Allele-specific and asymmetric polymerase chain reaction amplification in combination: a one step polymerase chain reaction protocol for rapid diagnosis of familial defective apolipoprotein B-100.
We have combined the asymmetric polymerase chain reaction (PCR) with allele-specific PCR to detect a single point mutation. A set of two priming oligonucleotides and a third allele-specific primer were used to identify heterozygotes for a G to A mutation at nucleotide 10,708 in the apolipoprotein B (apo B) gene. The system requires neither restriction enzyme digestion nor allele-specific oligonucleotides as conventionally applied for allele-specific hybridization of slot blots. This method clearly allows for the detection of the mutant allele by inspection, after agarose gel electrophoresis of a single PCR reaction. DNA from 40 patients with familial defective apo B-100 due to the G to A mutation at nucleotide 10,708 in the apo B gene and their normal relatives was analyzed. Complete agreement with allele-specific hybridization of slot blots confirms supposition that the system is effective to screen a larger population.
Schuster
H
H
Medizinische Poliklinik, Universität, München, Germany.
Rauh
G
G
Müller
S
S
Keller
C
C
Wolfram
G
G
Zöllner
N
N
D000483:Alleles; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D005260:Female; D006801:Humans; D006951:Hyperlipoproteinemias / Q000175:diagnosis / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D010375:Pedigree; D016133:Polymerase Chain Reaction / Q000379:methods*
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D004247:DNA