Nucleic Acids Research, Vol 26, Issue 3 810-815, Copyright © 1998 by Oxford University Press
P Vaughan and TV McCarthy
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.
ARTICLES
A novel process for mutation detection using uracil DNA-glycosylase
Department of Biochemistry and National Food Biotechnology Centre, University College, Cork, Ireland.
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