磁珠法半自动提取全血基因组DNA条件的优化

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。

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

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 min of lysis time,100 μl of blood volume,80 μl 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 μl of magnetic beads,15 min of lysis time and 100 μl 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.