Purification of DNA-derived deoxynucleotides from leukocytes involving nuclease elution of an ion-exchange column
| Autor: | Amr Nour Al-Deen, Roger W. Giese, Douglas J. Cecchini |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
Nuclease
Chromatography Lysis biology Elution Organic Chemistry Ion chromatography Deoxyribonucleotides Phenol extraction General Medicine DNA Chromatography Ion Exchange Biochemistry Analytical Chemistry chemistry.chemical_compound chemistry biology.protein Leukocytes Humans Micrococcal Nuclease Spectrophotometry Ultraviolet Sodium dodecyl sulfate Chromatography High Pressure Liquid Micrococcal nuclease |
| Zdroj: | Journal of chromatography. 600(2) |
| Popis: | 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). |
| Databáze: | OpenAIRE |
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