Whole genome amplification of DNA from laser capture-microdissected tissue for high-throughput single nucleotide polymorphism and short tandem repeat genotyping
Whole genome amplification of DNA from laser capture-microdissected tissue for high-throughput single nucleotide polymorphism and short tandem repeat genotyping
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DOI：
10.1016/s0002-9440(10)63092-1
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发表时间：
2004-01-01
影响因子：
6
通讯作者：
Wolfe, JL
中科院分区：
文献类型：
--
作者：
Rook, MS;Delach, SM;Wolfe, JL
全基因组筛选正常细胞和癌细胞之间以及肿瘤亚群之间的遗传改变，对于建立癌症的分子机制和分类具有重要意义。通过分析单核苷酸多态性和/或短串联重复序列标记的等位基因缺失，可以在癌细胞中广泛观察到杂合性缺失导致的基因沉默。为了利用癌细胞激光捕获显微解剖（LCM）所能获得的微量DNA，一种维持位点和等位基因平衡的全基因组扩增方法是必不可少的。我们已经成功地使用了基于phi29聚合酶的等温全基因组扩增方法，使用蛋白酶K裂解程序和池化策略来扩增LCM DNA。通过单核苷酸多态性和短串联重复基因型分析，我们证明使用来自两个或三个单独扩增反应的混合DNA可以显著减少扩增过程中引入的任何等位基因偏倚。这种策略在使用少量源DNA时特别有效。虽然在1500 - 3000个LCM细胞中，方便的碱解DNA提取方法提供了令人满意的结果，但在细胞数量较少的情况下，蛋白酶K消化效果更好。准确的基因分型是实现与少至100个细胞时，既蛋白酶K提取和池应用。
Genome-wide screening of genetic alterations between normal and cancer cells, as well as among subgroups of tumors, is important for establishing molecular mechanism and classification of cancer. Gene silencing through loss of heterozygosity is widely observed in cancer cells and detectable by analyzing allelic loss of single nucleotide polymorphism and/or short tandem repeat markers. To use minute quantities of DNA that are available through laser capture microdissection (LCM) of cancer cells, a whole genome amplification method that maintains locus and allele balance is essential. We have successfully used a phi29 polymerase-based isothermal whole genome amplification method to amplify LCM DNA using a proteinase K lysis procedure coupled with a pooling strategy. Through single nucleotide polymorphism and short tandem repeat genotype analysis we demonstrate that using pooled DNA from two or three separate amplification reactions significantly reduces any allele bias introduced during amplification. This strategy is especially effective when using small quantities of source DNA. Although a convenient alkaline lysis DNA extraction procedure provided satisfactory results from using 1500 to 3000 LCM cells, proteinase K digestion was superior for lower cell numbers. Accurate genotyping is achieved with as few as 100 cells when both proteinase K extraction and pooling are applied.