High sensitivity EndoV mutation scanning through real-time ligase proofreading

Published online October 28, 2004 Nucleic Acids Research, 2004, Vol. 32, No. 19 e148 doi:10.1093/nar/gnh150 High sensitivity EndoV mutation scanning through real-time ligase proofreading Hanna Pincas, Maneesh R. Pingle, Jianmin Huang, Kaiqin Lao1, Philip B. Paty2, Alan M. Friedman3 and Francis Barany* Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021, USA, 1 Applied Biosystems, Foster City, CA 94404, USA, 2Colorectal Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY 10021, USA and 3Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA Received September 9, 2004; Revised and Accepted October 11, 2004 The ability to associate mutations in cancer genes with the disease and its subtypes is critical for understanding oncogenesis and identifying biomarkers for clinical diagnosis. A two-step mutation scanning method that sequentially used endonuclease V (EndoV) to nick at mismatches and DNA ligase to reseal incorrectly or nonspecifically nicked sites was previously developed in our laboratory. Herein we report an optimized single-step assay that enables ligase to proofread EndoV cleavage in real-time under a compromise between buffer conditions. Real-time proofreading results in a dramatic reduction of background cleavage. A universal PCR strategy that employs both unlabeled gene-specific primers and labeled universal primers, allows for multiplexed gene amplification and precludes amplification of primer dimers. Internally labeled PCR primers eliminate EndoV cleavage at the 50 terminus, enabling high-throughput capillary electrophoresis readout. Furthermore, signal intensity is increased and artifacts are reduced by generating heteroduplexes containing only one of the two possible mismatches (e.g. either A/C or G/T). The single-step assay improves sensitivity to 1:50 and 1:100 (mutant:wild type) for unknown mutations in the p53 and K-ras genes, respectively, opening prospects as an early detection tool. INTRODUCTION Multiple somatic alterations lie at the root of cancer development and tumor heterogeneity. Of cancer genes reported in the current databases, 90% have somatic mutations, 20% have germline mutations and 10% have both (1). A major challenge in cancer biology is to unravel the molecular anatomy of individual cancers and tumor subtypes, which would allow a better understanding of the role of genetic alterations in tumor progression, and provide diagnostic and prognostic markers. The molecular basis for the development and progression of colorectal cancers is well developed in the scientific literature. Colorectal tumors have been classified as either demonstrating chromosomal instability (CIN) or microsatellite instability (MIN). The former is associated with sequential sporadic mutations in the APC (70%), K-ras (40%) and p53 (50%) genes, while the latter contains additional sporadic mutations in the TGFBRII (90%) and BAX (50%) genes (2–6). Mutational patterns may reflect alternative routes to activating/ inactivating signaling, growth and apoptosis pathways. APC mutations disrupt the association of APC and b-catenin, resulting in excessive amounts of b-catenin and overactivation of the Wingless/Wnt signaling pathway. Oncogenic mutations in b-catenin were also observed in some colorectal cancers that lacked APC mutations (3). Likewise, colorectal tumors lacking K-ras mutations may have their ras pathway activated through B-raf mutations (7). In addition to these shared mutations, a recent study showed a high frequency of mutations of the PIK3CA gene (a subunit of the phosphatidylinositol 3-kinase; PI3K) in colon cancers, making this gene a potential biomarker for early detection and/ or prognosis of the disease (8). Similarly, the discovery of mutations in various tyrosine kinase and tyrosine phosphatase genes in colorectal cancers suggests that these could become targets for personalized therapy (9,10). As the number of genes linked to cancer grows, there is an increasing demand for the development of new methods for high-throughput detection of unknown mutations. Different mutation detection technologies have been developed to identify known mutations: these include DNA microarrays (11–13), the polymerase chain reaction/ligase detection reaction (PCR/LDR) (14), now used in combination with the universal DNA microarray (15–17) and primer extension assays (18). Different technologies are used for detection of unknown mutations: hybridization analysis using high-density oligonucleotide arrays (12), denaturing high-performance liquid chromatography (DHPLC) (19), capillary electrophoresis-based single strand conformation polymorphism (CE-SSCP) (20), denaturing gradient gel electrophoresis (DGGE) (21) and heteroduplex analysis (HA) (22) *To whom correspondence should be addressed. Tel: +1 212 746 6509; Fax: +1 212 746 7983; Email: Nucleic Acids Research, Vol. 32 No. 19 ª Oxford University Press 2004; all rights reserved ABSTRACT e148 Nucleic Acids Research, 2004, Vol. 32, No. 19 MATERIALS AND METHODS Materials All routine chemical reagents were purchased from Sigma Chemicals (St. Louis, MO, USA) or Fisher Scientific (Fair Lawn, NJ, USA). GeneScanTM –500 (LIZTM) Size Standard, Hi-Di formamide, polymer POP-7, AmpliTaq and AmpliTaq Gold DNA polymerases, and deoxyribonucleoside triphosphates (dNTPs) were purchased from Applied Biosystems (Foster City, CA). BSA and ATP were purchased from Boehringer-Mannheim (Indianapolis, IN, USA). Proteinase K was purchased from QIAGEN (Valencia, CA, USA). Lambda exonuclease and T4 polynucleotide kinase were purchased from New England Biolabs (Beverly, MA). Unlabeled deoxyoligonucleotides were purchased from Integrated DNA Technologies Inc. (Coralville, IA, USA), while HPLCpurified VIC- and NED-labeled deoxyoligonucleotides were obtained from Applied Biosystems. Thermotoga maritima Endonuclease V and Thermus species AK16D DNA ligase were purified as previously described (50,53). Tumor and normal tissue were obtained from surgical resection of colon cancers at Memorial Sloan Kettering Cancer Center, which were snap-frozen in liquid nitrogen within 15 min of tumor removal. DNA from these tissues was extracted and purified using a proteinase-K/lithium chloride/ethanol protocol as described (QIAGEN). Tumor samples known to contain Q192Ter (C!T) or Y205F (A!T) mutations in exon 6 of p53 were used, as well as their matched normal colonic mucosa. Genomic DNA from cell lines was extracted using DNeasy tissue kit from QIAGEN. HT-29 cell line contains the wild-type K-ras gene, while SW480 and SW620 contain pure exon 1 G12V (G!T) mutation. LoVo cell line contains wild-type p53 gene, while HT-29, SW480 and SW620 cell lines contain exon 8 R273H (G!A) mutation. PCR amplification and 50 phosphorylation of deoxyoligonucleotides DNA sequences of PCR primers used in this study are listed in Table 1. Fifty-microliter PCR (...truncated)