Selective 'stencil'-aided pre-PCR cleavage of wild-type sequences as a novel approach to detection of mutant K-RAS

Abstract

The enriched PCR widely used for detection of mutant K-RAS in either tumor tissues or circulating DNA was modified so that abundant wild-type K-RAS alleles are cleaved prior to PCR. We took advantage of an AluI recognition site located immediately upstream of the K-RAS codon 12. The site was reconstituted upon DNA denaturation followed by annealing with a 'stencil', a 16-bp synthetic oligonucleotide complementary to the wild-type sequence. As opposed to normal K-RAS, the mutant allele forms, upon annealing with the stencil, a mismatch at the codon 12 which lies within the AluI enzyme binding site and partially inhibits its activity. The mismatch also lowers the melting temperature of the stencil-mutant K-RAS double helix as compared to stencil-wild-type duplex, so that only the latter is double stranded and selectively digested by AluI at elevated temperatures. The proposed method of stencil-aided mutation analysis (SAMA) based on selective pre-PCR elimination of wild-type sequences can be highly advantageous for detection of mutant K-RAS due to: (i) an enhanced sensitivity because of reduced competition with a great excess of normal K-RAS, and (ii) a decrease in a number of false-positive results from Taq polymerase errors. Application of SAMA for generalized detection of DNA mutations is discussed.

Figure 1

Schematic presentation of stencil-aided selective cleavage of wild-type K-RAS. (A) The fragment (157 bp) of K-RAS gene amplified by PCR. Positions of AluI recognition sites as well as the left and right primers are indicated. The primer K-RAS-L is immediately upstream of codon 12. (B) Denaturation-annealing of wild-type DNA (plus-strand) with the excess of complementary minus-stencil spanning AluI recognition site plus 6 bp on both sides restores an adequate substrate for the enzymatic cleavage. (C) Denaturation-annealing of mutant DNA with the same stencil restores the duplex with a mismatch at codon 12 (mutant nucleotides at either first or second positions are indicated as X and Y).

Figure 2

AluI digestion of perfectly matched and mismatched 16 bp oligonucleotides. Oligonucleotides (50 pmol in 50 µl of AluI buffer) used in this experiment were wild-type plus-stencil (5′-AGTTGGAGCTGGTGGC), wild-type minus-stencil (5′-GCCACCAGCTCCAACT) and mutant minus-stencil (5′-GCCATCAGCTCCAAC) with a substitution (C→T, underlined) of the second nucleotide in codon 12. Oligonucleotides were taken either alone (lanes 1–3), or in pairs, perfectly matched (lanes 4) or mismatched (lanes 5). They were denatured at 94°C for 5 min, annealed at 47°C for 10 min and incubated for 60 min under different conditions: at 37°C with no enzyme added (panel I); at 37 and 42.5°C, with 5 U of AluI (panels II and III, respectively). Aliquots of the samples were subjected to electrophoresis in 20% PAAG and stained with ethidium bromide (0.5 µg/ml). Positions of single-stranded, double-stranded and cleaved oligonucleotides are indicated by arrows. Single-stranded and double-stranded oligonucleotides are distinguished by electrophoretic mobility and intensity of ethidium bromide staining.

Figure 3

Pre-PCR digestion of wild-type or mutant K-RAS followed by enriched PCR. Normal DNA from blood cells of healthy donors (10–1000 ng) and mutant DNA from the SW480 cell line (3–12 ng) were separately subjected to the procedure of stencil-aided AluI cleavage. The target sequences were thereafter amplified by the enriched PCR. The samples with or without AluI restriction enzyme treatment are indicated above the lanes. Note that the signal of mutant K-RAS even taken in low amounts remains unchanged after AluI digestion, while wild-type K-RAS is fully destroyed, except for 1000 ng sample (see text).

Figure 4

SAMA of mutant K-RAS. Mixtures of normal DNA (ranging from 1 to 1000 ng) and constant amount (1 ng) of mutant SW480 cell DNA were subjected to the stencil-aided AluI cleavage (untreated mixtures were used as controls). The target sequences were amplified by the enriched PCR. Note that wild-type sequences are completely removed by the stencil-aided treatment and the signal of mutant K-RAS becomes stronger at the highest K-RAS^(W)/K-RAS^(M) ratio used.

Figure 5

Detection of mutant K-RAS in colorectal cancers. DNA samples isolated from ‘normal’ tissues surrounding a tumor and removed during surgery (Materials and Methods) were subjected to enriched PCR (A) or SAMA (B). Lanes 1, 2 and 3, DNA samples from individual patients.