Recovery of genomic DNA from residual frozen archival blood clots suitable for amplification and use in genotyping assays
Recovery of genomic DNA from residual frozen archival blood clots suitable for amplification and use in genotyping assays
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DOI：
10.1089/gte.2006.10.44
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发表时间：
2006-03-01
期刊：
影响因子：
--
通讯作者：
Shaw, GM
中科院分区：
文献类型：
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作者：
Iovannisci, DM;Ha, TT;Shaw, GM
对遗传学或法医学研究可能有用的DNA的一个非常被忽视的来源是从收集用于血清化学测量的血液样品中残留的凝块。我们研究了静脉穿刺后残留的血凝块在加州扩大母体血清甲胎蛋白筛查项目中的应用。我们报告了一个协议的基础上的盐析方法提取DNA的样品已被存档和冷冻长达2.5年。如通过PicoGreen（Molecular Probes，Inc.，尤金，OR）荧光测量。通过其在聚合酶链反应（PCR）扩增中作为模板的能力评价纯化DNA的质量，使用分布在整个人类基因组中的6个药物遗传学相关基因多态性区域侧翼的引物。成功扩增的基因区域的大小范围从215 bp到2064 bp，使用少至10 ng的模板DNA。由于许多基因分型方案通常建议设计100-200 bp范围内的扩增子和10-50 ng模板，因此我们得出结论，从采集用于血清化学测量的血液中去除血清后剩余的凝块可以作为基因分型研究的可靠DNA来源。
A greatly neglected source of DNA potentially useful for genetic or forensic studies is the clot remaining from blood samples collected for serum chemistry measurements. We have investigated the utility of residual clots remaining from venipunctures collected for California's Expanded Maternal Serum Alpha-Fetoprotein Screening Program. We report a protocol based on the salting out method for the extraction of DNA from samples which have been archived and frozen for up to 2.5 years. As much as 57 mu g of high-quality DNA can be obtained from a 2-ml clot as determined by PicoGreen (R) ( Molecular Probes, Inc., Eugene, OR) fluorescence measurements. Quality of the purified DNA was evaluated by its ability to serve as template in polymerase chain reaction (PCR) amplifications, using primers that flank the polymorphic regions of six genes of pharmacogenetic interest distributed throughout the human genome. Sizes of the gene regions successfully amplified range from 215 bp to 2064 bp, using as little as 10 ng of template DNA. Because many genotyping protocols routinely recommend the design of amplicons in the 100-200 bp range, and 10-50 ng of template, we conclude that the clot remaining after serum has been removed from blood collected for serum chemistry measurements can serve as a reliable source of DNA for genotyping studies.