kern S, Cavenee W K
Ludwig Institute for Cancer Research, Department of Pathology, University of California at San Diego 92093-0660, USA.
Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6704-9. doi: 10.1073/pnas.93.13.6704.
Detection of loss of heterozygosity (LOH) by comparison of normal and tumor genotypes using PCR-based microsatellite loci provides considerable advantages over traditional Southern blotting-based approaches. However, current methodologies are limited by several factors, including the numbers of loci that can be evaluated for LOH in a single experiment, the discrimination of true alleles versus "stutter bands," and the use of radionucleotides in detecting PCR products. Here we describe methods for high throughput simultaneous assessment of LOH at multiple loci in human tumors; these methods rely on the detection of amplified microsatellite loci by fluorescence-based DNA sequencing technology. Data generated by this approach are processed by several computer software programs that enable the automated linear quantitation and calculation of allelic ratios, allowing rapid ascertainment of LOH. As a test of this approach, genotypes at a series of loci on chromosome 4 were determined for 58 carcinomas of the uterine cervix. The results underscore the efficacy, sensitivity, and remarkable reproducibility of this approach to LOH detection and provide subchromosomal localization of two regions of chromosome 4 commonly altered in cervical tumors.
通过使用基于聚合酶链反应（PCR）的微卫星位点比较正常和肿瘤基因型来检测杂合性缺失（LOH），相对于传统的基于Southern印迹的方法具有相当大的优势。然而，目前的方法受到几个因素的限制，包括在单个实验中可用于评估LOH的位点数量、区分真实等位基因与“拖带”以及在检测PCR产物时使用放射性核苷酸。在这里，我们描述了在人类肿瘤中多个位点同时高通量评估LOH的方法；这些方法依赖于通过基于荧光的DNA测序技术检测扩增的微卫星位点。通过这种方法产生的数据由几个计算机软件程序处理，这些程序能够自动进行线性定量和等位基因比率的计算，从而快速确定LOH。作为对这种方法的测试，对58例子宫颈癌的4号染色体上一系列位点的基因型进行了测定。结果强调了这种LOH检测方法的有效性、敏感性和显著的可重复性，并提供了4号染色体上两个在宫颈肿瘤中常见改变区域的亚染色体定位。
