CAJ | 학술논문

目的评估经非缓冲福尔马林固定不同时间后的人体组织STR分型有效性,了解各种人体组织在非缓冲福尔马林固定剂中可获得完全STR分型位点的时限.方法市售40%福尔马林溶液经1:9稀释后在室温(15～20℃)下固定人体组织,不同时间后取样.以QIAamp~(R)DNA法和IQ~(TM) DNA System法提取DNA,用quantifiler humanTaqman探针法进行DNA定量,用常规16 STR位点的AmpFSTR identifiler kit和短小片段9 STR位点的AmpFSTR MiniFiler kit进行PCR扩增,在3100遗传分析仪进行扩增DNA片段长度检测,用GeneMapper ID v3.2对STR位点榆出率进行分析.结果福尔马林固定时间、组织类型以及DNA提取方法、PCR的DNA模板终浓度均影响非缓冲福尔马林固定后人体组织STR分型效能.DNA提取用QIAgen法为优,DNA模板终浓度的最佳范围在1～3ng/μL.各类型组织在非缓冲福尔马林固定剂中的降解速率有差异,肺组织的降解速率最慢,肝、肠组织最快.固定时间在4d内的组织可以获得常规STR的完整位点数;固定时间在15d内的组织可以获得miniSTR的完整位点数.结论非缓冲福尔马林固定人体组织时间是影响STR分型的最主要因素,其次组织类型、提取方法、DNA模板浓度及STR基因座的选择也是此类降解样品成功检测的关键因素.
목적 평고경비완충복이마림고정불동시간후적인체조직STR분형유효성,료해각충인체조직재비완충복이마림고정제중가획득완전STR분형위점적시한.방법 시수40%복이마림용액경1:9희석후재실온(15～20℃)하고정인체조직,불동시간후취양.이QIAamp~(R)DNA법화IQ~(TM) DNA System법제취DNA,용quantifiler humanTaqman탐침법진행DNA정량,용상규16 STR위점적AmpFSTR identifiler kit화단소편단9 STR위점적AmpFSTR MiniFiler kit진행PCR확증,재3100유전분석의진행확증DNA편단장도검측,용GeneMapper ID v3.2대STR위점유출솔진행분석.결과 복이마림고정시간、조직류형이급DNA제취방법、PCR적DNA모판종농도균영향비완충복이마림고정후인체조직STR분형효능.DNA제취용QIAgen법위우,DNA모판종농도적최가범위재1～3ng/μL.각류형조직재비완충복이마림고정제중적강해속솔유차이,폐조직적강해속솔최만,간、장조직최쾌.고정시간재4d내적조직가이획득상규STR적완정위점수;고정시간재15d내적조직가이획득miniSTR적완정위점수.결론 비완충복이마림고정인체조직시간시영향STR분형적최주요인소,기차조직류형、제취방법、DNA모판농도급STR기인좌적선택야시차류강해양품성공검측적관건인소.
Objective To evaluate the STR profiling availability of formalin-fixed tissues and the detectable time limit of intact STR profile.Methods The different human tissues were fixed with 10-fold diluted commercial 40%formalin fixative for different duration under 15～20℃,and then DNA was extracted using the method of QIAamp~(R)DNA and IQ~(TM) DNA System.The extracted DNA was quantified with QuantifilerTM kit and amplified by both AmpFSTR identifiler kit and AmpFSTR MiniFiler kit.The STR profile was analyzed by GeneMapper ID v3.2 on 3100-Avant.Resuls The STR profiling availability of formalin-fixed tissues was relevant to the formalin fixing duration mainly.as well as the type of tissues and the template concentration and protocol of DNA extracting.The optimal ranges of template concentration is 1～3ng/μL and the QIAamp extracting method was preferable.There are differences in the degradation rate between various types of tissues in the unbuffered formalin fixative,and the lung tissue showed the slowest rate and liver and intestine tissues the fastest.Intact miniSTR profile of all the tissues detected could be obtained within 15 days duration of formalin fixing while intact STR profile could be obtained within 4 days.Conclusion The major factor that impact the availability of STR profiling of formalin-fixed tissues is the fixing duration in unbuffered formalin,as well as the type of tissues,method of extraction,concentration of PCR template and the kinds of STR loci.