DNA Polymerase Reaction Research Articles

A simple DNA extraction and rapid specific identification technique for single cells and early embryos of two breeds of Bos taurus

In the process of nuclear transfer (NT), different cytoplasm from a donor cell and a recipient oocyte are mixed. However, it is unclear what effect the donor cytoplasm has upon the dedifferentiation of donor nuclei in enucleated ooplasm and upon subsequent production of live cloned offspring. Mitochondria are component parts of cytoplasm so the detection of mitochondrial DNA is helpful to reveal changes of donor cytoplasm in the NT reconstructed embryos. In this study, the experiments were designed to develop efficient DNA extraction techniques and specific primer pairs for mitochondrial DNA of Holstein and Chinese Yellow breeds in order to identify the changes of donor cytoplasm in early stage embryos. Firstly, by adding Triton X-100 and Taq DNA polymerase reaction buffer to the DNA extraction mixture, DNA was rapidly isolated from single diploid cells, single oocytes, early stage embryos and from single hairs. Secondly, two specific primer pairs for the two breeds were designed to detect the cytoplasmic DNA in a different amount of single cells and in early stage embryos. The results show that two specific fragments were successfully amplified from single somatic cells, single oocytes, parthenogenetic embryos and from NT reconstructed embryos. As a result, the techniques provide a powerful tool for studying the developmental mechanism in NT reconstructed embryos.

The READIT assay as a method for genotyping NAT1*10 polymorphisms.

Polymorphisms in the N-acetyltransferases (NATs) have been associated with increased risks for the development of a variety of cancers. The NAT1*10 allele, for example, has been reported to be associated with an increased risk of colon and urinary bladder cancers, among others. Therefore, considerable effort is being placed on the development of genotyping methodologies for NAT activities both for pharmacological as well as disease preventative applications. Most NAT genotyping approaches are gel based and consist of PCR-restriction fragment length polymorphism (RFLP) analysis, allele-specific PCR, or both. Although these approaches have their utility, they are slow, labor intensive, and are not amenable to automation. Recently, a novel approach to genotyping known as the READIT Assay has been introduced. The READIT methodology involves a reversal of the DNA polymerase reaction to generate dNTPs through the phosphorolytic cleavage of oligonucleotide probe molecules annealed to target DNAs. In a coupled reaction, kinase converts the resulting dNTPs to ATP. ATP production is then monitored by the addition of luciferase, generating a light signal proportional to the amount of dNTPs generated through probe depolymerization. We describe the development of a READIT genotyping protocol for the analysis of NATs using the NAT1*10 allele as a model system and demonstrate its utility for the analysis of archival dried blood specimens. We applied this technology to genotype 678 DNAs at the NAT-1088T --> A polymorphic site, and 680 DNAs for the 1095C --> A polymorphism. We report complete concordance for the 1088T --> A polymorphism for all 678 genotypes previously determined by RFLP analysis.

Syntheses and structure-activity relationships of nonnatural beta-C-nucleoside 5'-triphosphates bearing an aromatic nucleobase with phenolic hydroxy groups: inhibitory activities against DNA polymerases.

Five nonnatural beta-C-nucleoside 5'-triphosphates bearing a 3,4-dihydroxyphenyl (1TP), a 2-hydroxyphenyl (2TP), a 3-hydroxyphenyl (3TP), a 4-hydroxyphenyl (4TP), or a phenyl (5TP) group were synthesized, and their structure-activity relationships were examined for a series of DNA polymerase reactions in vitro under typical polymerase chain reaction conditions. We found that the 5'-triphosphates (1TP-5TP) are not incorporated into DNA strands but inhibit the DNA polymerase reactions in the presence of natural nucleoside 5'-triphosphates (dNTPs). 1TP having two phenolic hydroxy groups at the nucleobase moiety showed the most potent inhibitory effect against DNA synthesis by Ex Taq polymerase (IC(50) = 30 microM). The competition assay indicated that 1TP and dNTPs are most likely to affect DNA polymerase reactions competitively. This finding may raise the appealing possibility that artificial nucleoside 5'-triphosphates having phenolic hydroxy groups could exhibit potent inhibitory activity against DNA-directed enzymatic reactions.

A molecular beacon for quantitative monitoring of the DNA polymerase reaction in real-time.

An assay for real-time monitoring of DNA synthesis by DNA polymerases is presented. With the opening of a dual dye-labeled probe, triggered by the progressing enzymatic DNA synthesis, a fluorescent signal is generated (see scheme), enabling the high-throughput quantitative characterization of inhibitors and monitoring of the DNA polymerase reaction in cell lysates.

Synthesis and properties of oligonucleotides containing 5-formyl-2'-deoxycytidine: in vitro DNA polymerase reactions on DNA templates containing 5-formyl-2'-deoxycytidine.

Oligodeoxynucleotides (ODNs) containing 5-formyl-2'-deoxycytidine (fC) were synthesized by the phosphoramidite method and subsequent oxidation with sodium periodate. The stabilities of duplexes containing A, G, C or T opposite fC were studied by thermal denaturation. It was found that fC:A, fC:C or fC:T base pairs significantly reduce the thermal stabilities of duplexes. Next, single nucleotide insertion reactions were performed using ODNs containing fC as templates and the Klenow fragment of Escherichia coli DNA polymerase I. It was found that: (i) insertion of dGMP opposite fC appears to be less efficient relative to insertion opposite 5-methyl-2'-deoxycytidine (mC); (ii) dAMP is misincorporated more frequently opposite fC than mC, although the frequency of misincorporation seems to be dependent on the sequence; (iii) TMP is misincorporated more frequently opposite fC than mC. These results suggest that fC may induce the transition mutation C.G-->T.A and the transversion mutation C.G-->A.T during DNA synthesis.

Optimization of the PCR for Detection of Staphylococcus aureus nuc Gene in Bovine Milk

Staphylococcus aureus is an economically important and a major mastitis-causing pathogen that also poses food safety and antimicrobial resistance threats. Substances in mastitic milk inhibit the Taq DNA polymerase reaction (Taq PCR) making it of limited use for detecting S. aureus mastitis. In the study reported here, a set of oligonucleotide primers of 21 and 24 bases was used in Taq-PCR to amplify DNA from S. aureus (isolates from bovine mastitis). A specific amplicon of 270 bp was generated as predicted. Replacing Taq DNA polymerase with Thermus thermophilus (Tth) DNA polymerase alone (Tth-PCR) raised the sensitivity of S. aureus detection in milk from experimentally infected cows from 65 to 80%. Combining the use of Tth DNA polymerase and the purification of crude DNA extract using Chelex-100 before PCR raised the sensitivity to 100%. In a random survey involving 100 milk samples from cattle not infected with S. aureus, the test was 100% specific. With milk samples from clinical cases of bovine mastitis, 100% sensitivity and specificity were also observed. It is concluded that Tth-PCR on milk samples with the purification of crude DNA extracts using Chelex-100 is as sensitive as but faster than conventional milk bacteriological culture techniques and is highly specific. The modified PCR correlates with elevated somatic cell counts, detects evidence of chronic and resolving infection based on S. aureus-specific DNA and circumvents the endogenous inhibitory effects of milk.

Enzymatic properties of Escherichia coli and human 7,8-dihydro-8-oxoguanine DNA glycosylases.

Oxidative damage to DNA generates aberrant guanine bases such as 2,6-diamino-4-hydroxy-formamido-pyrimidine (Fapy) and 7,8-dihydro-8-oxoguanine (8-oxoG). Although synthetic oligonucleotides containing a single 8-oxoG have been widely used to study enzymatic processing of this lesion, the synthesis of oligonucleotides containing Fapy as a unique lesion has not been achieved to date. In this study, an oligonucleotide containing a single 2,6-diamino-4-hydroxy-5-(N-methyl)formamido-pyrimidine (me-Fapy, a methylated derivative of Fapy) was prepared by a DNA polymerase reaction and the subsequent alkali treatment. The repair activity of Fpg and hOGG1 proteins were compared using oligonucleotide substrates containing me-Fapy and 8-oxoG.

Preparation and enzymatic recognition of guanine lesions induced by nitrogen oxides.

Xanthine (Xan) and oxanine (Oxa) are the major deamination products of guanine formed by the treatment with nitrogen oxides (e.g., NO and HNO2). In this study, 2'-deoxyribonucleoside 5'-triphosphates of Xan and Oxa were prepared by the NaNO2 treatment of dGTP. These modified nucleotides were incorporated into oligonucleotides by DNA polymerase reactions. The repair activities of various DNA N-glycosylases for Xan and Oxa were examined using these substrates.

An optimized recipe for cloning of the polymerase chain reaction-amplified DNA inserts into plasmid vectors.

This study compares a number of parameters that are important in the ligation of the polymerase chain reaction-amplified DNA inserts into plasmid vectors and their efficient transformation to bacterial cells. The parameters covered were: T4 polynucleotide kinase treatment followed by either the large fragment of E. coli DNA polymerase or T4 DNA polymerase reactions, the amount of T4 DNA ligase, temperature and duration of ligation, molar ratio of insert to vector as well as the total DNA concentration. The results show that the T4 polynucleotide kinase-treated group without further enzymatic manipulation, at an insert to vector ratio of 3:1 gave the highest recombination efficiency when 10 microg/ml DNA and 20 units T4 DNA ligase were applied for ligation for 12 h at 4 degrees C.

Photoregulation of the DNA Polymerase Reaction by Oligonucleotides Bearing an Azobenzene

Zwei DNA-Moleküle von festgelegter Länge werden selektiv an einem DNA-Templat gebildet. Die templatabhängige Reaktion der T7-DNA-Polymerase kann photochemisch mit einem Oligonucleotid gesteuert werden, das eine Azobenzoleinheit als Modulator trägt (siehe schematische Darstellung).

Photoregulation of the DNA Polymerase Reaction by Oligonucleotides Bearing an Azobenzene We should like to thank Professor Kazunari Taira for valuable comments. This work was partially supported by the Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (Molecular Synchronization for Design of New Materials System). The support of the grant from the "Research for the

Photoregulation of the DNA Polymerase Reaction by Oligonucleotides Bearing an Azobenzene We should like to thank Professor Kazunari Taira for valuable comments. This work was partially supported by the Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (Molecular Synchronization for Design of New Materials System). The support of the grant from the "Research for the

Preparation of DNA containing 7-methylguanine as unique lesions.

The predominant adduct produced by both endogenous and exogenous methylating agents is 7-methylguanine(m7G). Most studies on the repair of m7G reported so far used methylated DNA as substrates which contained other unintended lesions. In the presented study, DNA substrates containing m7G as unique lesions were prepared by DNA polymerase reactions. Using these substrates, damage recognition of E. coli 3-methyladenine DNA glycosylase II (AlkA) was analyzed. The obtained results suggested that the repair rate of m7G by AlkA was affected by the flanking sequence context of the lesion.

Interactions of High Affinity Anti (6-4) Photoproduct Antibody Fragments with Damaged DNA

The interactions between chemically synthesized DNA fragments containing a T(6-4)T and antigen binding fragments (Fab) or single-chain antibodies (scFv) were investigated by X-ray crystallography, NMR, and surface plasmon resonance. The high affinity scFv protein was found to bind to the template DNA near the (6-4) photoproduct site and to interfere with DNA polymerase reactions in vitro.

Sequence Analysis of Genomic Regions Containing Trinucleotide Repeats Isolated by a Novel Cloning Method

A method was developed for effective isolation of trinucleotide repeats from genomic DNA. This method is based on the DNA polymerase reaction, which is restricted with only two or three nucleotide substrates and primed by biotinylated oligonucleotide probes. Sequences are then isolated by a streptavidin biotin-trapping method. More than 80% of the clones from each library contained more than eight trinucleotide repeats. Sequence analysis showed that the characteristic dinucleotide flanking sequences usually confronting various trinucleotide repeats are not found in the vicinity of CAG repeats, suggesting that CAG repeats may have been generated through a mechanism different from that of other trinucleotide repeats.

Effects of a High‐Affinity Antibody Fragment on DNA Polymerase Reactions Near a (6–4) Photoproduct Site

Pyrimidine (6-4) pyrimidone photodimers are major photoproducts that have mutagenic and carcinogenic consequences. One major reason for these biological effects of (6-4) photoproducts may be base mispairing/DNA replication errors due to hydrogen bonding to bases opposite these damaged sites. We synthesized a modified 41-mer DNA containing a (6-4) photoproduct using a preformed building block, then employed it as a template for primer extension reactions catalyzed by Klenow fragment and DNA polymerases alpha, beta and delta (pol alpha, pol beta and pol delta). None of these DNA polymerases were able to bypass the (6-4) photoproduct and elongation terminated at or near the 3'-pyrimidone of the photoproduct, depending on the dNTP concentration. When a single-chain Fv (scFv) with high affinity for the (6-4) photoproduct was included in the polymerization reaction, DNA synthesis was inhibited at base positions four, six, eight or eight nucleotides prior to the 3'-pyrimidone by Klenow fragment, pol alpha, pol beta or pol delta, respectively. These results suggest that the scFv can bind to the template DNA containing a (6-4) photoproduct and inhibit extension reactions by polymerases.

Sanger DNA-Sequencing Reactions Performed in a Solid-Phase Nanoreactor Directly Coupled to Capillary Gel Electrophoresis

A miniaturized, solid-phase nanoreactor was developed to prepare Sanger DNA-sequencing ladders which was directly interfaced to a capillary gel electrophoresis system. A biotinylated fragment of the rat brain actin gene (1 kbp) was amplified by PCR and attached to the interior wall of an (aminoalkyl)silane-derivatized fused-silica capillary tube via a biotin/streptavidin/biotin linkage. Coverage of the capillary wall with the biotinylated DNA averaged 77 +/- 10%. Stability of the anchored template under pressure (33 nL/s) and electroosmotic flows (11.3 nL/s) were favorable, requiring rinsing for > 150 h to reduce the surface coverage by only 50%. In addition, the immobilized template was stable toward temperatures required for preparing sequencing ladders, even under cycling conditions. Standard Sanger dideoxynucleotide termination performed in a large-volume (approximately 8 microL) solid-phase reactor using the thermally stable polymerase enzymes Taq and Vent and the polymerases T7 and Bst with off-line slab gel electrophoresis and autoradiographic detection indicated that acceptable fragment generation was achieved only in the case of the thermally stable polymerases. Banding was not apparent for T7 and Bst since all reagents were inserted into the column in a single plug at the beginning of the reaction. A small volume reactor (volume approximately 62 nL) was then used to perform DNA polymerase reactions and was coupled directly to a capillary gel column for separation. The capillary reactor was placed inside a thermocycler to control the temperature during chain extension and was directly connected to the gel column via zero dead volume fused-silica connectors. The complementary DNA fragments generated (C-track only) in the reactor were denatured using heat and directly injected onto the gel-filled capillary for size separation with detection accomplished using near-IR laser-induced fluorescence. Extension and single-base separation resolution of the C-track, which was directly injected onto the gel column, was estimated to be > 450 bases from the primer annealing site with plate numbers ranging from 1 x 10(6) to 2 x 10(6)/m.

Inhibition of DNA polymerase reactions by pyrimidine nucleotide analogues lacking the 2-keto group.

To investigate the influence of the pyrimidine 2-keto group on selection of nucleotides for incorporation into DNA by polymerases, we have prepared two C nucleoside triphosphates that are analogues of dCTP and dTTP, namely 2-amino-5-(2'-deoxy-beta-d-ribofuranosyl)pyridine-5'-triphosphate (d*CTP) and 5-(2'-deoxy- beta-d-ribofuranosyl)-3-methyl-2-pyridone-5'-triphosphate (d*TTP) respectively. Both proved strongly inhibitory to PCR catalysed by Taq polymerase; d*TTP rather more so than d*CTP. In primer extension experiments conducted with either Taq polymerase or the Klenow fragment of Escherichia coli DNA polymerase I, both nucleotides failed to substitute for their natural pyrimidine counterparts. Neither derivative was incorporated as a chain terminator. Their capacity to inhibit DNA polymerase activity may well result from incompatibility with the correctly folded form of the polymerase enzyme needed to stabilize the transition state and catalyse phosphodiester bond formation.

A Simple and Sensitive Ribonucleotide Reductase Assay

Ribonucleotide reductase (RR) is a key regulatory enzyme in the DNA synthesis pathway and is the target of the cancer chemotherapeutic agent hydroxyurea. The study of RR is significantly hindered by the tedious and labor-intensive nature of enzymatic assay. In this report, we present a novel RR assay in which detection of the deoxyribonucleotides produced by RR occurs via coupling to the DNA polymerase reaction, and is enhanced by using RNase to degrade endogenous RNA. Cell extracts from various cell lines were treated with RNase and then reacted with ATP and radioactive ribonucleotide diphosphate as the substrate. Incorporation of the radioactive substrate [¹⁴C]CDP into DNA was linear over 30 min and was linear with the amount of extract, which provided RR activity. The reaction was inhibited by hydroxyurea and required Mg²⁺ and ATP, suggesting that the assay is specific to RR activity. While RR activities determined by our method and by a conventional method were comparable, this novel method proved to be simpler, faster, more sensitive and less expensive. In addition, assay of the RR activity for multiple samples can easily be performed simultaneously. It is superior to other RR assays in all aspects.

Kinetic equation for template-dependent polymerase reactions and its application to Thermus thermophilus DNA polymerase

A kinetic equation was derived for complex DNA polymerase reactions. The complex repertoire of polymerase reactions includes the facts that the four monomeric substrates involved in the polymerase reactions may also work as competitive inhibitors depending on their incorporation sites, the incorporation rates of each deoxynucleotide are different, and polymerization proceeds in parallel at various sites on each template DNA. Using the equation, the kinetic properties of Thermus thermophilus DNA polymerase were investigated. The reaction at the A-incorporation site is strongly inhibited by dTTP and dGTP with inhibition constants of 9.9 and 7.1 μM, respectively. The reaction at the T-incorporation site is also inhibited by dATP with an inhibition constant of 7.6 μM, while the G- and C-incorporation sites are less sensitive to competitive inhibition. When substrate concentrations are low, the incorporation rate of A is the fastest and that of C is the slowest. Accordingly, the C-incorporation step largely limits the overall reaction rate. Using the kinetic parameters determined experimentally, the optimum ratio of the concentrations of the four nucleotides was calculated.

A simple and sensitive ribonucleotide reductase assay.

Ribonucleotide reductase (RR) is a key regulatory enzyme in the DNA synthesis pathway and is the target of the cancer chemotherapeutic agent hydroxyurea. The study of RR is significantly hindered by the tedious and labor-intensive nature of enzymatic assay. In this report, we present a novel RR assay in which detection of the deoxyribonucleotides produced by RR occurs via coupling to the DNA polymerase reaction, and is enhanced by using RNase to degrade endogenous RNA. Cell extracts from various cell lines were treated with RNase and then reacted with ATP and radioactive ribonucleotide diphosphate as the substrate. Incorporation of the radioactive substrate [14C]CDP into DNA was linear over 30 min and was linear with the amount of extract, which provided RR activity. The reaction was inhibited by hydroxyurea and required Mg2+ and ATP, suggesting that the assay is specific to RR activity. While RR activities determined by our method and by a conventional method were comparable, this novel method proved to be simpler, faster, more sensitive and less expensive. In addition, assay of the RR activity for multiple samples can easily be performed simultaneously. It is superior to other RR assays in all aspects.