Molecular and genetic targets in early detection.
ABSTRACT Recent research has revealed the existence of specific mutations in cancer. These mutations are being investigated as targets to find subjects at high risk for cancer, to detect early cancer, to detect the early recurrence of established cancer, and to find micrometastasis. These mutations are reviewed for the major anatomic sites. Some of the clinical issues related to the application of these mutations and the limitations of using molecular targets are also considered. Current methods for determining the risk of cancer are reviewed. Risk assessment is essential for defining cohorts for chemoprevention and other interventions. The concept of using surrogate anatomic and functional sites for estimating risk is introduced. Finally, the increasing complexity of molecular genetic analysis and the biologic heterogeneity of cancer are discussed in relation to early detection.
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ABSTRACT: The efficacy of capillary electrophoresis for detecting DNA mutations via heteroduplex analysis (HDA) is dependent upon both the effective passivition of the capillary surface and the choice of the correct polymer network for sieving. Using HDA with laser-induced fluorescence detection of fluorescently labeled DNA fragments, an effective coating and optimal polymer matrix were sought. Optimized separation conditions were determined through the methodological evaluation of a number of different silanizing reagents, polymeric coatings, and polymer networks for resolving the PCR-amplified DNA fragments associated with five mutations (185delAG, 1294del40, 4446C>G, 5382insC, 5677insA) in the breast cancer susceptibility gene (BRCA1). For capillary coating, allyldimethylchlorosilane, 4-chlorobutyldimethylchlorosilane, (γ-methacryloxypropyl)trimethoxysilane, chlorodimethyloctylsilane (OCT), and 7-octenyltrimethoxysilane were evaluated as silanizing reagents in combination with poly(vinylpyrrolidone) (PVP) and polyacrylamide (PA) as the polymeric coat. The HDA results were compared with those obtained using a commercial (FC) coated capillary. Of these, the OCT−PVP combination was found to be most effective. Using this modified capillary, HDA with polymer networks that included hydroxyethylcellulose (HEC), linear polyacrylamide, and PVP showed that a PVP-, PA-, or FC-coated capillary, in combination with HEC as the sieving polymer, could be used effectively to discriminate the mutations in less than 10 min. However, optimal performance was observed with the OCT−PVP-coated capillary and HEC as the polymer network.Analytical Chemistry 10/2000; 72(21). DOI:10.1021/ac0004916 · 5.83 Impact Factor
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ABSTRACT: Dietary nutrients can influence cancer risk by inhibiting or enhancing carcinogenesis through diverse mechanisms of action. The identification and elucidation of their sites of action have been a focus of nutrition and cancer research for more than four decades. Transforming nutrition and cancer research from a predominantly observational to a molecular approach offers exciting opportunities for truly identifying those who will and will not benefit from dietary intervention strategies. The emerging field of nutritional genomics, defined here as the study of any genetic or epigenetic interaction with a nutrient, will be key to this evolution. Unraveling which genetic upregulation or downregulation leads to subsequent phenotype changes will not be easy. There is evidence that genetic polymorphisms can influence the dynamics between nutrients and molecular targets and, thus, contribute to variation in response among individuals. Because many molecular targets will likely be identified, it may be necessary to credential nutrients, that is, to determine which specific nutrient-related genetic and epigenetic changes bring about phenotypic changes, to establish which interactions are the most important and under what circumstances. Vitamin D, calcium, folate, selenium, genistein, and resveratrol are highlighted, because they represent specific classes of nutrients and illustrate the need to credential various nutrients to understand their physiological significance in cancer prevention. As the science of nutrition unfolds, a clearer understanding will emerge about how nutrients can modulate cancer risk through molecular interactions and how foods might be changed by agronomic approaches and/or biotechnology. Undeniably, embracing new genomic technologies offers exciting opportunities for advances in the broad area of nutrition, especially those related to cancer prevention.Nutrition and Cancer 02/2001; 41(1-2):1-16. DOI:10.1080/01635581.2001.9680606 · 2.47 Impact Factor
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ABSTRACT: Detection of mutations by gel electrophoresis and allele-specific amplification by PCR (AS-PCR) is not easily scaled to accommodate a large number of samples. Alternative electrophoretic formats, such as capillary electrophoresis (CE) and microchip electrophoresis, may provide powerful platforms for simple, fast, automated, and high-throughput mutation detection after allele-specific amplification. DNA samples heterozygous for four mutations (185delAG, 5382insC, 3867G-->T, and 6174delT) in BRCA1 and BRCA2, and homozygous for one mutation (5382insC) in BRCA1 and two mutations (16delAA and 822delG) in PTEN were chosen as the model system to evaluate the capillary and microchip electrophoresis methods. To detect each mutation, three primers, of which one was labeled with the fluorescent dye 6-carboxyfluorescein and one was the allele-specific primer (mutation-specific primer), were used to amplify the DNA fragments in the range of 130-320 bp. AS-PCR was combined with heteroduplex (HD) analysis, where the DNA fragments obtained by AS-PCR were analyzed with the conditions developed for CE-based HD analysis (using a fluorocarbon-coated capillary and hydroxyethylcellulose). The CE conditions were transferred into the microchip electrophoresis format. Three genotypes, homozygous wild type, homozygous mutant, and heterozygous mutant, could be identified by CE-based AS-PCR-HD analysis after 10-25 min of analysis time. Using the conditions optimized with CE, we translated the AS-PCR-HD analysis mutation detection method to the microchip electrophoresis format. The detection of three heterozygous mutations (insertion, deletion, and substitution) in BRCA1 could be accomplished in 180 s or less. It is possible to develop a CE-based method that exploits both AS-PCR and HD analysis for detecting specific mutations. Fast separation and the capacity for automated operation create the potential for developing a powerful electrophoresis-based mutation detection system. Fabrication of multichannel microchip platforms may enable mutation detection with high throughput.Clinical Chemistry 02/2001; 47(2):173-85. · 7.77 Impact Factor