Screening for point mutations by semi-automated DNA sequencing using sequenase and magnetic beads.
We have established an improved method for detecting point mutations by semi-automated DNA sequencing of PCR fragments generated from genomic DNA. The method employs magnetic beads to create immobilized single-stranded DNA templates, and the sequencing reaction is performed with Sequenase. This method is superior to sequencing with Taq DNA polymerase because the uniform peak height with Sequenase makes heterozygosity easily detectable as double peaks that are half the normal height. Detection of heterozygosity by this method is illustrated by sequencing a 180-bp fragment of the human apolipoprotein B gene. This fragment contains codon 3500, where a point mutation (3500CGG-->CAG) is found in subjects with the autosomal dominant disease familial defective apolipoprotein B. The nonuniform peak height with Taq DNA polymerase makes it more difficult to detect heterozygosity. This is also illustrated by sequencing a 278-bp fragment of the low-density lipoprotein receptor gene.
Leren
T P
TP
Department of Medical Genetics, Ullevål University Hospital, Oslo, Norway.
Rødningen
O K
OK
Røsby
O
O
Solberg
K
K
Berg
K
K
D001055:Apolipoproteins B / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004259:DNA-Directed DNA Polymerase; D005069:Evaluation Studies as Topic; D006801:Humans; D008280:Magnetics; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011973:Receptors, LDL / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D019914:Taq Polymerase
D001055:Apolipoproteins B; D011973:Receptors, LDL; D004247:DNA; D019914:Taq Polymerase; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase