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

Nucleic Acids Res. 1994 May 11;22(9):1541-7. doi: 10.1093/nar/22.9.1541.


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.

MeSH terms

  • Base Sequence
  • DNA
  • DNA Mutational Analysis / methods*
  • HLA-DP Antigens / analysis
  • HLA-DP Antigens / genetics*
  • HLA-DP beta-Chains
  • Heterozygote
  • Histocompatibility Testing / methods*
  • Humans
  • Molecular Sequence Data
  • Nucleic Acid Heteroduplexes
  • Polymerase Chain Reaction / methods*


  • HLA-DP Antigens
  • HLA-DP beta-Chains
  • HLA-DPB1 antigen
  • Nucleic Acid Heteroduplexes
  • DNA