Hydroxyapatite-mediated transfer of DNA on nitrocellulose filters in dot-hybridization experiments
Moiseev, V.L.; Zaboĭkin, M.M.; Likhtenshteĭn, A.V.
Molekuliarnaia Biologiia 21(3): 737-742
1987
ISSN/ISBN: 0026-8984 PMID: 2821377 Document Number: 287208
The method of hydroxylapatite-mediated rapid and effective transfer of DNA onto nitrocellulose filters for following dot-hybridization was elaborated. The analysed for DNA occurred initially in diluted and large volume solutions (from 1 to 10 ml) with various composition (2 M NaCl; 4 M LiCl-8 M urea; 4 M CsCl; 5 and 20% sucrose) was adsorbed on hydroxylapatite and quantitatively transferred onto nitrocellulose after hydroxylapatite solubilization in a small volume of acid (usually, 200 microliters of 10% TCA). As examplified by the hybridization of total rat liver DNA with the plasmid ph22 DNA containing a cluster of sea urchin histone genes, the method presented appears to be not only simple and useful for handling multiple probes of diluted DNA solutions with high concentrations of salts, sucrose and urea but also more sensitive than some convenient DNA dot-hybridization methods.