Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples

Abstract

The enriched polymerase chain reaction (PCR) assay has been used extensively in the detection of ras gene mutations in many types of human malignancies. Although it is very sensitive, it has a number of features that limit its use in the routine diagnostic laboratory. The aim of this study was to develop a novel enriched PCR strategy, in which the concurrent activity of the restriction enzyme BstNI and Taq polymerase allowed the amplification of mutant K-ras while inhibiting the formation of wild-type product. This restriction endonuclease-mediated selective PCR assay uses three sets of primers, together with BstNI, in the reaction mix, and the amplification products are analyzed by gel electrophoresis. The reliability of the restriction endonuclease-mediated selective PCR assay to detect activated K-ras was determined in a variety of clinical samples, including 139 fresh colorectal carcinomas and 113 paraffin-embedded blocks from 80 separate tumors of the colon and rectum, pancreas, breast, or kidney. Codon 12 mutations of the K-ras oncogene were identified in DNA from both fresh and paraffin-embedded tumors in a rapid, sensitive, and reproducible manner. Mutations were detected in 33 (24%) of the fresh colorectal cancers and 16 (20%) of the paraffin-embedded tumors. These results were 97% concordant in cases in which paraffin blocks and fresh specimens from the same tumor were available for analysis. We conclude that restriction endonuclease-mediated selective PCR is a sensitive, rapid, and robust assay for the detection of point mutations in a variety of clinical samples. Importantly, there is no need for manipulation of the sample once the PCR has been set up, and therefore, the chance of contamination is significantly reduced. In contrast to previous assays, restriction endonuclease-mediated selective PCR is not labor intensive, and its format is suitable for use in routine diagnostic laboratory.

Figure 1.

Schematic representation of diagnostic primers (5BKIT and 3K2) used in REMS-PCR amplification of K-ras.

Figure 2.

REMS-PCR detection of mutant K-ras in DNA from fresh colorectal tumor tissue. Results from six representative colorectal cancers are shown (C1 to C6). DNA from the wild-type cell line K562 analyzed with (+) and without (−) BstN1 in the reaction, and DNA from the heterozygous mutant cell line Calu 1 (Calu) are also shown. Mutant K-ras (81-bp amplicon) is present in three tumors (C1 to C3). All samples contain the template control band (214 bp). Only the K562 DNA analyzed without BstN1 contains the enzyme control band. Absence of this band in the other lanes indicates that the restriction enzyme was active in all other reactions. Products are shown on a 5% NuSieve gel stained with ethidium bromide. MW, phiX174/Hinf.

Figure 3.

REMS-PCR detection of mutant K-ras in DNA from paraffin-embedded tumor tissue. DNA from paraffin-embedded sections of the colorectal tumors analyzed in Figure 2 ▶ are shown (C1 to C6). Control samples (+), (−), and Calu are as for Figure 2 ▶ . Mutant K-ras is present in tumors C1 to C3, whereas tumors C4 and C5 are wild type. Although a weak control amplicon is present in C6, the sample was designated as indeterminate because of insufficient amplification. Also shown is analysis of DNA from two pancreatic tumors (P1 and P2) that contain mutant K-ras. Products are shown on a 5% NuSieve gel stained with ethidium bromide. MW, phiX174/Hinf.