Polymerase Chain Reaction

Validation of Roche LightCycler Epstein-Barr Virus Quantification Reagents in a Clinical Laboratory Setting

DNA strands containing an unnatural T-triazole-T linkage have been synthesized by click DNA ligation between oligonucleotides with 3'-AZT and 5'-propargylamido dT and amplified efficiently by polymerase chain reaction (PCR) using several different polymerases. DNA sequencing of PCR amplicons and clones in two different sequence contexts revealed the presence of a single thymidine at the ligation site. The remarkable ability of thermostable polymerases to reproducibly copy DNA templates containing such an unnatural backbone opens up intriguing possibilities in gene synthesis, genetic analysis, biology, and nanotechnology.

Synthesis of high molecular weight DNA strands during S phase

Automation Highlights from the Literature

Covalently closed circular DNA from polyoma virus (component I) was injected into enucleated unfertilized eggs of Xenopus laevis together with [3H]thymidine. Polyoma DNA stimulated [3H]thymidine incorporation and part of the labelled product had the sedimentation characteristics of component I polyoma DNA. Polyoma component I DNA was prepared with a density label (5‐bromo deoxyuridine) in one strand and tritium in the other. After injection into egg cytoplasm, part of the labelled material changed buoyant density to that of normal (light) double‐stranded polyoma DNA, indicating the synthesis of new DNA strands rather than terminal addition or partial strand repair. 14C‐labelled component I polyoma DNA of hybrid buoyant density was prepared and injected together with [3H]thymidine. Part of the newly synthesized material was complementary to, and hydrogen bonded to, the heavy parental strands. Degradation of the injected DNA could not be detected suggesting that the strand synthesis observed was due to replication of the injected DNA rather than nick translation.

DNA primase synthesizes short RNA oligonucleotides that DNA polymerase alpha further elongates in order to initiate the synthesis of all new DNA strands during eukaryotic DNA replication. To develop potent and specific primase inhibitors, we combined 2'-modified sugars with bases incapable of normal Watson-Crick hydrogen bonding. The presence of a 2'-hydroxyl in either the ara or ribo configuration greatly enhances the ability of primase to polymerize a nucleotide. Further modifying the 2'-position by including both a hydroxyl and methyl group at this position greatly reduced the ability of primase to polymerize the resulting nucleotides. Replacing the base of the NTP with analogues incapable of normal Watson-Crick hydrogen bonding (benzimidazole, nitrobenzimidazole, and dichlorobenzimidazole) resulted in compounds that inhibited primase quite well and with similar potency. We synthesized arabinofuranosylbenzimidazole triphosphate (araBTP) and found that this sugar change increased inhibition by 2-4-fold relative to the ribofuranosyl analogue. AraBTP inhibited polymerization of both purines and pyrimidines, although primase polymerized only small amounts of the compound. Interestingly, even though araBTP was not readily polymerized by primase, it inhibited primase almost as potently as araATP, a compound that primase polymerizes extremely rapidly and that results in very strong chain termination. Importantly, this compound was a very weak inhibitor of and only slowly polymerized by DNA polymerase alpha, indicating that it is a specific primase inhibitor. The potential utility and mechanistic implications of these inhibitors are discussed.

Malignant tumors of the rectum are treated by neoadjuvant radiochemotherapy. This involves a combination of 5-fluorouracil (5-FU) and double stranded DNA-break (DSB)-inducing radiotherapy. Here we explored how 5-FU cooperates with DSB-induction to achieve sustainable DNA damage in colorectal cancer (CRC) cells. After DSB induction by neocarzinostatin, phosphorylated histone 2AX (γ-H2AX) rapidly accumulated but then largely vanished within a few hours. In contrast, when CRC cells were pre-treated with 5-FU, gammaH2AX remained for at least 24 hours. GFP-reporter assays revealed that 5-FU decreases the efficiency of homologous recombination (HR) repair. However, 5-FU did not prevent the initial steps of HR repair, such as the accumulation of RPA and Rad51 at nuclear foci. Thus, we propose that 5-FU interferes with the continuation of HR repair, e. g. the synthesis of new DNA strands. Two key mediators of HR, Rad51 and BRCA2, were found upregulated in CRC biopsies as compared to normal mucosa. Inhibition of HR by targeting Rad51 enhanced DNA damage upon DSB-inducing treatment, outlining an alternative way of enhancing therapeutic efficacy. Taken together, our results strongly suggest that interfering with HR represents a key mechanism to enhance the efficacy when treating CRC with DNA-damaging therapy.

A metal-mediated base pair, composed of two ligand-bearing nucleotides and a bridging metal ion, is one of the most promising components for developing DNA-based functional molecules. We have recently reported an enzymatic method to synthesize hydroxypyridone (H)-type ligand-bearing artificial DNA strands. Terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase, was found to oligomerize H nucleotides to afford ligand-bearing DNAs, which were subsequently hybridized through copper-mediated base pairing (H–CuII–H). In this study, we investigated the effects of a metal cofactor, MgII ion, on the TdT-catalyzed polymerization of H nucleotides. At a high MgII concentration (10 mM), the reaction was halted after several H nucleotides were appended. In contrast, at lower MgII concentrations, H nucleotides were further appended to the H-tailed product to afford longer ligand-bearing DNA strands. An electrophoresis mobility shift assay revealed that the binding affinity of TdT to the H-tailed DNAs depends on the MgII concentration. In the presence of excess MgII ions, TdT did not bind to the H-tailed strands; thus, further elongation was impeded. This is possibly because the interaction with MgII ions caused folding of the H-tailed strands into unfavorable secondary structures. This finding provides an insight into the enzymatic synthesis of longer ligand-bearing DNA strands.

Introduction. Retroviruses are single-stranded RNA viruses of eukaryotes. Different subfamilies have been described. Avian or murine oncoviruses induce neoplasms, whereas lentiviruses, typified by human immunodeficiency virus and spumaviruses, produce persistent infections. Lentiviral infections may cause chronic disease whereas spumaviruses are apparently non-pathogenic. The retroviral life cycle is characterized by reverse transcription of their single-stranded plus (i.e. coding) RNA genome into a double-stranded DNA intermediate that integrates into the host genome. Over the last 25 years the mechanism of reverse transcription has been studied in great detail and these studies have led to the model shown in Fig. I. The result of reverse transcription is a linear double-stranded DNA molecule with a long terminal repeat (LTR) at each extremity. Synthesis of each DNA strand by the virus-encoded reverse transcriptase requires one template switch, also called a jump: the first jump is needed for synthesis of a minus DNA strand complementary to the viral RNA, the second jump for plus DNA strand synthesis.

We have carried out a solution study of the local conformation in a hybrid-chimeric duplex of the [sequence: see text] type (where r and D represent RNA and DNA). The object of this study was to investigate the sugar conformations at the internal junction in the hybrid-DNA octamer duplex (gccaCTGC). (GCAGTGGC)--where the lower-case letters represent RNA residues. Such duplexes represent good models for Okazaki fragments in which RNA primers are covalently extended into DNA strands during DNA replication of the lagging strand. Furthermore, this particular sequence occurs during HIV-1 retrovirus reverse transcription. The chimeric RNA-DNA strand and the complementary pure DNA strand chosen for this study result from the priming of (-)-strand DNA synthesis by tRNA(Lys) and subsequent (+)-strand DNA synthesis by reverse transcriptase prior to HIV-1 retrovirus integration. Despite the unusual specificity of the RNase H activity of reverse transcriptase, which cleaves the RNA c-a phosphodiester rather than the junction a-C linkage, we found no major structural differences among the RNA c-a phosphodiester rather than the junction a-C linkage, we found no major structural differences among the RNA sugar conformations--all RNA sugars were found in the normal C3'-endo A-form conformation. Instead, we find that the first DNA residue of the chimeric strand (5C) assumes a sugar conformation in the C4'-exo to O4'-endo range (P = 54-90 degrees). Furthermore, the hybrid segment of this duplex is more heteronomous than previously assumed for duplexes of the [sequence: see text] type.(ABSTRACT TRUNCATED AT 250 WORDS)

The G-quadruplex is a non-canonical DNA structure biologically significant in DNA replication, transcription and telomere stability. To date, only G4s with all guanines originating from the same strand of DNA have been considered in the context of the human nuclear genome. Here, I discuss interstrand topological configurations of G-quadruplex DNA, consisting of guanines from both strands of genomic DNA; an algorithm is presented for predicting such structures. I have identified over 550,000 non-overlapping interstrand G-quadruplex forming sequences in the human genome—significantly more than intrastrand configurations. Functional analysis of interstrand G-quadruplex sites shows strong association with transcription initiation, the results are consistent with the XPB and XPD transcriptional helicases binding only to G-quadruplex DNA with interstrand topology. Interstrand quadruplexes are also enriched in origin of replication sites. Several topology classes of interstrand quadruplex-forming sequences are possible, and different topologies are enriched in different types of structural elements. The list of interstrand quadruplex forming sequences, and the computer program used for their prediction are available at the web address http://moment.utmb.edu/allquads.

Genetic material present in hospital wastewaters: Evaluation of the efficiency of DNA denaturation by ozonolysis and ozonolysis/sonolysis treatments

Background:It is estimated that about 370 million people are chronic carriers of HBV worldwide. Apparently 3% of Iranian populations are chronic carriers of this virus. We aimed to evaluate the viral DNA in biological fluids of chronic hepatitis patients compared to a control group.Methods:The current case–control study was designed to evaluate the viral DNA in biological fluids of 70 chronic hepatitis patients compared to a control group using ELISA, PCR and Real Time.Results:All individuals (100%) in case group were HBsAg positive while in control group only 2 individuals (2.8%) were HBsAg positive. Three individuals, in control group were positive using PCR and Real Time PCR indicating that about 7% of those in control group were chronic carriers of HBV. The interesting point was the copy of viral DNA; (5.49 ×104, 2.162×103 and 7.26×106) for 3 chronic carriers using sera while it was about (5.71×103, 1.45×102 and 2.56×105) using ear cerumen confirming the necessity of investigating for the carriers of HBV in different biological fluid and by different methods.Conclusion:It can be concluded chronic carriers of hepatitis B are much more than what is diagnosed by routine diagnostic tests. On the other hand ELISA alone can not be relied on as a complete test for screening of chronic carriers in hepatitis B. PCR and Real Time PCR are more reliable tests for this purpose.

C8-Arylamine-dG and C8-arylamine-dA adducts have been prepared using palladium cross-coupling chemistry. These adducts were subsequently converted into the corresponding 5'-O-DMTr-C8-arylamine-3'-O-phosphoramidites and then used for the automated synthesis of different site-specifically modified oligonucleotides. These "damaged" oligonucleotides have been characterized by ESI-MS, UV thermal stability assays, and circular dichroism, and they have been used in EcoRI assays as well as in primer extension studies using various DNA polymerases.

Detection of genetically modified material in feed and foodstuffs containing soy and maize in Jordan