We have developed a simple and flexible mutation detection technology for the discovery and mapping of both known and unknown mutations. This technology is based on a new mismatch-specific DNA endonuclease from celery, Surveyor nuclease, which is a member of the CEL nuclease family of plant DNA endonucleases. Surveyor nuclease cleaves with high specificity at the 3' side of any mismatch site in both DNA strands, including all base substitutions and insertion/deletions up to at least 12 nucleotides. Surveyor nuclease technology involves four steps: (i) PCR to amplify target DNA from both mutant and wild-type reference DNA; (ii) hybridization to form heteroduplexes between mutant and wild-type reference DNA; (iii) treatment of annealed DNA with Surveyor nuclease to cleave heteroduplexes; and (iv) analysis of digested DNA products using the detection/separation platform of choice. The technology is highly sensitive, detecting rare mutants present at as low as 1 in 32 copies. Unlabeled Surveyor nuclease digestion products can be analyzed using conventional gel electrophoresis or high-performance liquid chromatography (HPLC), while end labeled digestion products are suitable for analysis by automated gel or capillary electrophoresis. The entire protocol can be performed in less than a day and is suitable for automated and high-throughput procedures.

Acquisition of death-resistance is critical in the evolution of neoplasia. Our aim was to model the early stages of carcinogenesis by examining intracellular alterations in cells that have acquired apoptosis-resistance after exposure to a complex genotoxin. We previously generated sub-populations of BJ-hTERT human diploid fibroblasts, which have acquired death-resistance following exposure to hexavalent chromium [Cr(VI)], a broad-spectrum genotoxicant. Long-term exposure to certain forms of Cr(VI) is associated with respiratory carcinogenesis. Here, we report on the death-sensitivity of subclonal populations derived from clonogenic survivors of BJ-hTERT cells treated with 5 μM Cr(VI) (DR1, DR2), or selected by dilution-based cloning without treatment (CC1). Following Cr(VI) treatment, CC1 cells downregulated expression of the anti-apoptotic protein Bcl-2 and exhibited extensive expression of cleaved caspase 3. In contrast, the DR cells exhibited no cleaved caspase 3 expression and maintained expression of Bcl-2 following recovery from 24 h Cr(VI) exposure. The DR cells also exhibited attenuated mitochondrial-membrane depolarization and mitochondrial retention of cytochrome c and SMAC/DIABLO following Cr(VI) exposure. The DR cells exhibited less basal mtDNA damage, as compared to CC1 cells, which correlates with intrinsic (non-induced) death-resistance. Notably, there was no difference in p53 protein expression before or after treatment among all cell lines. Taken together, our data suggest the presence of more resilient mitochondria in death-resistant cells, and that death-resistance can be acquired in normal human cells early after genotoxin exposure. We postulate that resistance to mitochondrial-mediated cell death and mitochondrial dysregulation may be an initial phenotypic alteration observed in early stage carcinogenesis.

Conserved motifs found in known bacterial polI DNA polymerase sequences were identified, and degenerate PCR primers were designed for PCR amplification of an internal portion of polI genes from all bacterial divisions. We describe here a method that has allowed the rapid identification and isolation of 13 polI genes from a diverse selection of thermophilic bacteria and report on the biochemical characteristics of nine of the purified recombinant enzymes. Several enzymes showed significant reverse-transcriptase activity in the presence of Mg2+, particularly the polymerases from Bacillus caldolyticus EA1, Caldibacillus cellovorans CompA.2, and Clostridium stercorarium.

Site-directed mutagenesis and polymerase chain reaction (PCR)-based cloning are well-established methods carried out routinely in most modern molecular biology laboratories. Application of these methods requires confirmation of the DNA sequence of the target gene by sequencing of DNA purified from multiple colonies, a laborious process. We have developed an alternative approach to screen DNA amplified directly from colony DNA for both desired and undesired mutations. This approach is based on the use of a plant mismatch DNA endonuclease, Surveyor Nuclease, to directly screen clones derived by site-directed mutagenesis. We have also used this approach to identify error-free clones of three genes from celery cDNA produced by PCR and TOPO cloning. Sequence confirmation using Surveyor Nuclease provides a fast and simple approach to obtain desired clones from site-directed mutagenesis and PCR-based cloning methods without the necessity of sequencing DNAs purified from multiple clones.

Therapeutic agents such as cetuximab and panitumumab are epidermal growth factor receptor (EGFR) antagonists that can be effective in the treatment of colorectal cancer. However some 40% of these tumors have activating K-RAS exon 2 codon 12 and 13 mutations that have a strong association with poor response to EGFR antagonists. Attempts have been made to detect the presence of K-RAS exon 2 mutations in free circulating DNA from plasma/serum from colorectal cancer patients with K-RAS exon 2 mutations in their tumors. The small amount of circulating DNA in serum and the low sensitivity of the detection techniques used have resulted in sporadic success in establishing this correlation. Co-amplification at Lower Denaturation Temperature-PCR (COLD-PCR) is a novel form of PCR that preferentially amplifies mutant alleles in a mutant/wild-type mixture with wild-type alleles in vast excess. Fast COLD-PCR, one of two forms of the method, is applied to point mutations or insertion/deletions that lower the melting temperature (Tm) of the mutant allele. In Fast COLD-PCR, preferential amplification of a mutant allele is achieved by setting the PCR denaturation temperature during each cycle at the critical temperature (Tc) at which mutant DNA duplexes but not wild-type duplexes are denatured. COLD-PCR is normally carried out in a thermocycler with independent temperature control of each well, ensuring that Tc is precisely achieved. Well-to-well temperature variation of standard, bench thermocyclers is often not within the narrow range required to achieve efficient COLD-PCR mutation enrichment. We have developed a variation of the Fast COLD-PCR method that reduces the need for precise well-to-well temperature control. In this study we have taken advantage of the Tm lowering of most of the K-RAS exon 2 mutations in codons 12 and 13 (G>A or G>T), and have successfully applied our variation of Fast COLD-PCR to their enrichment. A plasmid-based model system was established to measure the Limit of Detection (LOD) of the K-RAS exon 2 codon 12 mutation G12S (GGT>GAT). Mixtures of two plasmids, one bearing a wild-type copy of exon 2 and the other the G12S mutation were prepared at ratios of mutant/wild-type ranging from 1:10 to 1:1,000 (0.1%). A total of 10,000 plasmid copies alone or combined with purified human serum DNA were enriched. Two rounds of PCR were performed: standard PCR producing a 218-bp amplicon followed by enrichment using nested Fast COLD-PCR at a Tc of 80.9 °C producing a 161-bp amplicon. SURVEYOR® Nuclease/WAVE® HPLC and Sanger DNA sequencing were used to identify the G>A mutation in the enriched PCR product. The LOD for the G12S mutation before enrichment was 1% by SURVEYOR/WAVE and 5% by sequencing. After Fast COLD-PCR enrichment, the LOD was <0.1% in the absence and <0.4% in the presence of serum with both detection methods. This represents detection of less than 1 mutant in 1000 wild-type K-RAS copies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2119.