full digestion of plasmid to get only dNTPs - (Apr/15/2009 )

Hello everybody,
I want to totally digest a plasmid, so that I end up only with dNTPs. There are several enzymes (exonucleases) that partially do the job, but until now I could not find one that will only produce dNTPs. DNAse I for example will produce also di- and oligonucleotides which is not suitable for my application.
A pure chemical method would also be fine, but I need the nucleotides to be intact in the end. So something that will also lead to free bases and sugars will not help.
I hope that someone can help me on this one. There has to be something easy to get this done.

Thank you in advance,
cheers,
Martin

Hi
Here is a protocol I used
You may omit the treatment with CIAP if You don't want to remove the phosphate groups.

10 μg DNA dissolved in 10 μl Tris�HCl (25 mM, pH 8.0) was treated with 10 U DNase I for 1 h at 37 �C, denatured by heating at 100 �C for 3 min, and cooled rapidly in ice. Then 1 μl nuclease P1 (5 μg), 2 μl sodium acetate (0.5 M, pH 5.2), and 2 μl ZnSO4 (1 M) were added, and mixtures were incubated at 37 �C for 16 h. Following the addition of 3 μl Tris�HCl (1 M, pH 8.0), 1.5 μl (1.5 U) CIAP, and 2 μl CIAP buffer, mixtures were incubated at 37 �C for 2 h. Samples were kept at −20 �C until analysis.

This protocol can be found in this article:
Anal Biochem. 2008 Mar 15;374(2):378-85. Epub 2007 Nov 28. High-performance liquid chromatography determination of 5-methyl-2'-deoxycytidine, 2'-deoxycytidine, and other deoxynucleosides and nucleosides in DNA digests.Maga�a AA, Wrobel K, Caudillo YA, Zaina S, Lund G, Wrobel K.
Instituto de Investigaciones Cientificas, Universidad de Guanajuato, 36000 Guanajuato, Mexico.
Good luck
Michael

Thank you very much Michaelro.
I definitely will give it a try. haven't read the paper in detail yet, but it might be exactly what I was looking for.

cheers,
Martin

A quibble, but perhaps important: You end up with dNMPs, not dNTPs. No 5' triphosphate.