Enzymatic assay for deoxyribonucleotides?

Andrew Hobbs andrewh at uniwa.uwa.edu.au
Thu May 6 21:40:17 EST 1993


T.J.R. Cutts (tjrc1 at cus.cam.ac.uk) wrote:
: 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

Hi,  
	Sorry I haven't ever tried the technique but I would imagine you
would get quite a reasonable incorporation as background anyway, since the
poly(dA.dT) would self anneal and provide the primer.  If the 'primer'
ended with an 'A' then a 'T' would be added anyway without any extra
dATP added.  

I would imagine it would be better to design a better template/primer
combination.   Either that or use the assay as it is but using labelled
dATP in a competition type assay.



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