连接酶反应技术检测冠心病三个相关基因的单核苷酸多态性

目的 利用聚合酶链反应-连接酶反应（polymorphism chain reaction-ligase detection reaction,PCR-LDR）技术检测瘦素受体基因Lys109Arg （A/G）、Gln223Arg （A/G）,脂联素基因G276T、T45G,护骨素基因T950C、G1181C六个多态性位点的单核苷酸多态性（single nucleotide polymorphism,SNP）.方法 参照待测多态性位点所在DNA序列设计并合成一对引物及3条探针,先通过PCR反应获得含有待检测突变位点的基因片段,再进行LDR,根据测序电泳结果所显示的含荧光标记的LDR产物的片段长度来判断基因型别.结果 采用PCR技术成功扩增出包含瘦素受体基因（110 bp,130 bp）、脂联素基因（400 bp）、护骨素基因（118 bp,163 bp）多态性位点的基因片段.根据LDR产物片段长度不同进行基因分型,纯合子只有一种片段长度,杂合子包含两种纯合子的片段长度,如瘦素受体基因SNP Lys109Arg （A/G）的PCR产物长度为110 bp,LDR产物片段的长度为110 bp时则判断为AA基因型,112 bp则判断为GG基因型,AG基因型则具有110 bp、112 bp两种片段长度.PCR-LDR基因分型结果与DNA测序技术的检测结果一致（Kappa=1,P=0.00）.结论 PCR-LDR技术简单、快速、准确、成本低,适用于大批量SNP检测.

The ligase detection reaction for detecting single-nucleotide polymorphism of three gene correlated with coronary heart disease

] Objective To develop a method based on the polymorphase chain reaction-ligase detec- tion reaction （PCR-LDR） for detection of single-nueleotide polymorphism （SNP） of leptin receptor gene Lysl09Arg, Gln223Arg, adiponectin gene G276T, T45G and osteoprotegerin gene T950C, Gll81C. Methods Primers and probes were designed according to target DNA sequence. The LDR test was performed after PCR amplification and the results were analyzed according to the length of fragments of LDR products on a se- quencer. Results The fragments containing polymorphism sites of leptin receptor gene （110 bp, 130 bp）, adiponeetin gene （400 bp） and osteoprotegerin gene （118 bp, 163 bp） were amplified successfully by PCR. The genotype was detected by the length and number of LDR products. There was only one kind fragment in ho- mozygote, and two kinds fragments in heterozygoe. For example, the length of PCR product of leptin receptor gene SNP Lysl09Arg was 110 bp. The appearance of 110 bp fragment of LDR product means genotype AA, 112 bp for GG. The LDR product of genotype AG included 110 bp and 112 bp fragments. DNA sequencing analysis validated that PCR-LDR technique could provide unbiased genotyping results（Kappa= 1, P= 0.00）. Conclusion The PCR-LDR technique is simple, fast, accurate, economic and suitable for large-scale SNP studies.