Large scale isolation of genes as DNA fragment lengths by continuous elution electrophoresis through an agarose matrix.
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 CEE should also prove useful in other research areas requiring gene isolation, such as genomics and molecular biology.
Rodriguez
H
H
Department of Biology, Beckman Research Institute of the City of Hope, Duarte, CA, USA.
Akman
S A
SA
D004247:DNA / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D016158:Genes, p53; D006801:Humans; D008297:Male; D012150:Polymorphism, Restriction Fragment Length; D012685:Sepharose
D004247:DNA; D012685:Sepharose