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378 results on '"Thilly, William G."'

102. Scanning Low-Frequency Point Mutants in the Mitochondrial Genome Using Constant Denaturant Capillary Electrophoresis.

Author

Walker, John M., Copeland, William C., Zheng, Weiming, Marcelino, Luisa A., and Thilly, William G.

Abstract

The ability to measure rare mutational events in mitochondrial genomes from human blood and tissues without resorting to phenotypic selection is invaluable. It is essential for the study of the cause(s) of mitochondrial mutation and for the identification of mutations associated with diseases. We have developed a highly sensitive technique termed constant denaturant capillary electrophoresis (CDCE) (1), permitting measurement of mitochondrial point mutations at fractions as low as 106 in human cells and tissues (2). CDCE is based on the earlier development of denaturing gradient gel electrophoresis (DGGE) by Fischer and Lerman (3) and constant denaturing gradient gel electrophoresis (CDGE) by Hovig (4). When a DNA consists of two contiguous "isomelting domains" with different melting temperatures, under partially denaturing condition it alternates in rapid equilibrium between the double helix state and the partially melted state. The later form has an Y-shaped configuration and, thus, lower mobility through the gel matrix than the double helix. The distribution of the "fast" and "slow" states governs the velocity under electrophoretic conditions. Base changes in the low-melting domain result in changes in the melting behavior and, hence, the mobility of the sequence under the denaturing condition of electrophoresis, allowing variants to be separated from each other. Unlike DGGE and CDGE, CDCE employs replaceable linear (uncrosslinked) polyacrylamide and capillary electrophoresis (1). A zone of elevated temperature along the capillary maintains the constant denaturing condition necessary for DNA separation. The result is fast separation, improved resolution, and higher sensitivity (1). [ABSTRACT FROM AUTHOR]

Published
2002
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103. Applications of Constant Denaturant Capillary Electrophoresis and Complementary Procedures.

Author

Walker, John M., Mitchelson, Keith R., Cheng, Jing, Kim, Andrea S., Li-Sucholeiki, Xiao-Cheng, and Thilly, William G.

Abstract

Constant denaturant capillary electrophoresis (CDCE) separates macromolecules based on differences in their melting temperatures. The specific apparatus and operating conditions have been described previously that allow CDCE to separate point mutants among 100-150-bp iso-melting DNA sequences (1,2). CDCE coupled with high-fidelity DNA polymerase chain reaction (PCR) has been applied to the measurement of point mutational spectra in human cell and tissue samples (3-4). For reviews of this field seerefs. 7,8. This chapter describes additional techniques which when combined with CDCE and high-fidelity PCR allow point mutation detection at fractions as low as 10−6. [ABSTRACT FROM AUTHOR]

Published
2001
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104. High Resolution Analysis of Point Mutations by Constant Denaturant Capillary Electrophoresis (CDCE).

Author

Walker, John M., Mitchelson, Keith R., Cheng, Jing, Khrapko, Konstantin, Coller, Hilary A., Li-Sucholeiki, Xiao-Cheng, André, Paulo C., and Thilly, William G.

Abstract

In recent years, the need for techniques capable of detecting and identifying point mutants has increased dramatically in the fields of genomics, cancer research, and molecular diagnostics. The large arsenal of methods for mutation detection ranges from direct sequencing and array hybridization to allele-specific polymerase chain reaction (PCR). CDCE (1) holds a unique position within this list of techniques due to its high flexibility. Having set up a CDCE system, one can use it to solve a wide range of tasks. At one extreme, CDCE is capable of detecting mutants at very low frequencies (as low as 10-6) (2,3), in which it rivals the high sensitivity mutation detection methods like allele specific PCR. At the other extreme, CDCE is capable of identifying mutants with precision comparable to that of direct sequencing (4). In addition, the instrument for CDCE does not need to be dedicated to one purpose. In fact, most capillary DNA electrophoresis systems, such as DNA sequencing capillary instruments, can be adapted for CDCE separations. [ABSTRACT FROM AUTHOR]

Published
2001
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107. Single nucleotide polymorphism spectra in newborns and centenarians: identification of genes coding for rise of mortal disease1Published in conjunction with A Wisconsin Gathering Honouring Waclaw Szybalski on the occasion of his 75th year and 20 years of Editorship-in-Chief of Gene, 10–11 August 1997, University of Wisconsin, Madison, WI, USA.1

Author

Tomita-Mitchell, Aoy, primary, Muniappan, Brindha P., additional, Herrero-Jimenez, Pablo, additional, Zarbl, Helmut, additional, and Thilly, William G., additional

Published
1998
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108. Mutation analyses of KRAS exon 1 comparing three different techniques: temporal temperature gradient electrophoresis, constant denaturant capillaryelectrophoresis and allele specific polymerase chain reaction

Author

Bjørheim, Jens, primary, Lystad, Sigrid, additional, Lindblom, Annika, additional, Kressner, Ulf, additional, Westring, Sophia, additional, Wahlberg, Siobhan, additional, Lindmark, Gudrun, additional, Gaudernack, Gustav, additional, Ekstrøm, Per, additional, Røe, Janne, additional, Thilly, William G., additional, and Børresen-Dale, Anne-Lise, additional

Published
1998
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122. The Human Genomic Melting Map.

Author

Fang Liu, Tøstesen, Eivind, Sundet, Jostein K., Jenssen, Tor-Kristian, Bock, Christoph, Jerstad, Geir Ivar, Thilly, William G., and Hovig, Eivind

Subjects
GENOMICS, CELLS, DNA, MACROMOLECULES, CHROMATIN, DNA replication, DNA repair, GENOMES
Abstract

In a living cell, the antiparallel double-stranded helix of DNA is a dynamically changing structure. The structure relates to interactions between and within the DNA strands, and the array of other macromolecules that constitutes functional chromatin. It is only through its changing conformations that DNA can organize and structure a large number of cellular functions. In particular, DNA must locally uncoil, or melt, and become single-stranded for DNA replication, repair, recombination, and transcription to occur. It has previously been shown that this melting occurs cooperatively, whereby several base pairs act in concert to generate melting bubbles, and in this way constitute a domain that behaves as a unit with respect to local DNA single-strandedness. We have applied a melting map calculation to the complete human genome, which provides information about the propensities of forming local bubbles determined from the whole sequence, and present a first report on its basic features, the extent of cooperativity, and correlations to various physical and biological features of the human genome. Globally, the melting map covaries very strongly with GC content. Most importantly, however, cooperativity of DNA denaturation causes this correlation to be weaker at resolutions fewer than 500 bps. This is also the resolution level at which most structural and biological processes occur, signifying the importance of the informational content inherent in the genomic melting map. The human DNA melting map may be further explored at http://meltmap.uio.no. [ABSTRACT FROM AUTHOR]

Published
2007
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132. Optimization of environment for high density vero cell culture: effect of dissolved oxygen and nutrient supply on cell growth and changes in metabolites

Author

Nahapetian, Ara T., Thomas, James N., and Thilly, William G.

Abstract

This study was initiated for optimization of the environment of a technologically useful mammalian cell line for high density production. Cultures of Vero cells on microcarriers were perfused with 100%, 50%, 25% and 12-5% modified L15 media (galactose was replaced with 10 mM-fructose, with 4 HIM-glutamine and 5% foetal bovine serum) in phosphate-buffered saline at either 4 or 8 vol. day−1. Cell growth, pH, dissolved oxygen, and changes in the metabolites, lactate to pyruvate and lactate to ammonia indices, demonstrated that under the conditions used in the present study, perfusion of cultures with 50% L15 medium in PBS at 8 vol. day−1’ provided the optimum microenvironment for Vero cell growth. The highest cell density in the perfused cultures was 3×107 cells ml−1, which at these conditions was ten times higher than the maximum cell density (3×106 cells ml−1’) obtained in a batch culture. Nutrient supply and conditioning factors were the most probable growth-limiting factors in cultures that were perfused with 12·5% and 25% L15 media, while multilayering, limitation of available oxygen, and accumulation of metabolic endproducts in the cellular microenvironment were the most probable causes of a densitydependent inhibition of cell growth observed under the optimized and overfed (supply of 100% L15 medium at the rate of 8 vol. day−1’) culture conditions. Under the optimized environmental condition, the major source of energy was probably glutamine during the first week. However, significant utilization of fructose became evident at higher cell densities during the second week, when lactate production dramatically declined and reached an almost undetectable level, while respiration progressively assumed the predominant role in energy production. It is postulated that ‘available’ oxygen in the multicell-layered microenvironment of the optimized cultures was higher than in the overfed culture due to the greater utilization rate of oxygen for oxidation of excess nutrients in the overfed culture.

Published
1986
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134. Use of constant denaturant capillary electrophoresis of pooled blood samples to identify single-nucleotide polymorphisms in the genes (Scnn1a and Scnn1b) encoding the alpha and beta subunits of the epithelial sodium channel

Author

Xue, Ming-Zhan, Bonny, Olivier, Morgenthaler, Stephan, Bochud, Murielle, Mooser, Vincent, Thilly, William G., Schild, Laurent, and Leong-Morgenthaler, Phaik-Mooi

Subjects
urogenital system, respiratory system, Polymorphism, Single Nucleotide
Abstract

The epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. Mutations in the Scnn1b and Scnn1g genes, which encode the beta and the gamma subunits of ENaC, cause a severe form of hypertension (Liddle syndrome). The contribution of genetic variants within the Scnn1a gene, which codes for the alpha subunit, has not been investigated.

136. Multistage carcinogenesis and the fraction at risk

Author

Morgenthaler, Stephan, Herrero, Pablo, Thilly, William G., Morgenthaler, Stephan, Herrero, Pablo, and Thilly, William G.

Abstract

Multistage carcinogenesis models describe the evolution of the cells in an individual's organ from a normal stage to a pre-neoplastic stage to a neoplastic stage. The triggers for the passage from one stage to the next one are presumed to be genetic alterations, which are not only governed by purely random events but also by individual environmental and genetic factors. We generalize existing models of carcinogenesis to populations composed of heterogeneous individuals, thus taking the environmental and genetic variability into account

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