Comparison of eleven methods for genomic DNA extraction suitable for large-scale whole-genome genotyping and long-term DNA banking using blood samples.
Comparison of eleven methods for genomic DNA extraction suitable for large-scale whole-genome genotyping and long-term DNA banking using blood samples.
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比较使用血液样本的11种基因组DNA提取方法的提取方法。
DOI：
10.1371/journal.pone.0115960
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Banos G
中科院分区：
文献类型：
--
作者：
Psifidi A;Dovas CI;Bramis G;Lazou T;Russel CL;Arsenos G;Banos G
近年来，新一代测序和微阵列技术应用于生物系统的高通量分析，给科学研究带来了革命性的变化。分离大量纯、完整、双链、高浓度、未受污染的基因组DNA是成功和可靠的大规模基因分型分析的先决条件。创建DNA库也需要大量的纯DNA。在本研究中，研究了11种不同的DNA提取方法，包括苯酚-氯仿、二氧化硅和磁珠提取，以确定它们从绵羊血液样本中提取DNA的相对有效性。随后通过分光光度法测量、量子比特测量、实时PCR扩增和凝胶电泳评估差异提取DNA的质量和数量。并对每种方法的加工时间、劳动强度和成本进行了评价。结果显示，11种方法之间存在显著差异，只有4种方法产生满意的结果。这四种方法，包括三种改性二氧化硅商业试剂盒（改性血液，改性组织，改性Dx试剂盒）和内部开发的基于磁珠的方案，最适合提取高质量和高数量的DNA，适合大规模微阵列基因分型，也适合长期DNA存储，成功应用于600个人。
Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals.
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来自1,092个人基因组的遗传变异的综合图。
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
9.4
作者：
Fahle, GA;Fischer, SH
通讯作者：
Fischer, SH
影响因子：
2.4
作者：
Clements, Dylan N.;Wood, Shona;Ollier, William E. R.
通讯作者：
Ollier, William E. R.
影响因子：
5.6
作者：
Arif IA;Bakir MA;Khan HA;Ahamed A;Al Farhan AH;Al Homaidan AA;Al Sadoon M;Bahkali AH;Shobrak M
通讯作者：
Shobrak M
影响因子：
6.4
作者：
Jaluria P;Konstantopoulos K;Betenbaugh M;Shiloach J
通讯作者：
Shiloach J