The purpose of this article is to develop a new high throughput method for detecting genetic polymorphism of warfarin metabolism-related genes rapidly in a single tube. Genomic DNA from human peripheral blood was extracted, and amplified with biotinylated primer to obtain single-stranded templates for pyrosequencing. Then, the single-stranded tem-plates were subjected to Pyrosequencing analysis using PyroMark ID instrument. Simultaneously, Sanger sequencing was also applied to sequence the products as a control to check the reliability of the pyrosequencing result.. The results dis-played that three variants of the warfarin metabolism-related genetic polymorphism (CYP2C9*2, CYP2C9*3, and VKORC1(-1693)) could be simultaneously detected using three different sequencing primers in a single-tube (one test), and 96 tests could be carried out each time. Repeat test and reliability test indicated that the agreement between the pyrosequencing and the Sanger sequencing methods was 100%. . All of these demonstrated that pyrosequencing could accurately and rapidly detect the genetic polymorphism of the warfarin drug metabolism-related genes with high throughput. Compar-ing with simplex pyrosequencing, the method established in the present study was much more economical and timesaving. It has a great value in personalized medical treatment and could be extended to the other genetic diseases.
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