Multiplex-PCR-based single-strand conformation polymorphism protocol for simultaneous analysis of up to five fragments of the low-density-lipoprotein receptor gene.
Single-strand conformation polymorphism has become a screening method for the detection of mutations in different genes. For analysis of the promotor region and the coding sequence of the low-density-lipoprotein receptor gene by standard protocols, 21 radiolabeled PCRs and electrophoreses have to be performed. To accelerate this procedure, we developed a nonradioactive multiplex approach of the single-strand conformation polymorphism analysis. Multiplex PCRs were established, each resulting in the amplification of 4 or 5 fragments of this gene. The heat-denatured, single-stranded multiplex-PCR products were electrophoresed, blotted on a nylon membrane and visualized using a chemiluminescence detection system. The simultaneously amplified fragments were clearly resolved by their different mobility on the gel. Comparing the pattern of bands of each separately amplified PCR product and the multiplex-PCR products allowed identification of each band as one exon, part of an exon or the promotor region of the gene. To determine the sensitivity of this method, the low-density-lipoprotein receptor gene of 11 patients with 11 different mutations was analyzed. All mutations could be identified in the multiplex reactions. We conclude that a multiplex-PCR-based, single-strand conformation polymorphism protocol is much faster but equally sensitive compared to standard protocols.
Weiss
N
N
Ludwig-Maximilians-Universität München, Germany.
Eggersdorfer
I
I
Keller
C
C
D003062:Codon / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons / Q000235:genetics; D005796:Genes; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000235:genetics; D008163:Luminescent Measurements; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D011401:Promoter Regions, Genetic; D011973:Receptors, LDL / Q000235:genetics*
D003062:Codon; D011973:Receptors, LDL