A NOVEL PROCESS FOR MUTATION DETECTION USING URACIL DNA-GLYCOSYLASE

A novel process is presented for the detection of known mutations-and polymorphisms in DNA. This process, termed glycosylase mediated polymo rphism detection (GMPD) involves amplification of the target DNA using three normal dNTPs and a fourth modified dNTP, whose base is a substr ate for a specific DNA-glycosylase once incorporated into the DNA. The work described here utilises uracil DNA-glycosylase as the specific g lycosylase and dUTP as the modified dNTP, Primers are designed so that during extension, the position of the first uracil incorporated into the extended primers differs depending on whether a mutation is presen t or absent. Subsequent glycosylase excision of the uracil residues fo llowed by cleavage of the apyrimidinic sites allows detection of the m utation in the amplified fragment as a fragment length polymorphism. V ariation in the sizes of the fragment length polymorphisms generated, can be readily achieved through the use of inosine bases in place of a denine bases in the upper and/or lower primers. The GMPD process is al so adaptable to solid phase analysis. The use of the process for detec tion of mutations in the RYR1 and CFTR genes is demonstrated. Overall, the simplicity, specificity, versatility and flexibility of the GMPD process make it an attractive candidate for both small and large scale application in mutation detection and genome analysis.