DHPLC和PCR-SSCP两种方法筛查基因变异的比较  

Comparison of DHPLC and PCR-SSCP in Screening Gene Variation

作　　者：赵芳芳 张鸣明 罗玉柱 李少斌 Zhao Fangfang;Zhang Mingming;Luo Yuzhu;Li Shaobin(College of Animal Science and Technology of Gansu Agricultural University,Gansu Key Laboratory of Herbivorous AnimalBiotechnology,Gansu Engineering Lab of Genetic Improvement in Ruminants,Lanzhou 730070,China;Gansu Institute of Business & Technology,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物科学技术学院,甘肃省草食动物生物技术重点实验室,甘肃省牛羊基因改良工程实验室,兰州730070 [2]甘肃省商业科技研究所有限公司,兰州730070

出　　处：《中国草食动物科学》2019年第4期11-14,共4页China Herbivore Science

基　　金：甘肃省基础研究创新群体计划项目(17JR5RA137,18JR3RA190);甘肃省科技计划项目(18YF1WA082);甘肃省高等学校科研项目(2015A-078);甘肃省草食动物生物技术重点实验室开放课题(GKLAB-201702)

摘　　要：SNP(Single nucleotide polymorphism)被认为是揭示遗传变异最理想的遗传标记,广泛应用于疾病相关的基因筛查、动植物经济性状遗传标记、群体遗传进化等。目前,筛查SNP的方法很多,文章旨在比较DHPLC(Denaturing high performance liquid chromatography)和PCR-SSCP(Polymerase chain reaction-single stranded conformational polymorphism)两种方法在筛查同一基因片段变异的效果,为筛查基因突变的方法提供选择依据。以绵羊孕酮受体基因(PGR)第4外显子为突变筛查对象,以DHPLC和PCR-SSCP两种方法筛查基因变异,以直接测序作为标准,对比分析两种方法在检出率、准确率、经济性和自动化程度等方面的优劣。结果表明:在对391份藏绵羊血液样品的PGR基因进行分析后,SSCP法检出率100%,而DHPLC法检出率仅83.63%;DHPLC法准确率97.25%,高于SSCP法的94.37%;同时操作性和自动化方面DHPLC法要优于SSCP法,但其检测成本比SSCP法高20倍左右;SSCP法应用的广泛性比DHPLC高许多,其中在畜牧和动物医学这两门学科中应用最多,而DHPLC法在人类医学研究中的应用较多。结论:DHPLC法适合运用于基因突变率低和已完全测出基因序列的物种,具有准确率高、操作简单和自动化程度高等优点;SSCP法对基因序列是否已知及突变率高低无要求,具有检出率高、成本低等优点,此法在动物育种领域中应用更为广泛。SNP(Single nucleotide polymorphism)is considered to be the most ideal genetic marker to reveal genetic variation,which is widely used in disease-related gene screening,genetic markers for economic traits of animals and plants,and population genetic evolution.At present,there are many methods for screening SNP.This paper aims to compare the effects of DHPLC(Denaturing high performance liquid chromatography)and PCR-SSCP(Polymerase chain reaction-single stranded conformational polymorphism)in screening the mutation of the same gene fragment,so as to provide a basis for selection of methods for screening gene mutation.DHPLC and PCR-SSCP were used to screen mutationof sheep progesterone receptor gene(PGR)exon 4,then the advantages and disadvantages of the two methods were compared in detection rate,accuracy,economy and automation,and direct sequencing was used as the standard.The results showed that after analyzing the PGR gene of 391 Tibetan sheep samples,the detection rate of SSCP method was 100%,while that of DHPLC method was only 83.63%.The accuracy of DHPLC was 97.25%higher than that of SSCP 94.37%.At the same time,DHPLC was superior to SSCP in operability and automation,but its detection cost was about 20 times higher than SSCP. SSCP is more widely used than DHPLC,among which animal husbandry and animal medicine were the two most widely used disciplines,while DHPLC was more widely used in human medical research.Conclusion:DHPLC is suitable for low mutation rate and species with complete detection of gene sequence,which has the advantages of high accuracy,simple operation and high automation. SSCP does not require whether the gene sequence is known or not and whether the mutation rate is high or low,and it also has many advantages,such as high detection rate and low cost.It is widely used in animal breeding.

关 键 词：基因 突变 DHPLC PCR-SSCP 

分 类 号：S826.2[农业科学—畜牧学]