磁珠法半自动提取全血基因组DNA条件的优化  

Optimization of genomic DNA extraction with magnetic bead-based semi-automatic system

作　　者：凌洁[1] 王昊[2] 张帅[2] 张丹丹[2] 来茂德[2] 朱益民[1] 

机构地区：[1]浙江大学医学院流行病与卫生统计学系,浙江杭州310058 [2]浙江大学医学院病理学与病理生理学系,浙江杭州310058

出　　处：《浙江大学学报（医学版）》2012年第3期320-326,共7页Journal of Zhejiang University(Medical Sciences)

基　　金：国家科技支撑项目(2009BAI80B02);中央高校基本科研业务费专项资金(2010QNA7022)

摘　　要：目的:探讨磁珠法提取基因组DNA的影响因素,建立快速、高效、批量提取全血基因组DNA的优化方案。方法:使用磁珠法核酸自动提取系统从全血中提取基因组DNA,紫外分光光度计测定DNA浓度和纯度。采用正交试验设计分析裂解时间、全血量、磁珠量和乙醇浓度4个因素的主效应和全血量与裂解时间、磁珠量,裂解时间与磁珠量的二阶交互作用对所提取DNA浓度和纯度的影响。结果:裂解时间、全血量、磁珠量和乙醇浓度4个主效应对DNA浓度的影响均存在统计学差异(P<0.05),当裂解时间为15 min,全血量为100μl,磁珠量为80μl,乙醇浓度为80%时,DNA平均浓度最高。磁珠量、裂解时间和全血量的交互作用对DNA OD260/OD280的比值有影响(P=0.008和P=0.013)。当磁珠量为40μl,裂解时间为15 min,全血量为100μl时,OD260/OD280的比值较好。磁珠量和乙醇浓度影响DNA OD260/OD230的比值(P=0.017和P<0.05),磁珠量为40μl,乙醇浓度为80%时,OD260/OD230的比值更好。结论:当DNA得率优先考虑时,可以选择裂解时间15 min、全血量100μl、磁珠量80μl、乙醇浓度80%的提取条件;而对DNA纯度要求较高时,可以将磁珠量改为40μl,以获得最优DNA提取效果。Objective： To develop a rapid and effective method for genomic DNA extraction with magnetic bead-based semi-automatic system. Methods： DNA was extracted from whole blood samples semi-automatically with nucleic acid automatic extraction system. The concentration and purity of samples was determined by UV-spectrophotometer. Orthogonal design was used to analyze the main effect of lysis time, blood volume,magnetic bead quantity and ethanol concentration on the DNA yield; also the 2-way interaction of these factors. Results： Lysis time, blood volume, magnetic bead quantity and ethanol concentration were associated with DNA yield （ P 〈 0.05 ）, but no interaction existed. DNA yield was higher under the condition with 15 rain of lysis time, 100 ~xl of blood volume ,80 Ixl of magnetic beads and 80% of ethanol. A significant association was found between the magnetic bead quantity and DNA purity OD260/OD280 （P = 0. 008）. Interaction of blood volume and lysis time also existed （P = 0. 013）. DNA purity was better when the extracting condition was 40 ~1 of magnetic beads, 15 rain of lysis time and 100μl of blood volume. Magnetic beads and ethanol concentration were associated with DNA purity OD260/OD230（P =0.017 and P 〈 0.05）, the result was better when magnetic beads was 40 μl and ethanol concentration was 80%. Conclusion： The results indicate that the optimized conditions with 40 μl magnetic beads will generate higher quality of genomic DNA from the whole blood samples

关 键 词：自动化 磁石 DNA/分离和提纯 血液 基因组文库 正交试验 乙醇 细胞 培养的 

分 类 号：R440[医药卫生—诊断学]