一种简单、稳定、可靠的屏氧酶1基因多态性分析法

目的：建立一种简单、稳定、可靠的PON1基因多态性分析法。方法：取外周静脉血100μl，用ROSE法提取基因组DNA，用两对引物：P192F 5′-TAT TGT TGC TGT GGG ACC TGA G-3′,P192R 5′-CAC GCT AAA CCC AAA TAC ATC TC-3′；P55F 5′-GAA GAG TGA TGT ATA GCC CCA G-3′,P55R 5′-TTT AAT CCA GAG CTA ATG AAA GCC-3分别进行PCR扩增，用限制性内切酶AlwI,NlaⅢ对两种PCR产物分别进行酶切，3％琼脂糖凝胶电泳。结果：扩增的两个PCR产物在192、55多态性位点大小分别为99bp、170bp。Alw I酶切后，凝胶电脉得到完全酶切（66bp,33bp)，部分酶切（99bp、66bp、33bp)，未被酶切（99bp)三种类型DNA片段（RR，QR，QQ基因型）；NlaⅢ酶切后，凝胶电脉得到部分酶切（170bp,126bp,44bp)，未被酶切（170bp)两种类型DNA片段（LM，LL基因型）。经双盲重复检测，结果一致。应用此方法对胃癌患者血样标本检测，发现其PON1基因多态性在192位点频率较高。结论：采用一步PCR－RFLP技术可以建立简单、稳定、可靠的PON1基因多态性分析法。

A Simple,Stable,Reliable Method for Analysis of Paraoxonase 1 Gene Polymorphism

Objective: To setup a simple,stable and reliable method for analysis of PON1 gene polymorphism.Methods:Genomic DNA was extracted from peripheral vein blood by ROSE method. PCR amplification was done by two pairs primers: P192F 5'-TAT TGT TGC TGT GGG ACC TGA G-3',P192R 5'-CAC GCT AAA CCC AAA TAC ATC TC-3';P55F 5'-GAA GAG TGA TGT ATA GCC CCA G-3',P55R 5'-TTT AAT CCA GAG CTA ATG AAA GCC-3',respectively.PCR products were digested with restricted enzymes AlwI,NlaIII,respectively.To run electrophoresis on 3% agarose gel.Results:PCR products at position 192 and 55 were 99bp and 170bp, respectively.Three kinds of DNA fragment were seen when PCR products at position 192 digested with AlwI were run on 3% agarose gel, they were 66bp,33bp( whole digestion,RR genotype),99bp,66bp,33bp(part digestion,QR genotype)and 99bp( no digestion, QQ genotype);Only two kinds of DNA fragment in this study were seen when PCR products at position 55 digested with NlaIII were run on 3% agarose gel,they were 170bp,126bp,44bp( part digestion,LM genotype)and 170bp( no digestion,LL genotype).Moreover,results were confirmed by double-blind analysis.At the same time,to analysis by this method,we found that frequency at position 192 of PON1 gene polymorphism was higher in patients with gastric cancer.Conclusion:A simple,stable and reliable method for analysis of PON1 gene polymorphism was setup by one-step PCR-RFLP technique.