摘要
目的建立盐析法快速从口腔拭子中提取基因组DNA的方法。方法首先以细胞裂解液和蛋白酶K消化口腔上皮细胞,然后用5 mol/L NaCl沉淀蛋白质,用异丙醇沉淀DNA,最后用70%乙醇洗涤得到的DNA并将其溶于TE(10mmol/L Tris-HCl,1 mmol/L EDTA,pH 8.0)中。用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术对TP53基因的rs1042522和CHRNA3基因的rs12910984位点进行分型。对不同基因型的样本测序验证。结果单支口腔拭子提取得到的DNA量在0.68～2.56μg之间,D260/D280比值在1.77～1.94之间。经PCR扩增和酶切消化,10个样本的两个单核苷酸多态性都得到了清楚分型。酶切结果与测序结果吻合。结论本实验所建立的盐析法可以快速、简便、经济地从口腔拭子得到高质量的基因组DNA。
Objective To establish a rapid salting out method for extraction of genomic DNA from buccal swabs.Methods Buccal epithelial cells were digested with cell lysate solution and proteinase K solution.Then the proteins were removed by salting out and centrifugation and DNA was precipitated with isopropyl alcohol.Finally,the precipitations of DNA were washed with 70% ethanol and were resuspended in TE.The rs1042522 loci of TP53 gene and rs12910984 loci of CHRNA3 gene were genotyped by polymerase chain reaction-restriction fragment length polymorphism（PCR-RFLP） technique.The samples with different genotypes were confirmed by direct sequencing analysis.Results The DNA yield of single buccal swab ranged from 0.68 to 2.56 μg;the D260/D280 value ranged from 1.77 to 1.94.After PCR amplification and enzyme digestion,two single nucleotide polymorphisms（SNPs） of 10 samples were clearly genotyped.The results of PCR-RFLP agreed well with the results of direct sequencing.Conclusion The present salting out method is rapid,simple,and economical for DNA extraction from buccal swabs.The obtained genomic DNA is of high quality.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2011年第12期1370-1374,共5页
Academic Journal of Second Military Medical University
基金
国家自然科学基金(30860319)
江西省科技支撑计划(2009BSB09502)~~
关键词
盐析
口腔拭子
DNA提取
单核苷酸多态性
salting out
buccal swabs
DNA extraction
single nucleotide polymorphisms
