Influence of residual uracil-DNA glycosylase activity on the electrophoretic migration of dUTP-containing PCR products.

In diagnostic amplification protocols contamination by previously amplified nucleic acids is considered the major source of false positive results. Substituting dUTP for dTTP in the PCR and initial treatment with uracil-DNA glycosylase (UNG) virtually eliminates these carryover contaminations. Subsequent procedures to visualize the amplicons or to optimize sensitivity and specificity of the test are not always fully compatible with UNG-treated PCR products. Here we describe the more pronounced influence of residual UNG activity on the migration of PCR amplification products in polyacrylamide as compared to agarose gels.

Substrate and mispairing properties of 5-formyl-2'-deoxyuridine 5'-triphosphate assessed by in vitro DNA polymerase reactions.

5-Formyluracil (fU) is one of the thymine lesions produced by reactive oxygen radicals in DNA and its constituents. In this work, 5-formyl-2'-deoxyuridine 5'-triphosphate (fdUTP) was chemically synthesized and extensively purified by HPLC. The electron withdrawing 5-formyl group facilitated ionization of fU. Thus, p K a of the base unit of fdUTP was 8.6, significantly lower than that of parent thymine (p K a = 10.0 as dTMP). fdUTP efficiently replaced dTTP during DNA replication catalyzed by Escherichia coli DNA polymerase I (Klenow fragment), T7 DNA polymerase (3'-5'exonuclease free) and Taq DNA polymerase. fU-specific cleavage of the replication products by piperidine revealed that when incorporated as T, incorporation of fU was virtually uniform, suggesting minor sequence context effects on the incorporation frequency of fdUTP. fdUTP also replaced dCTP, but with much lower efficiency than that for dTTP. The substitution efficiency for dCTP increased with increasing pH from 7.2 to 9.0. The parallel correlation between ionization of the base unit of fdUTP (p K a = 8.6) and the substitution efficiency for dCTP strongly suggests that the base-ionized form of fdUTP is involved in mispairing with template G. These data indicate that fU can be specifically introduced into DNA as unique lesions by in vitro DNA polymerase reactions. In addition, fU is potentially mutagenic since this lesion is much more prone to form mispairing with G than parent thymine.

[Preparation of 5-(125)iodine-2'-deoxyuridine triphosphate and incorporation of the labeled nucleotide into DNA by polymerase chain reaction]. / Poluchenie 5-(125)-iod-2'-dezoksiuridintrifosfata i vkiuchenie mechenogo nukleotida v DNK metodom polimeraznoi tsepnoi reaktsii.

Radioiodine was substituted into deoxyuridine triphosphate by radioiodination of 5-mercuri-2'-deoxyuridine triphosphate. The purified 125I-nucleotide was incorporated into DNA in reactions by the protocol of the polymerase chain reaction. The incorporation of the radioactive nucleotide required added thymidine triphosphate for the synthesis of full length molecules during the one minute elongation period. The iodine substituent in the DNA remained covalently bound during both heat and alkaline denaturation.

Development of a trichloroacetic acid precipitation assay for covalent adducts of thymidylate synthase.

The use of trichloroacetic acid as a protein precipitant and denaturant in the quantitative measurement of covalent complexes of thymidylate synthase is described. Enzyme inactivated with N[3H]ethylmaleimide and inhibitory ternary complex (formed with native enzyme, 5-[6-3H]fluoro-2'-deoxyuridylate, and methylenetetrahydrofolate) served as reagents which were used to establish the conditions under which trichloroacetic acid precipitation, washing, and solubilization steps provided quantitative results. The ternary complex formed by dihydrofolate reductase with [3H]methotrexate and NADPH was used as a control to assess whether tight, but noncovalent, enzyme:ligand complexes survived trichloroacetic acid precipitation. The fact that no counts above background were detected in the pellet of precipitated protein demonstrated that the noncovalent complexes were completely dissociated by this treatment. The dynamic range of linear response for the inhibitory ternary complex of thymidylate synthase spanned five orders of magnitude, and the assay detected levels of enzyme as low as 10 fmol, a value which was essentially limited by the specific radioactivity of 5-[6-3H]fluoro-2'-deoxyuridylate. The ability of the enzyme to bind 5-[6-3H]fluoro-2'-deoxyuridylate specifically, as measured by the trichloroacetic acid assay, generated a specific binding value of 13.4 nmol of enzyme/mg protein (assuming a binding ratio of 1.5 for the inhibitory ternary complex). Specific binding values were compared to specific activity values (obtained from the spectrophotometric assay) at each stage of purification of the enzyme from Lactobacillus casei and were found to give parallel results. The characteristics of the trichloracetic acid assay procedure, which exclusively detects covalent enzyme-ligand adducts, are compared to those for other ligand binding assays for thymidylate synthase.

Improved assays for DNA-polymerizing enzymes by the use of enzymatically synthesized 5-[125I]iodo-2'-deoxyuridine triphosphate, illustrated by direct quantitation of anti-HIV reverse transcriptase antibody and by serum DNA polymerase analyses.

A one-step procedure which uses enzymes in a crude extract of herpes simplex virus (HSV) type 1-infected cells to synthesize 5-[125I]iodo-2'-deoxyuridine triphosphate [( 125I]dUTP) from [125I]dU is described. The design of a one-step procedure for the purification of the product is also presented. The recovery of [125I]dUTP from [125I]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 sensitivity and specificity of [125I]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 [3H]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 [125I]dUTP was accepted as a substrate equally as well [3H]dTTP by all of the specimens at all of the concentrations tested. When the same amount of radiolabel was used, [125I]dUTP gave a sensitivity 10- to 25-fold higher than that of [3H]dTTP. The gain in sensitivity was due to the higher specific activity and a higher counting efficiency of the 125I-label compound. The use of [125I]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 half-life of the nucleic also gives a reasonable shelf-life for use in routine assays. Activity of less than 0.3 pg of HIV RT could be detected when the new substrate was used, and this made it possible to quantitate HIV RT antibodies (abs) in diluted serum samples without purifying the immunoglobulin. Analysis of 31 HIV-infected individuals showed that all of them had anti-HIV RT ab and that the amount of serum needed for 50% inhibition of the HIV RT activity corresponded to an amount of immunoglobulin 100-fold smaller (i.e., 0.02-31.4 micrograms) than has been previously reported. With the substrate it was also possible to detect DNAp activity in serum from healthy individuals, although a long-duration assay was required. In a long-duration assay the DNAp activity found in sera from healthy individuals was linear with respect to time, whereas the DNAp activity found in many sera from tumor patients was not. [125I]dUTP is judged to be an excellent substrate for detecting and quantifying the activity of various DNA-synthesizing enzymes and their blocking abs.

Simple separation of tritiated water and [3H]deoxyuridine from [5-3H]deoxyuridine 5'-monophosphate in the thymidylate synthase assay.

A simple micromethod was developed for the accurate measurement of the activity of dTMP synthase in rat liver crude extracts. The reaction product of dTMP synthase activity assay, i.e., tritiated water, generated by the release of tritium from carbon-5 of [5-3H]deoxyuridine 5'-monophosphate (dUMP), was separated simply by 100% KOH absorption from [5-3H]deoxyuridine (dUrd), which is the side-product by dephosphorylation of [5-3H]deoxyuridine (dUrd), which is the side-product by dephosphorylation of [5-3H]dUMP during the enzyme reaction. Tritiated water was trapped in three droplets of 100% KOH deposited on the underside of the vessels' lids, while [3H]dUrd remained in the bottom of vessels after absorption of the substrate, [5-3H]dUMP, from the reaction mixture by charcoal treatment. Under standard assay conditions in the crude extract of rat liver, the specific activities of dTMP synthase and dUMP phosphatase were 0.092 +/- 0.002 and 0.351 +/- 0.013 nmol/h/mg protein, respectively. This method was also adapted for dTMP synthase assay in crude extracts of rat hepatoma 3924A. The major advantages of this procedure are the elimination of the phosphatase activity which interferes with the estimation of dTMP synthase activity in crude extracts, one-step separation of 3H2O, high sensitivity (with a limit of detection of 10 pmol of 3H2O production), high reproducibility (less than +/- 4.3%), and capability to measure activity in small amounts of sample (30-45 micrograms protein).

High-performance liquid chromatographic purification of deoxynucleoside 5'-triphosphates and their use in a sensitive electrophoretic assay of misincorporation during DNA synthesis.

This paper describes techniques and strategies for semi-preparative high-performance liquid chromatographic (HPLC) purification of 2'-deoxynucleoside 5'-triphosphates (dNTPs). The procedure yields dNTPs that are sufficiently pure for use in a sensitive electrophoretic assay of misincorporation during DNA synthesis. Anion-exchange HPLC was used to purify the four normal dNTPs (dATP, dGTP, dCTP and dTTP), plus the chemically modified analogues, 5-BrdUTP, 5-IodUTP and 1,N6-etheno-dATP (epsilon dATP). Baseline separations were achieved by isocratic elution of dNTPs with potassium dihydrogen phosphate mobile phase. In general, the resolution of dNTPs was highly dependent on pH, although the influence of mobile phase composition on separation of dNTPs was not the same for all three HPLC packing materials used. A Hewlett-Packard diode array detector was extremely valuable in the identification of contaminating peaks and in the development of optimal mobile phase conditions for dNTP purification. The pure dNTPs were used in the electrophoretic assay of misincorporation, yielding information about the mispairing potential of the modified dNTPs. BrdUMP and IodUMP were misincorporated in place of dCMP during chain elongation catalyzed by purified DNA polymerase I of Escherichia coli. epsilon dAMP was incorporated into DNA in place of dAMP, although at much lower efficiency than dAMP.

Isolation of the covalent binary complex of 5-fluorodeoxyuridylate and thymidylate synthetase by trichloroacetic acid precipitation.

Strong chemical evidence for the existence of a covalent binary complex between 5-fluorodeoxyuridylate and thymidylate synthetase was provided by the isolation of the complex by trichloroacetic acid precipitation. This result together with that of a control experiment with N-ethymaleimide inactivated thymidylate synthetase demonstrated that only nucleotide covalently bound to the protein survived repeated washings of the precipitate. Under the conditions used, a maximum binding stoichiometry of about 0.9 was obtained for the covalent binary complex, Kd = 1.1 X 10(-5) M. Also, a binding ratio of 1.7 was obtained for the methylenetetrahydrofolate-5-fluorodeoxyuridylate-thymidylate synthetase ternary complex.

Evaluation of folylpolyglutamates by electrophoretic separation of fluorodeoxyuridylate-thymidylate synthase-methylenetetrahydrofolate complexes.

The chain length of specific reduced folylpolyglutamates has been estimated by incorporation of the 5,10-methylenetetrahydrofolate form of the cofactor into a stable ternary complex with L. casei thymidylate synthase and tritiated fluorodeoxyuridylate followed by electrophoretic separation based on charge differences in complexed polyglutamates. The method is also applicable to tetrahydrofolate polyglutamates after conversion to the active cofactor form by introduction of formaldehyde. The method can be used to analyze less than one pmole of folylpolyglutamate and can be applied to evaluation of tissue polyglutamates or to monitor relevant enzyme catalyzed reactions.