比较全血DNA提取法适用单核苷酸多态性分析

Comparison of two methods for Genomic DNA extraction from whole blood and its application in SNP analysis

目的比较两种提取冰冻全血基因组DNA的方法,即分离白细胞法和裂解红细胞法所获得基因组DNA的质量。方法分别测定所获DNA的效率和纯度,同时,笔者用PCR/SNP敏感性分子开关技术,对两种方法获得的模板DNA进行单核苷酸多态性(single-nucleotidepolymorphism,SNP)分析。结果结果显示分离白细胞法DNA产量与纯度均高,裂解红细胞法获得的DNA产量、纯度相对较低;PCR/SNP敏感性分子开关检测表明:分离白细胞法和裂解红细胞法制备的DNA模板,均可在体外进行有效的PCR扩增,但分离白细胞法获得的目的DNA带特异性凸显,而裂解红细胞法获得的目的带特异性相对较差。结论预先分离纯化白细胞是从冰冻全血中获得较高纯度DNA和有效提高DNA产量的重要操作步骤。

[Objective] To compare the purities and yields of the DNA extracted from prior purified leukoeytes with those surviving from intentional RBC breakage of blood samples. [Methods] Frozen blood samples were used in this study. The DNA yields and the OD260/OD280 ratio were determined with UV spectrometer. DNA templates isolated by the two methods were analyzed using the sensitive on/off switch of PCR/SNP. [Results] Our results show that the yield and purity of DNAs extracted from frozen blood with prior purified leukoeytes are higher than those from prior erythroeyte lysis. The DNAs from the two methods were both effectively amplifiable, but more sharp bands were observed using DNA templates from the method of isolated leukoeytes. Some non-specific bands were seen at the PCR products when DNAs from the method of lysising, red cells were used as templates. [Conclusion] Prior purification of leukoeytes is a simple and useful step in DNA extraction method. In our experience, this step is more important for frozen blood sample.