Detection of the apoB-3500 mutation (glutamine for arginine) by gene amplification and cleavage with MspI.
A single primer-template mismatch 2 bp from the apoB-3500 (G to A) mutation permits introduction of a cleavage site for MspI (C/CGG) in normal alleles but not in mutant alleles (CCAG). After amplification, cleavage, and polyacrylamide gel electrophoresis, normal and mutant alleles could be unambiguously distinguished. We constructed a positive (homozygous mutant) standard by site-directed mutagenesis. A negative standard was DNA from a homozygous normal subject. The method enables us to screen for the mutation with 12 microliters of spotted whole blood as the source of DNA.
Hansen
P S
PS
University Department of Internal Medicine and Cardiology, Arhus Amtssygehus, Copenhagen, Denmark.
Rüdiger
N
N
Tybjaerg-Hansen
A
A
Faergeman
O
O
Gregersen
N
N
D001055:Apolipoproteins B / Q000097:blood / Q000172:deficiency / Q000235:genetics*; D001120:Arginine / Q000235:genetics*; D001483:Base Sequence; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific; D005784:Gene Amplification; D005973:Glutamine / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation
D001055:Apolipoproteins B; D005973:Glutamine; D001120:Arginine; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific