Sizing of single-stranded regions in double-stranded DNA by preparative benzoylated DEAE-cellulose chromatography.
The concentration of caffeine required to elute wholly single-stranded DNA from benzoylated DEAE-cellulose is proportional to the polynucleotide length. The use of benzoylated DEAE-cellulose chromatography for isolating and sizing single-stranded regions in double-stranded DNA has been examined using a series of hybrid molecules. Restriction fragments of the replicating form of bacteriophage luminal diameter X174 were hybridized to the intact 'plus' strand, thereby forming hybrids having single- and/or double-stranded regions in the kilobase range. A series of such hybrid preparations were subject to caffeine concentration gradient elution from benzoylated DEAE-cellulose. After logarithmic transformation, a linear relationship (R = 0.94) could be demonstrated between eluting caffeine concentration and single-stranded length, irrespective of the length of associated double-stranded regions or the location, within a given fragment, of unpaired nucleotides. Benzoylated DEAE-cellulose chromatography may therefore be used to separate and characterize, on a preparative scale, double-stranded DNA containing single-stranded regions.
Norris
M D
MD
Children's Leukaemia and Cancer Research Unit, Prince of Wales Children's Hospital, Randwick, NSW, Australia.
Stewart
B W
BW
D010584:Bacteriophage phi X 174; D002110:Caffeine; D002848:Chromatography, DEAE-Cellulose; D003636:DEAE-Cellulose / Q000031:analogs & derivatives; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis*; D009693:Nucleic Acid Hybridization
D004277:DNA, Single-Stranded; D004279:DNA, Viral; C055068:benzoylated DEAE-cellulose; D002110:Caffeine; D003636:DEAE-Cellulose; D004262:DNA Restriction Enzymes