High-Throughput Mitochondrial Genome Screening Method for Nonmelanoma Skin Cancer Using Multiplexed Temperature Gradient Capillary Electrophoresis

Abstract

Background:We explored the utility of multiplexed temperature gradient capillary electrophoresis (TGCE) as a screening tool for identifying genetic changes in the human mitochondrial genome. We examined changes in mitochondrial DNA (mtDNA) in nonmelanoma skin cancers (NMSCs), using TGCE to resolve genetic differences contained within the tumors compared with the control DNA. Methods: The entire mtDNA from NMSC tissue samples was amplified in 17 overlapping amplicons averaging 1.1 kb in size. Fourteen of these amplicons were digested with restriction endonucleases into as many as five smaller analyzable fragments. Digested tumor mtDNA amplicons were annealed with digested amplicons from the control DNA to form heteroduplexes in regions of DNA mismatch. TGCE was performed in a 96-well parallel format to detect mtDNA changes in a high-throughput fashion. Results: TGCE resolved heteroduplexes from homoduplexes in singlet reactions and in multiplexed assays. Using a single programmed temperature gradient, we detected 18 of 20 mtDNA changes contained within the specimens. This system was also able to detect a single nucleotide change in a fragment as large as 2 kb. Conclusion: Multiplexed TGCE is a sensitive and highthroughput screening tool for identifying mtDNA variations.

Carbonic Anhydrase-Related Protein VIII Deficiency Is Associated With a Distinctive Lifelong Gait Disorder in Waddles Mice

“…As described in materials and methods, our strategy includes identifying a target genomic region on the basis of linkage mapping, identifying every gene and biologically functional element within the candidate region, TGCE heteroduplex analysis (Chou et al 2005;Girald-Rosa et al 2005), confirmation of suspected mutations by cDNA sequencing, and, finally, investigatation of the encoded protein.…”

Mitochondrial DNA mutations in renal cell carcinomas revealed no general impact on energy metabolism

“…Recently, a high incidence of specific mtDNA alterations has been reported for gastric (Maximo et al, 2001;Wu et al, 2005), prostate (Jeronimo et al, 2001;Petros et al, 2005), pancreatic (Jones et al, 2001), skin (Girald-Rosa et al, 2005), colorectal (Polyak et al, 1998;Hibi et al, 2001a;Lievre et al, 2005), urinary bladder (Fliss et al, 2000), thyroid (Yeh et al, 2000), oesophageal (Hibi et al, 2001b;Kumimoto et al, 2004), liver (Nishikawa et al, 2001), breast (Richard et al, 2000;Tan et al, 2002;Zhu et al, 2005), uterine cancers (Pejovic et al, 2004) as well as chromophobe renal cell carcinoma (Nagy et al, 2002). Of all mtDNA mutations reported in cancer tissues, only a few are known to be of pathological relevance as shown for patients with disorders of the mitochondrial energy metabolism.…”

Detecting low-abundance p16 and p53 mutations in pancreatic juice using a novel assay: Heteroduplex analysis of limiting dilution PCRs

“…The most likely explanation for the low-frequency heteroduplexes in control DNA samples is Taq polymerase-induced amplification errors. To minimize DNA polymerase errors during PCR, we used a high-fidelity DNA polymerase (Pfu, Platinum Taq) 29 which has a low error-rate (1.3 errors/10 -6 base pair/cycle). With this high specificity, one could theoretically detect mutations at lower concentrations by screening more PCR products for mutations.…”

“…With refinement of our assay, it should be possible to evaluate multiple different sized PCRs during the same TGCE run which would reduce assay cost. 29 The sensitivity of TGCE for detecting mutations could also be improved by using GC clamps in the PCR primers, by using fluorescently labeled primers. 43 Our results also suggest that limiting dilution PCR analysis could be used as part of the diagnostic evaluation of other cancers, such as for detecting p16 and p53 mutations in lung or biliary tract cancers, or mutations in other genes particularly when mutations that are not limited to a few single nucleotide hotspots, such as mutations of the EGFR gene found in nonsmall cell lung carcinomas.…”

“…Samples are then subjected to temperature gradient capillary electrophoresis (TGCE) Novel Assay for Detecting Low Abundance Mutations using a SCE9610 automated sequencer (SpectruMedix Corporation, State College, PA). 28,29 Heteroduplex analysis of PCR products is performed twice, so that PCR products are subjected to two different temperature protocols during capillary electrophoresis. The temperature protocol used varied with the size of the PCR product (e.g., 400 bp PCR products underwent a temperature ramp between 50-60˚C over 30 minutes).…”