Enzymatic assay for deoxyribonucleotides?

T.J.R. Cutts tjrc1 at cus.cam.ac.uk
Thu May 6 04:35:58 EST 1993


I am trying to assay the levels of deoxypurine nucleotides in mammalian cells,
following the method outlined in (1, 2).  The assay works by incoroprating
labelled nucleotides into DNA (using Pol I), the limiting factor being your
supplied sample of one nucleotide, so if you're measuring dATP, you use a
poly[dA.dT] template with hot dTTP.  The amount of dATP in your sample is thus
proportional to the amount of labelled DNA produced, once precipitated, washed
and counted.  The assay also includes AMP to inhibit any phosphatases.

At present I am trying to get some standard curves before attempting to measure
cell extracts, with little success.  I end up with large amounts of label on
the filters, even if no dATP is supplied.  I'm using 3MM filters, and washing
three times with 5%TCA/1%Na4P2O7, and then twice with 95% ethanol, all washes
ice cold.

Has anyone tried this assay and got it to work?  Thanks all...

Tim.

References:

1.  Hunting D, Henderson JF (1982) Methods Cancer Res 20 p. 245
2.  Wilkinson YA, McKenna PG (1989) Leukemia Research 13/7 pp. 615-620



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