改良凝胶电泳方法在基因多态性检测中的应用  

Application of improved gel electrophoresis to the detection of genotype polymorphisms

作　　者：夏沛格 张茜[1] 时赞扬[1] 程欣茹[1] 文建国[2] 

机构地区：[1]郑州大学第一附属医院新生儿科,450052 [2]郑州大学第一附属医院重点实验室,450052

出　　处：《中华实用儿科临床杂志》2014年第15期1174-1176,共3页Chinese Journal of Applied Clinical Pediatrics

基　　金：河南省科技厅基金（083SGYS33263-5）

摘　　要：目的 探讨改良聚丙烯酰胺凝胶电泳（PAGE）-溴化乙啶（EB）显色法在基因型多态性检测中的应用特点.方法 采用病例-对照研究方法,选取颅内出血新生儿167例作为病例组,同期因住院观察的非颅内出血的新生儿163例作为对照组,分别使用PAGE-银染、琼脂糖凝胶电泳（AGE）-EB以及PAGE-EB 3种方法进行聚合酶链式反应-限制性片段长度多态性（PCR-RFLP）分析技术检测所有研究对象μ-阿片受体A118G（OPRM1 A118G）基因型,比较3种方法的应用特点.结果 PAGE-银染法及改良PAGE-EB电泳法均能清晰检测OPRM1 A118G基因型：突变纯合子GG（169 bp）,突变杂合子AG（193 bp,169 bp）,野生纯合子AA（193 bp）,DNA片段分辨率比较差异无统计学意义（P=0.884）,AGE-EB则只能检测出原始条带（193 bp）,而不能准确分辨OPRM1 A118G的具体基因型.PAGE-银染法共13个步骤,需4～5h,12种化学试剂,相机采集图片,技术要求高,操作难度大,耗时;改良PAGE-EB法共6个试验步骤,历时2h,使用8种试剂,自动凝胶成像仪采集图片.结论 在基因型多态性检测中,改良PAGE-EB法简便、快捷、分辨率高,值得推广.Objective To explore the traits of improved polyacrylamide gel electrophoresis （PAGE）-ethidium bromide（EB） staining in the detection of genotype polymorphisms.Methods The methods of PAGE-silver staining,agarose gel electrophoresis （AGE）-EB staining and improved PAGE-EB staining in polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） were used to detect the μ-opioid receptor （OPRM1） A118G genotypes in case group （n =167） infants with intracranial hemorrhage （ICH）,and control group（n =163） infants without ICH,to conduct a case study analysis.And the application traits of three methods were compared.Results Genotypes of OPRM1 A118G were GG （169 bp）,AG （193 bp,169 bp）,AA （193 bp）.Both the electrophoresis methods of PAGE-silver staining and PAGE-EB could be used to detect the genotypes of OPRM1 A118G clearly in this study.There was no statistically significant difference between the resolutions of DNA fragments （P =0.884）.The first method,which had 13 experiment steps,consuming 4-5 hours,involving 12 kinds of chemical reagents,and the pictures were taken with the camera,was complex,with difficult operation,more time consuming; Compared with the first method,the secondmethod was simple,which had 6 test procedures,consuming 2 hours with 8 reagents,and the pictures were taken by using an automatic gel imager.AGE-EB could not be used to detect genotypes of OPRM1 A118G.Conclusions The method of improved PAGE-EB has the advantages of fast,easy operation,and high resolution,which is worthy of wider application.

关 键 词：聚丙烯酰胺凝胶电泳-银染 琼脂糖凝胶电泳-EB 改良凝胶电泳 基因多态性 

分 类 号：R440[医药卫生—诊断学]