The use of dUTP also offered technical advantages over alternative substrates available, such as product separation without acid precipitation and exclusion of the need for scintillation cocktails. The gain in sensitivity was due to the higher specific activity and a higher counting efficiency of the 125I-label compound. When the same amount of radiolabel was used, dUTP gave a sensitivity 10- to 25-fold higher than that of dTTP. The dUTP was accepted as a substrate equally as well dTTP by all of the specimens at all of the concentrations tested. The sensitivity and specificity of dUTP as a substrate for both DNA-dependent DNA polymerase (DNAp) and RNA-dependent DNA polymerase (reverse transcriptase, RT) were evaluated and compared to those of dTTP for the following specimens: purified cloned Klenow fragment, crude extracts of HeLa-, BHK-, and HSV-2-infected BHK cells, purified avian myeloblastosis virus RT, and purified cloned human immunodeficiency virus (HIV) RT. The recovery of dUTP from dU varied between 50 and 75%, the radiochemical purity of the product was greater than 90%, and both synthesis and purification were completed within 8 h. The design of a one-step procedure for the purification of the product is also presented. A one-step procedure which uses enzymes in a crude extract of herpes simplex virus (HSV) type 1-infected cells to synthesize 5-iodo-2'-deoxyuridine triphosphate dUTP) from dU is described.
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