Mutational analysis using enriched PCR and cycle sequencing.

During the course of our research on chemically induced mutational spectra in mouse skin and liver tumors, we found the need to develop a sensitive method that would screen out nonmutated genes, related genes and pseudogenes. Although cold single-strand conformation polymorphism methods that used intron-specific primers existed, these methods were only sensitive to the point of 1 mutant in 33 normal molecules (7,8). We were able to substantially modify an enriched polymerase chain reaction (PCR) screening method, originally used to screen for K-ras codons 12 and 13 in human colon tumors (5,6), to be used for H-ras codons 12 and 13 as well as codon 61 mutations in chemically induced mouse skin and liver tumors. Briefly, this method consisted of four steps: (i) primary amplification, (ii) intermediate digestion, (iii) secondary amplification and (iv) final digestion. Aliquots of the final digests were resolved on agarose gels rather than polyacrylamide gels as in the original procedure (5,6). Mutant PCR products are cut once, whereas normal PCR products are cut twice by the codon-specific restriction endonuclease. This allows for an internal control upon digestion with the enzymes. The procedure is outlined briefly in Table 1. Although this method was used to detect specific mouse H-ras mutations, it could easily be adapted to detect point mutations within other genes and species such as the suspected aflatoxin-induced codon 249 mutation in the p53 gene in human liver or the suspected benzo[a]pyrene-induced codon 157 mutation in p53 in human lung tissue using the wide array of commercially available restriction endonucleases. DNA was isolated from both normal and tumor-laden mouse skin and liver using Puregene reagents (Gentra Systems, Minneapolis, MN, USA) and proteinase K and quantitated by UV spectroscopy at 260 nm. All PCR primers were based on the mouse H-ras genomic sequence of Brown et al. (2). All PCRs were carried out in a PTC-100 Programmable Thermal Controller (MJ Research, Watertown, MA, USA). Positive controls were prepared by amplifying 200 ng of normal DNA using mutated 5′ primers and normal 3′ primers (Table 2; codons 12 and 13: primers 1 and 2; codon 61: primers 3 and 4) under the following conditions: 50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25°C), 0.1% Triton X-100, 0.2 mM each dNTP, 2.0 mM MgCl2, 1 U Taq DNA Polymerase (Promega, Madison, WI, USA) and 0.2 mM each primer in a 50μL final volume, covered with 25 μL of Chill-out 14 (MJ Research) and subjected to the following amplification protocol: initial denaturation at 94°C for 5 min and 35 cycles of denaturation at 94°C for 1 min, annealing at 62°C for 2 min and extension at 72°C for 1 min, with a final extension at 72°C for 2 min. PCR products (combined codons 12 and 13: 252 bp and codon 6: 226 bp) were purified on 2% MetaPhor agarose gels (FMC BioProducts, Rockland, ME, USA), extracted from the agarose using QIAEX (Qiagen, Chatsworth, CA, USA), quantitated on 2% MetaPhor agarose gels using a Low DNA Mass Ladder (Life Technologies, Gaithersburg, MD, USA) and diluted to 15 pg/μL for further use. These positive controls contained a codon 12 mutation of GGA→GAA, a codon 13 mutation of GGC→GAC and a codon 61 mutation of CAA→CGA. Tumor DNA was screened for codon 12, 13 and 61 mutations using the following protocol. Positive control (7.5