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Factors affecting the kinetics of DNA reassociation in phenol–water emulsion at high DNA concentrations
Author(s) -
Wieder Robert,
Wetmur James G.
Publication year - 1982
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.360210313
Subject(s) - chemistry , dna , kinetics , reaction rate constant , emulsion , heterologous , drop (telecommunication) , endonuclease , biochemistry , biophysics , biology , gene , telecommunications , physics , quantum mechanics , computer science
DNA reassociation kinetics using the phenol emulsion reassociation technique (PERT) [Kohne, D. E., Levison, S. A. & Byers, M. J. (1977) Biochemistry 16 , 5329–5341] has been investigated at high DNA concentrations using an endonuclease S1 assay of reaction progress. Apparent second‐order rate constants fall on two intersecting straight lines when presented as a function of DNA concentrations on a log–log plot. In the low DNA concentration range, the rate constants drop about 10‐fold when concentration increases 1000‐fold. In the high DNA concentration range, the rate constants drop more than 10‐fold when concentration increases 10‐fold. The slopes of these lines are the same in different solvents and at different temperatures. The intersection between the lines occurs when the available catalytic surface is saturated. At high DNA concentrations, high‐complexity heterologous denatured DNA apparently competes 2–4 times better for the surface than homologous DNA because it does not participate in a reassociation reaction. Native and partially native DNA molecules cannot compete with single‐stranded DNA for a saturated surface. At high DNA concentrations, reactions using PERT become dependent on the single‐strand DNA length. Increasing length lowers reassociation rates.