Purification of DNA-derived deoxynucleotides from leukocytes involving nuclease elution of an ion-exchange column.
A method has been developed in which the DNA of leukocytes (as the buffy coat from blood) is isolated in the form of its constituent deoxynucleotides. The steps in this method are as follows: (1) lyse the leukocytes with sodium dodecyl sulfate (SDS) and enzymatically digest the proteins and RNA, (2) remove the SDS on a non-polar adsorbent (Bio-Beads SM-4) and then trap the DNA on a quaternary amine silica cartridge, (3) wash the column with 1 M NaCl-buffer, (4) digest the DNA on the column with staphylococcal nuclease and (5) elute the digested DNA with 0.5 M NaCl-buffer and digest it further with bovine spleen phosphodiesterase II to deoxynucleotide-3'-monophosphates. From a 40-microliters sample of butty coat was obtained 126 +/- 14 micrograms (two experiments, eight sample total) of deoxynucleotides. Reversed-phase high-performance liquid chromatography, which removed the added enzymes, showed only peaks for deoxynucleotides. For comparison, the amount of deoxynucleotides obtained from the leukocytes by an automated phenol extraction procedure was 101 +/- 5.4 micrograms (one experiment in triplicate).
al-Deen
A N
AN
Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, Northeastern University, Boston, MA 02115.
Cecchini
D J
DJ
Giese
R W
RW
D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004247:DNA / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000302:isolation & purification*; D006801:Humans; D007962:Leukocytes / Q000737:chemistry*; D008836:Micrococcal Nuclease / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D003854:Deoxyribonucleotides; D004247:DNA; D008836:Micrococcal Nuclease