Phenolic extraction of DNA from mammalian tissues and conversion to deoxyribonucleoside-5'-monophosphates devoid of ribonucleotides.
Towards a goal of detecting scaled-up DNA adducts as altered deoxynucleotides by mass spectrometry, we have set up a practical and general method for isolating DNA-derived deoxyribonucleoside-5'-monophosphates devoid of ribonucleotides starting with a 1 g sample of mammalian tissue. The method is practical because costs have been minimized, and it is general because it can be applied to a more difficult sample such as mouse skin or non-fresh calf liver. The procedure, consisting of a series of steps that were largely gleaned and tuned from prior literature, proceeds as follows: (1) homogenize the tissue in sodium dodecyl sulfate; (2) digest with ribonuclease A, ribonuclease TI, alpha-amylase and proteinase K; (3) partition between water and phenol; (4) precipitate the DNA with ethanol followed by redissolving and dialysis; and (5) digest with nuclease P1 and phosphodiesterase I followed by ultrafiltration and boric acid gel chromatography. The yellow to brown color of DNA from difficult tissues only persisted up to the ultrafiltration step. Apparently this DNA was contaminated with iron-containing proteins. Residual ribonucleotides were not observable (<0.1%) by HPLC in the final sample. Without boric acid gel chromatography, residual contamination by ribonucleotides was about 1% even when the DNA was purified before digestion by phenol partitioning followed by use of a Genomic Tip kit from Qiagen.
Shimelis
Olga
O
Department of Pharmaceutical Sciences in the Bouve College of Health Sciences, Barnett Institute and Chemistry Department, Northeastern University, Boston, MA 02115, USA.
Zhou
Xiaojuan
X
Li
Guodong
G
Giese
Roger W
RW
D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000302:isolation & purification*; D003853:Deoxyribonucleosides / Q000737:chemistry*; D051379:Mice; D010636:Phenols / Q000737:chemistry*; D012265:Ribonucleotides / Q000737:chemistry*
D003853:Deoxyribonucleosides; D010636:Phenols; D012265:Ribonucleotides; D004247:DNA