A simple method for detecting single base substitutions and its application to HLA-DPB1 typing.
We have developed a simple and reliable method, PCR-PHFA (polymerase chain reaction dependent preferential homoduplex formation assay), for detection of single base substitutions within PCR amplicons. This technique is based upon strand competition during hybridization between a double labeled amplicon, prepared from biotin and DNP labeled primers, and an unlabeled amplicon. Under the precisely controlled temperature gradient, the preferential formation of a homoduplex over a heteroduplex occurs. After annealing, the identical sequence of the double labeled and unlabeled amplicon resulted in a low population of regenerated double labeled dsDNA due to strand exchange between them. Even when the two differed by only a single base substitution, double labeled molecule was regenerated efficiently because of preferential homoduplex formation. The regenerated double labeled molecule was captured onto a streptavidin coated microtiter plate and quantified enzymatically with a chromogenic substrate. The technique has been successfully applied in HLA-DPB1 typing. Furthermore, we detected a mutated gene even in the presence of a large excess of the corresponding normal gene.
Oka
T
T
Institute for Biotechnology Research, Wakunaga Pharmaceutical Co. Ltd., Hiroshima, Japan.
Matsunaga
H
H
Tokunaga
K
K
Mitsunaga
S
S
Juji
T
T
Yamane
A
A
D001483:Base Sequence; D004247:DNA; D004252:DNA Mutational Analysis / Q000379:methods*; D006682:HLA-DP Antigens / Q000032:analysis / Q000235:genetics*; D059868:HLA-DP beta-Chains; D006579:Heterozygote; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes; D016133:Polymerase Chain Reaction / Q000379:methods*
D006682:HLA-DP Antigens; D059868:HLA-DP beta-Chains; C071170:HLA-DPB1 antigen; D009692:Nucleic Acid Heteroduplexes; D004247:DNA