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Nucleic Acids Research, 1994, Vol. 22, No. 9 1541-1547
© 1994

METHODS

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

Takanori Oka*,, Hironari Matsunaga, Katsushi Tokunaga1, Shigeki Mitsunaga1, Takeo Juji1 and Akio Yamane

Institute for Biotechnology Research, Wakunaga Pharmaceutical Co. Ltd 1624 Shimokotachi, Koda-cho, Takata-gun, Hiroshima 729-64 1The Japanese Red Cross Central Blood Center Tokyo, Japan

*To whom correspondence should be addressed

Received January 3, 1994. Revised April 6, 1994. Accepted April 6, 1994.

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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