Search results for "DNA polymerase"
showing 10 items of 83 documents
Loop Mediated Isothermal Amplification: Principles and Applications in Plant Virology
2020
In the last decades, the evolution of molecular diagnosis methods has generated different advanced tools, like loop-mediated isothermal amplification (LAMP). Currently, it is a well-established technique, applied in different fields, such as the medicine, agriculture, and food industries, owing to its simplicity, specificity, rapidity, and low-cost efforts. LAMP is a nucleic acid amplification under isothermal conditions, which is highly compatible with point-of-care (POC) analysis and has the potential to improve the diagnosis in plant protection. The great advantages of LAMP have led to several upgrades in order to implement the technique. In this review, the authors provide an overview r…
Engineering of a DNA Polymerase for Direct m6A Sequencing
2017
Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N6-methyladenosine (m6A) is the most abundant RNA modification in mammalian mRNA and is involved in the regulation of gene expression. Current detection techniques are laborious and rely on antibody-based enrichment of m6A-containing RNA prior to sequencing, since m6A modifications are generally "erased" during reverse transcription (RT). To overcome the drawbacks associated with indirect detection, we aimed to generate novel DNA polymerase variants for direct m6A sequencing. Therefore, we developed a screen to evolve an RT-active KlenTaq DNA polymerase variant that sets a mark for…
An alloherpesvirus infection of European perch Perca fluviatilis in Finland
2018
The order Herpesvirales includes viruses that infect aquatic and terrestrial vertebrates and several aquatic invertebrates (i.e. mollusks), and share the commonality of possessing a double-stranded DNA core surrounded by an icosahedral capsid. Herpesviruses of the family Alloherpesviridae that infect fish and amphibians, including channel catfish virus and koi herpes - virus, negatively impact aquaculture. Here, we describe a novel herpesvirus infection of wild European perch from lakes in Finland. Infected fish exhibited white nodules on the skin and fins, typically in the spring when prevalence reached nearly 40% in one of the sampled lakes. Transmission electron microscopic examination o…
Regulatory Interaction between the Cellular Restriction Factor IFI16 and Viral pp65 (pUL83) Modulates Viral Gene Expression and IFI16 Protein Stabili…
2016
ABSTRACT A key player in the intrinsic resistance against human cytomegalovirus (HCMV) is the interferon-γ-inducible protein 16 (IFI16), which behaves as a viral DNA sensor in the first hours postinfection and as a repressor of viral gene transcription in the later stages. Previous studies on HCMV replication demonstrated that IFI16 binds to the viral protein kinase pUL97, undergoes phosphorylation, and relocalizes to the cytoplasm of infected cells. In this study, we demonstrate that the tegument protein pp65 (pUL83) recruits IFI16 to the promoter of the UL54 gene and downregulates viral replication, as shown by use of the HCMV mutant v65Stop, which lacks pp65 expression. Interestingly, at…
Analysis of DNA Polymerases Reveals Specific Genes Expansion in Leishmania and Trypanosoma spp.
2020
Leishmaniasis and trypanosomiasis are largely neglected diseases prevailing in tropical and subtropical conditions. These are an arthropod-borne zoonosis that affects humans and some animals and is caused by infection with protozoan of the genera Leishmania and Trypanosoma, respectively. These parasites present high genomic plasticity and are able to adapt themselves to adverse conditions like the attack of host cells or toxicity induced by drug exposure. Different mechanisms allow these adapting responses induced by stress, such as mutation, chromosomal rearrangements, establishment of mosaic ploidies, and gene expansion. Here we describe how a subset of genes encoding for DNA polymerases …
Manganese Ions Individually Alter the Reverse Transcription Signature of Modified Ribonucleosides
2020
Reverse transcription of RNA templates containing modified ribonucleosides transfers modification-related information as misincorporations, arrest or nucleotide skipping events to the newly synthesized cDNA strand. The frequency and proportion of these events, merged from all sequenced cDNAs, yield a so-called RT signature, characteristic for the respective RNA modification and reverse transcriptase (RT). While known for DNA polymerases in so-called error-prone PCR, testing of four different RTs by replacing Mg2+ with Mn2+ in reaction buffer revealed the immense influence of manganese chloride on derived RT signatures, with arrest rates on m1A positions dropping from 82% down to 24%. Additi…
Machine learning of reverse transcription signatures of variegated polymerases allows mapping and discrimination of methylated purines in limited tra…
2020
AbstractReverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to trai…
Use of the polymerase chain reaction to demonstrate hepatitis B virus DNA in serum of children with chronic hepatitis B.
1992
The polymerase chain reaction was used to investigate the presence of hepatitis B virus DNA in sera of 61 children with chronic hepatitis B and negative results on dot biot hybridization tests. Our results demonstrate that most chronic carriers of hepatitis B surface antigen in childhood have hepatitis B virus DNA detectable by polymerase chain reaction in their serum and must be considered infectious.
Induction of heme oxygenase-1 and adaptive protection against the induction of DNA damage after hyperbaric oxygen treatment.
2000
Hyperbaric oxygen (HBO) treatment of human subjects (i.e. exposure to 100% oxygen at a pressure of 2.5 ATA for a total period of 3 x 20 min) caused clear and reproducible DNA damage in lymphocytes, as detected with the comet assay (single cell gel electrophoresis). Induction of DNA damage was found only after the first HBO exposure and not after further treatments of the same individuals. Furthermore, blood taken 24 h after HBO treatment was significantly protected against the induction of DNA damage by hydrogen peroxide (H(2)O(2)) in vitro, indicating that adaptation occurred due to induction of antioxidant defenses. The cells were not significantly protected against the genotoxic effects …
Hepatitis B and C virus variants in long-term immunosuppressed renal transplant patients in Latvia.
2004
The incidence of genome variants of hepatitis B and hepatitis C viruses among 38 long-term (2–15 years) immunosuppressed patients after renal transplantation and 10 patients undergoing dialysis was investigated. Twelve patients had only HBV infection, 9 had only HCV infection and 14 were co-infected. Regions corresponding to the HBV X/EnII/BCP, preC/C, preS/S and to the HCV core were sequenced for molecular characterization of the HBV and HCV genomes. Fifty-seven percent of HBV DNA isolates belonged to genotype D and 42% to genotype A, whereas 77% of HCV RNA isolates belonged to genotype 1b and only 17% to genotype 3a. One sample (6%) was of genotype 2c. Detailed analysis of the above-menti…