Isolation of high-molecular-weight nucleic acids for copy number analysis using high-performance liquid chromatography.
The Nucleogen DEAE 4000-10 high-performance liquid chromatography (HPLC) column was shown to isolate tRNA, rRNA, and DNA from crude cell lysates in the presence of a denaturant. Chromosomal DNA and plasmid DNA coeluted and retention was independent of size. An HPLC technique for plasmid copy number determination was then developed, verified, and optimized. The assay was successfully applied to a recombinant yeast system. Similar results were obtained earlier for recombinant Escherichia coli. The copy number can be calculated from the ratio of plasmid DNA to rRNA, then calculated to a per-cell basis using literature and measured constants. Chromosomal DNA coeluted with plasmid DNA and so was removed prior to analysis. For recombinant systems were the rRNA content is not constant, the plasmid area along with cell concentration can be used to calculate plasmid copy number. Nucleic acids' responses were linear, and plasmid copy number results using peak ratios had a low percent standard deviation. Errors increased when the absolute plasmid area was used to calculate plasmid copy number.
Coppella
S J
SJ
Department of Chemical Engineering, University of Delaware, Newark 19716.
Acheson
C M
CM
Dhurjati
P
P
D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis*; D004274:DNA, Recombinant; D012335:RNA, Ribosomal / Q000032:analysis*; D012343:RNA, Transfer / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet
D004274:DNA, Recombinant; D012335:RNA, Ribosomal; D004247:DNA; D012343:RNA, Transfer