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 polymorphism detection (GMPD) involves amplification of the target DNA using three normal dNTPs and a fourth modified dNTP, whose base is a substrate for a specific DNA-glycosylase once incorporated into the DNA. The work described here utilises uracil DNA-glycosylase as the specific glycosylase 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 present or absent. Subsequent glycosylase excision of the uracil residues followed by cleavage of the apyrimidinic sites allows detection of the mutation in the amplified fragment as a fragment length polymorphism. Variation in the sizes of the fragment length polymorphisms generated, can be readily achieved through the use of inosine bases in place of adenine bases in the upper and/or lower primers. The GMPD process is also adaptable to solid phase analysis. The use of the process for detection 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.
Vaughan
P
P
Department of Biochemistry and National Food Biotechnology Centre, University College, Cork, Ireland.
McCarthy
T V
TV
D001483:Base Sequence; D003550:Cystic Fibrosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D045647:DNA Glycosylases; D004252:DNA Mutational Analysis / Q000379:methods*; D003856:Deoxyuracil Nucleotides; D005260:Female; D006801:Humans; D007292:Inosine Nucleotides; D008297:Male; D008305:Malignant Hyperthermia / Q000235:genetics; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D010375:Pedigree; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D019837:Ryanodine Receptor Calcium Release Channel; D017384:Sequence Deletion; D051981:Uracil-DNA Glycosidase
C505032:CFTR protein, human; D003856:Deoxyuracil Nucleotides; D007292:Inosine Nucleotides; D019837:Ryanodine Receptor Calcium Release Channel; C027078:deoxyuridine triphosphate; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase