碱裂解法快速提取口腔拭子DNA对CHRNA3基因多态性的研究

Rapid DNA extraction from buccal swabs by alkaline lysis method for study of CHRNA3 gene polymorphism

目的建立一种快速的从口腔拭子中提取DNA的方法,研究其在尼古丁乙酰胆碱受体α3(CHRNA3)基因多态性分析中的应用。方法以NaOH和乙二胺四乙酸(EDTA)配制碱裂解液,以三羟甲基氨基甲烷-EDTA(TE)为中和液,经加热裂解和中和两步提取口腔拭子DNA。以提取的DNA为模板,用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)分析对CHRNA3基因的rs6495309进行分型。对不同基因型的样本测序验证。结果 PCR扩增和酶切的靶带清晰,无非特异性条带。酶切结果与测序结果吻合。结论碱裂解法提取口腔拭子DNA具有快速、简便、经济、可靠的特点,可以用于CHRNA3基因多态性的分析。

Objective To establish a rapid method for DNA extraction from buccal swabs and investigate its application in analysis of cholinergic receptor nicotinic alpha3 （CHRNAd） gene polymorphism. Methods Lysis solution was made from NaOH and ethylenediaminetetraacetic acid（EDTA）, and tris （hydroxymethyl） aminomethane EDTA （TE） was served as neutralizing solution. DNA was extracted from buccal swabs via two steps：heating and neutralizing, rs6495309 in CHRNA3 gene was genotyped by polymerase chain reaction-restriction fragment length polymorphism（PCR-RFLP） assay using templates of extracted DNA. The samples of different genotypes were confirmed by direct sequencing analysis of PCR products. Results The target DNA bands obtained by PCR amplification and enzyme digestion were clear,and no nonspecifie band was detected. The results of PCR-RFLP agreed well with the results of direct sequencing. Conclusion The alkaline lysis method for preparation of DNA from buccal swabs is rapid, simple,economical, reliable,and can be used for analysis of CHRNA3 gene polymorphism.