Nonradioisotopic PCR Heteroduplex Analysis: A Rapid, Reliable Method of Detecting Minor Gene Mutations

Gene mutations play an important role in the molecular basis of diseases including cancer. In addition to sequencing, there are several methods to detect minor mutations, even singlebase changes (1,3). However, none of these detect all of the minor molecular changes that can occur. White et al. (3) used 32P-labeled heteroduplex polymorphism to visualize single-base substitutions. We have modified their method by making use of ethidium bromide fluorescence to detect re-annealed heteroduplexes. The method requires only small amounts of polymerase chain reaction (PCR)-amplified DNA, is easy to handle because radioactivity is not involved and readily resolves differences between mutant and control DNA as we report here. The gpt gene in PL61/1 cells was mutated by γ-rays, and mutants were selected by resistance to 6-thioguanine (6-TG) challenge (2,4). DNA from controland mutant-cell lysates were separately amplified by using PCR using the same pair of primers (2). From lysates, these primers yielded a 561-bp fragment of the gpt gene. The PCR amplification was verified by DNA electrophoresis in a gel of 1% agarose in 1× TBE buffer (0.095 M Tris-borate, 0.002 M EDTA, pH 8.0) containing 0.5 μg/mL ethidium bromide. For heteroduplex analysis (HDA), 3.0 μL of the PCR products from both control and mutant cells were mixed and cut with 6.0 U of KpnI restriction endonuclease. This cutting resulted in 395and 166-bp fragments of double-stranded DNA in the normal gpt gene. Denaturations and renaturations were performed in a programmed PCR thermal cycler (Perkin-Elmer, Norwalk, CT, USA); 95°C for 8 min, followed by 1 min at 90°C, 1 min at 80°C, 1 min at 70°C and so on until room temperature was reached. For polyacrylamide gel electrophoresis (PAGE), the renatured sam-