Quantification of Cellular dNTP Levels

Cellular deoxynucleoside triphosphates were extracted from batches of 4x10⁶ differentiated native and SAMHD1 transduced U937 cells according to [40 (link)]. The dNTP levels were quantified by radiolabel incorporation assays performed using oligonucleotide templates detailed in [41 (link)] and the procedures described in [42 (link)] with the following modifications. Standard curves ranged from 0.05 to 4 pmole, 1 unit of KOD polymerase (Merck Millipore) was used in place of Thermo-Sequenase (GE Healthcare) and 2.5 μM of α-³²P-dATP was employed as an incorporation label.

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Arnold L.H., Groom H.C., Kunzelmann S., Schwefel D., Caswell S.J., Ordonez P., Mann M.C., Rueschenbaum S., Goldstone D.C., Pennell S., Howell S.A., Stoye J.P., Webb M., Taylor I.A, & Bishop K.N. (2015). Phospho-dependent Regulation of SAMHD1 Oligomerisation Couples Catalysis and Restriction. PLoS Pathogens, 11(10), e1005194.

Publication 2015

KOD polymerase Thermo-Sequenase

Assays Cellular Oligonucleotide Samhd1 Thermo sequenase Triphosphates U937 cells

Corresponding Organization : University of Manchester

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Variable analysis

independent variables

Differentiation status of U937 cells (native vs. SAMHD1 transduced)

dependent variables

Cellular deoxynucleoside triphosphate (dNTP) levels

control variables

Number of cells (4x10^6) used for dNTP extraction
Radiolabel incorporation assay using oligonucleotide templates
Standard curves ranging from 0.05 to 4 pmole
Use of 1 unit of KOD polymerase instead of Thermo-Sequenase
Use of 2.5 μM of α-32P-dATP as an incorporation label

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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