半定量聚合酶链反应在PCR-RFLP酶切法测定基因多态性中的应用

目的探索将半定量聚合酶链反应方法，应用于PCR-RFLP酶切法以提高基因多态性测定精确性和可重复性。方法PCR扩增81例人维生素D受体基因BsmI位点多态性片段，分别在酶量不同的两种酶切体系中测定基因型；PCR产物经半定量后，再次用上述两种体系酶切。考察4次测定的一致性。结果经产物半定量后用PCR-RFLP法测定VDR基因BsmI多态性，不同酶切系统测得基因多态性的一致性明显优于未经半定量的结果。结论应用半定量PCR方法可明显提高PCR-RFLP酶切法的精确性和可重复性。

Application of semi-quantitative polymerase chain reaction in the PCR - RFLP analysis to detect gene polymorphism

Objective To explore the effect of using semi-quantitative polymerase chain reaction in the restriction fragment length polymorphism(RFLP) to improve the accuracy and the repeatability of the assay for gene polymorphism.Methods VDR BsmI polymorphism restriction fragments of 81 individuals were amplified with PCR.VDR BsmI polymorphism were test using different doses of restriction enzymes;After quantifying and classifying the PCR products,VDR BsmI polymorphism were analyzed again by adjusting the doses of PCR products according to the concentration of each product into the reaction system which mentioned above,the consistency of 4 tests was assessed. Results The results indicated that in the reaction system with different doses of restriction enzymes,the agreement and the repeatability of the detection using semi-quantitative PCR in PCR-RFLP assay were better than PCR-RFLP assay without semi-quantitative.Conclusion A higher level of consistency and repeatability was found from the method of semi-quantitative PCR-RFLP compared to PCR-RFLP.