Hydroxyapatite chromatography of short single-stranded DNA.
Short single-stranded segments of calf thymus DNA were obtained by random cleavage with DNAase I. After treatment with various concentrations of DNAase I, fragment sizes were estimated using the ratio of total to terminal phosphorus. DNA populations ranging from 4--180 bases were obtained. Fragments with lengths up to 1140 were generated by shearing in a Virtis homogenizer. The hydroxyapatite elution profiles of sized populations were determined by elution with phosphate gradients. A curve relating elution molarities to single-strand chain length was 'biphasic', with the elution molarity being extremely sensitive to chain lengths below 50 nucleotides but much less sensitive to chain lengths above 100 nucleotides. These results show that single-stranded fragments below 50 nucleotides elute from hydroxyapatite appreciably before high molecular-weight denatured DNA using phosphate gradients. This is an important consideration when using hydroxyapatite to fractionate DNA populations which contain short single strands.
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D004277:DNA, Single-Stranded / Q000302:isolation & purification*; D006882:Hydroxyapatites; D008970:Molecular Weight
D004277:DNA, Single-Stranded; D006882:Hydroxyapatites