Detection of 81 of 81 known mouse beta-globin promoter mutations with T4 endonuclease VII--the EMC method.
The enzyme mismatch cleavage (EMC) method relies on the use of the resolvase T4 Endonuclease VII to cleave and thus detect mismatches in heteroduplex DNA formed by annealing normal DNA with mutant DNA. Detection is based on cleavage 3' to the mismatch within a few nucleotides. We report the detection of all 81 different homozygous single-basepair changes tested and present in the mouse beta-globin promoter by using the EMC method with a single set of conditions. Efficiency of cleavage was rated as strong, medium, or weak based on the intensity of the cleavage product(s) compared with background bands on autoradiography. We expect this method to detect near 100% of mutations.
Youil
R
R
Olive Miller Laboratory, Murdoch Institute, Melbourne, Victoria, Australia.
Kemper
B
B
Cotton
R G
RG
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000032:analysis / Q000378:metabolism; D004252:DNA Mutational Analysis; D004706:Endodeoxyribonucleases / Q000378:metabolism; D005914:Globins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis / Q000378:metabolism; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*
D009692:Nucleic Acid Heteroduplexes; D005914:Globins; D004247:DNA; D004706:Endodeoxyribonucleases; C035571:endodeoxyribonuclease VII