Large scale isolation of genes as DNA fragment lengths by continuous elution electrophoresis through an agarose matrix

Electrophoresis
H Rodriguez, S A Akman

Abstract

A simple procedure for the isolation of genes as DNA fragment lengths is described. By using a commercial continuous elution protein electrophoresis apparatus and incorporating an agarose matrix, preparative scale amounts (300 microg) of DNA can be purified by fragment lengths from a mixture of genomic fragment lengths with high recovery yields. Fractions corresponding to unique fragment length ranges are screened for individual genes by dot-blot analysis. Using this technique, we have isolated two genes: PGK1, a single copy housekeeping gene; and p53 (exons 3-11), a tumor suppressor gene, whose DNA fragment lengths elute at 4 and 7.5 kbp, respectively, from a single preparative run. As an example of the utility of the technique, we applied it to improving the sensitivity of the ligation-mediated polymerase chain reaction (LMPCR)--a nucleic acid amplification technique used for the detection and mapping of DNA damage along genes. By eliminating excess nontargeted genomic DNA, the agarose matrix continuous elution electrophoresis (CEE) procedure provided a 24-fold increase in signal strength attributable to base damage caused by exposing DNA to reactive oxygen species. Genomic DNA fragment length purification by agarose matrix C...Continue Reading

References

Jan 1, 1979·Methods in Enzymology·E Southern
Mar 11, 1994·Science·S Tornaletti, G P Pfeifer

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Citations

May 28, 2003·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Maria Teresa C P RibelaPaolo Bartolini
Nov 20, 2001·Nucleic Acids Research·E V SokurenkoS Lory
Sep 6, 2008·Canadian Journal of Microbiology·Mysore V TejesviHunthrike S Shetty
Jul 12, 2008·Analytical Biochemistry·Yutaka SadakaneYasumaru Hatanaka
Oct 6, 2000·Methods : a Companion to Methods in Enzymology·H RodriguezS P LeDoux
Aug 21, 2010·Angewandte Chemie·Patrik Neuhaus, Wolfram Sander
Mar 1, 2019·Genome Génome / Conseil National De Recherches Canada·Alba P Suaste-DzulAdrien Gallou
Mar 31, 1999·Free Radical Research·H Rodriguez, S A Akman
Feb 14, 2007·Molecular Nutrition & Food Research·Mugur N Siddaraju, Shylaja M Dharmesh
Jun 23, 2000·Annals of the New York Academy of Sciences·S A AkmanH Rodriguez

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