Nucleic acid hybridization using DNA covalently coupled to cellulose.
We describe a method for linking RNA and DNA covalently to finely divided cellulose through a diazotized aryl amine, which reacts primarily with guanine and uracil (thymine) residues of single strands. The high efficiency of coupling and high capacity of the cellulose for nucleic acid make possible a product with as much as 67 mug of nucleic acid per mg of cellulose. The product is especially suitable for hybridization experiments where very low backgrounds are important, and it is stable in 99% formamide at 80 degrees C so that hybridized nucleic acid can be recovered easily. Full length linear Simian Virus 40 (SV40) DNA, produced by cleavage of SV40(I) DNA with S1 nuclease, can be coupled to diazo cellulose with an efficiency of 80-90%, and is effective in hybridization experiments with SV40 DNA, complementary RNA synthesized in vitro from SV40(I) DNA with E. coli RNA polymerase, and the SV40-specific fraction of total RNA from SV40-infected and transformed cells. In these experiments an excess of cellulose-bound DNA was used, and the efficiency of hybridization was about 90% when ribonuclease treatment of the hybrids was omitted.
D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002482:Cellulose / Q000031:analogs & derivatives*; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004279:DNA, Viral / Q000032:analysis / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007668:Kidney; D008722:Methods; D008854:Microscopy, Electron; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000032:analysis*; D012367:RNA, Viral / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013539:Simian virus 40
D004279:DNA, Viral; D012367:RNA, Viral; D012313:RNA; D002482:Cellulose; D012260:Ribonucleases