full digestion of plasmid to get only dNTPs - (Apr/15/2009 )
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,
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.Magaña AA, Wrobel K, Caudillo YA, Zaina S, Lund G, Wrobel K.
Instituto de Investigaciones Cientificas, Universidad de Guanajuato, 36000 Guanajuato, Mexico.
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.
A quibble, but perhaps important: You end up with dNMPs, not dNTPs. No 5' triphosphate.