模板DNA质量对单核苷酸多态性分析的影响

Influence of the quality of template DNA on the single nucleotide polymorphism (SNP) analysis

目的探索在单核苷酸多态性分析中模板DNA质量对实验的影响以及提高模板DNA质量的途径。方法用PCR-RFLP法研究SNP rs1024611(G/A)的基因分型,从样本保存方式、保存时间及DNA提取方法三方面进行对比实验,观察其对模板DNA质量及SNP分析的影响。结果从抗凝血中提取DNA模板的得率比非抗凝血高,在SNP分析中,EDTA抗凝血效果最好、肝素抗凝血效果不好;基因组DNA在全血中的有效保存时间比提纯的DNA有效时间长;试剂盒法提取DNA的得率和纯度均高于传统的酚-氯仿法。结论可通过以下途径提高SNP研究中DNA模板的质量:采集血样用EDTA抗凝、分装成小份在-20℃冻存,避免反复冻融。使用前在37℃水浴中快速解冻,用试剂盒法提取DNA更适合于SNP的研究,提取的模板DNA要及时实验或分装后冻存于-20℃,若需长期保存模板DNA,保存全血优于保存纯化的DNA。

Objective To optimize the conservation method and conservation time of blood sample and explore the way of improving the quality of the template DNA in single nucleotide polymorphism analysis. Methods Using PCR - RLFP to study the polymorphism of SNP rs1024611 （G/A）, to investigate the effects of decoagulant, DNA extraction method , and sample conservation time on the quality of template DNA and on the results of SNPs analysis. Results The use of decoagulant simplified the DNA extraction procedure and resulted in a high yield, and EDTA was best decoagulant for gene polymorphism analysis. Although heparin has a high yield as anticoagulation, it has a poor effect in the PCR - RFLP study. Genome DNA in freezed whole blood was more stable than purified DNA. Use of kit method to extract DNA could reach a better yield and purity than that of phenol - chloroform method. Conclusion the ways of improving the quality of template DNA in SNPs analysis are as follows ： anticoagulation with EDTA in blood sample; stored in subpackage at - 20℃; avoidance of repeated freeze thawing. Rapid defrost of the blood sample in 37 ℃ water bath and use of the kit method to extract DNA. After extraction, the template DNA should be utilized immediately or stored in subpackage at -20 ℃. It is better to conserve the template DNA in whole blood rather than to conserve the purified form.