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. 1994 May 11;22(9):1541–1547. doi: 10.1093/nar/22.9.1541

A simple method for detecting single base substitutions and its application to HLA-DPB1 typing.

T Oka 1, H Matsunaga 1, K Tokunaga 1, S Mitsunaga 1, T Juji 1, A Yamane 1
PMCID: PMC308027  PMID: 8202352

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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