载脂蛋白B-100 3500精氨酸→色氨酸突变的简便快速诊断

目的 建立一个简便快速方法，用于诊断家族性载脂蛋白B-100缺陷症R3500W。方法 设计一对寡核苷酸引物，其上游引物为突变引物。以PCR扩增目的DNA顺序，产物用限制酶Ncol酸解，酶解体系中加入含Ncol切口的DNA片段作内参照物，以排除酶切时假阴性的出现。结果 所设计引物能成功地用行PCR以扩增目的DNA片段，长144碱基对。产物与限制酶Ncol保温，正常基因产物不被切割，突变基因产物则由于引入一个人工限制酶切口而被切割，产生117碱基对的片段，这两长度不同的片段可被2%琼脂糖凝胶电泳所分离。利用这一方法，于162例高血浆胆固醇患中检出两例杂合R3500W突变基因携带。结论 本突变引物PCR法成功地检出Apo B-100R3500W突变基因，方法可靠，简便易行。

Rapid diagnosis of familial defective apolipoprotein B-100 R3500W by mutagenic PCR primers

Objective A method combining the mutagenic PCR primers and restriction enzyme digestion was designed to facilitate the detection of gene mutation in familial defective apolipoprotein B 100 R3500W. Methods A pair of primer was designed and a mismatch nucleotide was introduced in its upstream primer. A segment of target DNA including the possibly mutated nucleotide was amplified by PCR and the products were digested by restriction enzyme Nco1. To overcome the potential false negative results due to improper digestion conditions, a segment of DNA with Nco1 cut size was added as reference. Results The target sequence were successfully amplified by PCR, producting a 144 bp DNA fragment as expected. When incubated with Nco1, the enzyme could digest the DNA, producing a 117 bp segment, only if it was amplified from the mutated gene, but not from the normal allele. This difference in length of DNA could be separated by electrophoresis on a 2% agarose gel. Thus we successfully detected two carriers of heterozygous FDB R3500W in 162 hypercholesterolemic patients. Conclusion Mutagenic PCR primers could be used to detect the gene mutation of Apo B 100 R3500W, it is simple and can be easily applied by clinical laboratories.