TECHNIQUES FOR ACCELERATING DNA RENATURATION AND THEIR PRELIMINARY APPLICATION TO GENE ISOLATION METHODS.
Item
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Title
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TECHNIQUES FOR ACCELERATING DNA RENATURATION AND THEIR PRELIMINARY APPLICATION TO GENE ISOLATION METHODS.
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Identifier
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AAI8222990
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identifier
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8222990
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Creator
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WIEDER, ROBERT.
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Contributor
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James G. Wetmur
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Date
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1982
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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Optimum methods for the acceleration of DNA renaturation were investigated. Renaturation rates in solution are effectively accelerated a maximum of ten fold by the addition of NaCl, LiCl, dextran sulfate, NaCl and dextran sulfate or LiCl and dextran sulfate to 2.4 M tetraethylammonium chloride. Acceleration of 100-fold may be achieved by volume exclusion with 35-40% dextran sulfate in 1 M NaCl at 70(DEGREES)C. Renaturation kinetics remain second order and temperature dependent, but become independent of dextran sulfate concentration if sufficient dextran sulfate is used. Dextran sulfate may be selectively precipitated by use of 1 M CsCl.;Reassociation kinetics of DNA at high concentrations on the surface of a phenol emulsion were also investigated. Apparent second order rate constants fall on two intersecting straight lines when presented as a function of DNA concentration on a log-log plot. The intersection occurs when the available catalytic surface is saturated. The slopes of these lines -0.3 and -1.3 for the low and high concentration ranges respectively are the same in different solvents and at different temperatures. At high DNA concentration, high complexity heterologous denatured DNA apparently competes 2-4 times better for the surface than homologous DNA because it does not participate in the reassociation reaction. Native and partially native DNA molecules cannot compete with single-stranded DNA for a saturated surface. At high DNA concentrations, rates become inversely dependent on the single-strand DNA length.;Both methods of accelerating DNA reassociation rates were employed in experiments carried out in attempts to isolate sequences specific for the Y chromosome of the rat. Three potential batch methods for the isolation of any insertion were attempted. They were deamination of female rat DNA and endonuclease S1 cleavage of the male/female heteroduplexes, BND chromatography of sonicated female DNA/restricted male DNA heteroduplexes and CsCl density gradient centrifugation of the products of renaturation of density-labeled female rat DNA and unmodified male rat DNA. Extensive damage was suffered by the DNA during long renaturation steps with either rate acceleration technique. This prevented significant enrichment of Y-specific sequences. The CsCl density gradient method was found to be useful for separating less complex DNA fragments.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biomedical Sciences
