CAJ | 학술논문

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.
목적 탐토개량취병희선알응효전영(PAGE)-추화을정(EB)현색법재기인형다태성검측중적응용특점.방법 채용병례-대조연구방법,선취로내출혈신생인167례작위병례조,동기인주원관찰적비로내출혈적신생인163례작위대조조,분별사용PAGE-은염、경지당응효전영(AGE)-EB이급PAGE-EB 3충방법진행취합매련식반응-한제성편단장도다태성(PCR-RFLP)분석기술검측소유연구대상μ-아편수체A118G(OPRM1 A118G)기인형,비교3충방법적응용특점.결과 PAGE-은염법급개량PAGE-EB전영법균능청석검측OPRM1 A118G기인형:돌변순합자GG(169 bp),돌변잡합자AG(193 bp,169 bp),야생순합자AA(193 bp),DNA편단분변솔비교차이무통계학의의(P=0.884),AGE-EB칙지능검측출원시조대(193 bp),이불능준학분변OPRM1 A118G적구체기인형.PAGE-은염법공13개보취,수4～5h,12충화학시제,상궤채집도편,기술요구고,조작난도대,모시;개량PAGE-EB법공6개시험보취,력시2h,사용8충시제,자동응효성상의채집도편.결론 재기인형다태성검측중,개량PAGE-EB법간편、쾌첩、분변솔고,치득추엄.
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.