Genotyping of Methylenetetrahydrofolate Reductase Gene by Pyrosequencing Coupled with Polymerase Chain Reaction Using Human Whole Blood as Starting Material
The polymorphisms of 677CT and 1298AC in the methylenetetrahydrofolate reductase gene(MTHFR) play a crucial role in the pharmacogenetics of methotrexate(MTX) and 5-fluorouracil(5-FU).Detection of the MTHFR gene polymorphisms will give a rational guidance to personalized medicine.In order to further reduce the detection time and costs,we developed a method based on highly sensitive pyrosequencing coupled with PCR using human whole blood as starting material.This method can efficiently detect the two sites by using only 1 μL of human whole blood samples for amplification based on " HpH Buffer ".After optimizing the conditions,single strand DNA denatured by alkali from 5 μL of PCR products and 1μL of streptavidin-coated beads could be enough for pyrosequencing.To investigate the accuracy of this method,12clinical samples were detected,and all of the MTHFR 677CT and 1298AC polymorphisms were correctly typed.This study demonstrated an easily-operated,time-saving,inexpensive,and highly accurate method for genotyping clinical samples.The method can be well applied to personalized medicine of MTX and 5-FU.