CAJ | 학술논문

目的：探索高分辨率熔解曲线法（high resolution melting ，HRM）检测胱硫醚β-合酶（CBS）基因 SNP位点所需的最佳反应体系和条件，建立快速高效的对CBS基因分型的方法。方法通过普通 PCR初步确立引物的退火温度范围，在实时荧光定量PCR（qPCR）-HRM反应中调整确定最佳退火温度。对影响 qPCR-HRM的因素包括反应程序、模板DNA、MgCl2再分别进行优化，确立最佳反应体系和反应条件，对在此基础上得出的基因分型结果通过测序验证其准确性，从而建立系统的 HRM检测CBS基因SNP的方法。结果 HRM检测CBS基因该片段最佳退火温度为62℃，qPCR-HRM最佳反应体系为20μl，引物浓度为0．2μmol/L，Mg2＋为2．5μmol/L，模板DNA为60 ng。在优化的体系条件下筛选出的突变型样本经测序验证与实验结果一致。结论 HRM可以作为检测 SNP准确、经济、高效的方法。
목적：탐색고분변솔용해곡선법（high resolution melting ，HRM）검측광류미β-합매（CBS）기인 SNP위점소수적최가반응체계화조건，건립쾌속고효적대CBS기인분형적방법。방법통과보통 PCR초보학립인물적퇴화온도범위，재실시형광정량PCR（qPCR）-HRM반응중조정학정최가퇴화온도。대영향 qPCR-HRM적인소포괄반응정서、모판DNA、MgCl2재분별진행우화，학립최가반응체계화반응조건，대재차기출상득출적기인분형결과통과측서험증기준학성，종이건립계통적 HRM검측CBS기인SNP적방법。결과 HRM검측CBS기인해편단최가퇴화온도위62℃，qPCR-HRM최가반응체계위20μl，인물농도위0．2μmol/L，Mg2＋위2．5μmol/L，모판DNA위60 ng。재우화적체계조건하사선출적돌변형양본경측서험증여실험결과일치。결론 HRM가이작위검측 SNP준학、경제、고효적방법。
Objective To explore the optimal reaction condition of high resolution melting (HRM)for detecting SNPs of Cystathionine Beta Synthetase(CBS),and establish the fast and effective way of genotyping.Methods The annealing temperature range was firstly determined by common polymerase chain reaction,then optimal annealing tem-perature was adjusted by qPCR-HRM.The qPCR-HRM reaction system,including reaction process,DNA template,and MgCl2 concentration,were optimized to obtain the best result.Finally,the genotyping results were varified by Sanger sequencing.In this way,the qPCR-HRM of detecting CBS SNP was established.Results The optimal annealing tem-perature was 62℃;the best reaction system of qPCR-HRM includs primers 0.2μmol/L,Mg2+ 2.5μmol/L,and DNA template 60 ng in 20μl.The results of HRM and direct sequencing were consistent.Conclusion HRM could serve as an accurate,fast,and effective method for detecting SNPs of genes.