PMID: 8471617Apr 20, 1993Paper

Quantitative multiplex sequence analysis of mutational hot spots. Frequency and specificity of mutations induced by a site-specific ethenocytosine in M13 viral DNA

Biochemistry
V A PalejwalaM Z Humayun

Abstract

We describe an assay for determining the frequency and specificity of mutations occurring at hot spots within a population of DNA molecules. The procedure consists of (a) annealing the DNA population with a labeled oligonucleotide designed to prime DNA synthesis at the mutational hot spot; (b) DNA elongation in the presence of a single dideoxynucleoside triphosphate together with 1-3 deoxynucleoside triphosphates, and (c) quantitation of all limit elongation products by high-resolution gel electrophoresis followed by autoradiography and computing densitometry. Derivation of mutational frequency and specificity over a wide range of values is demonstrated for M13 viral DNA mixtures containing defined proportions of wild-type and mutant DNAs, as well as for M13 viral DNA populations obtained by transfection of DNA bearing a defined site-specific ethenocytosine lesion. The assay is shown to yield results similar to those obtained by laborious clone-by-clone sequencing of viral progeny. The method is not affected significantly by several tested variables and appears to be suitable for use as a quantitative assay for sequence microheterogeneity at defined positions within DNA populations. Application of the methodology demonstrates t...Continue Reading

References

Dec 15, 1977·Journal of Molecular Biology·C Coulondre, J H Miller
Feb 1, 1989·Genetics·J S JacobsenM Z Humayun
Feb 20, 1987·Journal of Molecular Biology·L M RefoloM Z Humayun
Mar 1, 1984·Proceedings of the National Academy of Sciences of the United States of America·T A Kunkel
Mar 1, 1961·Proceedings of the National Academy of Sciences of the United States of America·S Benzer

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Citations

Jul 19, 1996·Molecular & General Genetics : MGG·G Wang, M Z Humayun
Mar 5, 1999·Mutation Research·J NairH Bartsch
Jun 1, 1995·Trends in Microbiology·P PuccettiF Bistoni
May 25, 2011·Journal of the American Chemical Society·Camila Carrião M GarciaMarisa H G Medeiros
Apr 23, 2002·Molecular Microbiology·Robert DoraziM Zafri Humayun
Aug 5, 1997·Proceedings of the National Academy of Sciences of the United States of America·S P FinkL J Marnett
Nov 13, 1998·Proceedings of the National Academy of Sciences of the United States of America·B HangB Singer
Jun 3, 2009·Hepatology : Official Journal of the American Association for the Study of Liver Diseases·Ying WangHelmut K Seitz
Sep 3, 2005·Biochemical and Biophysical Research Communications·Jagadeesan NairHelmut Bartsch
Aug 15, 2012·The Journal of Physical Chemistry. B·Venkatesan SrinivasadesikanShyi-Long Lee
Jan 28, 1999·Journal of Bacteriology·L RenM Z Humayun
Dec 1, 1996·Journal of Bacteriology·H S MurphyM Z Humayun

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