NDLI: Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: evidence for solvent-induced bends in DNA heteroduplexes.

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Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: evidence for solvent-induced bends in DNA heteroduplexes.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Ganguly, A. Prockop, D. J. Rock, M. J.
Description	Author Affiliation: Ganguly A ( Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA 19107.);
Abstract	Several techniques have recently been developed to detect single-base mismatches in DNA heteroduplexes that contain one strand of wild-type and one strand of mutated DNA. Here we tested the hypothesis that an appropriate system of mildly denaturing solvents can amplify the tendency of single-base mismatches to produce conformational changes, such as bends in the double helix, and thereby increase the differential migration of DNA heteroduplexes and homoduplexes during gel electrophoresis. The best separations of heteroduplexes and homoduplexes were obtained with a standard 6% polyacrylamide gel polymerized in 10% ethylene glycol/15% formamide/Tris-taurine buffer. As predicted by the hypothesis of solvent-induced bends, when the concentration of either ethylene glycol or formamide was increased, the differential migration decreased. Also, single-base mismatches within 50 bp of one end of a heteroduplex did not produce differential migration. Sixty of 68 single-base mismatches in a series of PCR products were detected in some 59 different sequence contexts. The eight mismatches not detected were either within 50 bp of the nearest end of the PCR product or in isolated high-melting-temperature domains. Therefore, it was possible to predict in advance the end regions and sequence contexts in which mismatches may be difficult to detect. The procedure can be applied to any PCR products of 200-800 bp and requires no special equipment or preparation of samples.
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	21
Volume Number	90
Language	English
Publisher	National Academy of Sciences
Publisher Date	1993-12-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	DNA, Viral Chemistry DNA Nucleic Acid Conformation Nucleic Acid Heteroduplexes Oligodeoxyribonucleotides Polymerase Chain Reaction Bacteriophage M13 Base Composition Electrophoresis, Polyacrylamide Gel Escherichia Coli Genetics Exons Factor IX Genes, Bacterial Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Multidisciplinary
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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