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Search results for: TAQ DNA POLYMERASE, GC-RICH TEMPLATES, PCR
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Increased concentration of Taq DNA polymerase as a solution for GC-rich templates from clinical and environmental samples
PublicationDNA polymerase is an enzyme which plays crucial role in replication and DNA repair. It found application in PCR (polymerase chain reaction) where catalyses process of in vitro DNA synthesis. To meet the demands posed by mod- ern diagnostic, molecular biology or genetic engineering it is necessary to improve DNA polymerases to obtain new or better features useful in these fields. So far implemented modifications in majority are...
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Fusion of DNA-binding domain of Pyrococcus furiosus ligase with TaqStoffel DNA polymerase as a useful tool in PCR with difficult targets
PublicationThe DNA coding sequence of TaqStoffel polymer- ase was fused with the DNA-binding domain of Pyrococcus furiosus ligase. The resulting novel recombinant gene was cloned and expressed in E. coli. The recombinant enzyme was purified and its enzymatic features were studied. The fusion protein (PfuDBDlig-TaqS) was found to have enhanced processivity as a result of the conversion of the Taq DNA polymerase from a relatively low processive...
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Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization
PublicationDNA polymerases are present in all organisms and are important enzymes that synthesise DNA molecules. They are used in various fields of science, predominantly as essential components for in vitro DNA syntheses, known as PCR. Modern diagnostics, molecular biology and genetic engineering need DNA polymerases which demonstrate improved performance. This study was aimed at obtaining a new NeqSSB-TaqS fusion DNA polymerase from the...
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Taq DNA polymerase fused with DNA binding protein with increased resistance to inhibitors from clinical samples
PublicationNowadays PCR method is commonly used in molecular diagnostic. However, in many cases PCR is limited, by the presence of inhibitory substances in biological, soil or food samples Efficiency and fidelity of amplification is strongly connected with DNA polymerase and reaction conditions. To meet the requirements of modern diagnostic methods it is essential to seeking for new DNA polymerases with better properties useful in these field....
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Fuzyjne polimerazy DNA – otrzymywanie, charakterystyka i zastosowanie
PublicationObecnie reakcje PCR (ang. Polymerase Chain Reaction) wykazują bardzo szerokie zastosowanie w diagnostyce medycznej, biologii molekularnej czy inżynierii genetycznej. Efektywność tych reakcji rozumiana jako wydajność i wierność przeprowadzonej amplifikacji jest nieodłącznie związana ze stosowaną polimerazą DNA i warunkami prowadzenia reakcji PCR. Aby sprostać wymaganiom stawianym przez nowoczesne metody diagnostyczne oraz współczesną...
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A simple modification to improve the accuracy of methylation-sensitive restriction enzyme quantitative polymerase chain reaction
PublicationDNA digestion with endonucleases sensitive to CpG methylation such as HpaII followed by polymerase chain reaction (PCR) quantitation is commonly used in molecular studies as a simple and inexpensive solution for assessment of region-specific DNA methylation. We observed that the results of such analyses were highly overestimated if mock-digested samples were applied as the reference.We determined DNA methylation levels in several...
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Modified DNA polymerases for PCR troubleshooting
PublicationPCR has become an essential tool in biological science. However, researchers often encounter problems with difficult targets, inhibitors accompanying the samples, or PCR trouble related to DNA polymerase. Therefore, PCR optimization is necessary to obtain better results. One solution is using modified DNA polymerases with desirable properties for the experiments. In this article, PCR troubleshooting, depending on the DNA polymerase...
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PCR amplification with primers based on IS2404 and GC-rich repeated sequence reveals polymorphism in Mycobacterium ulcerans
PublicationOpracowano prostą w wykonaniu metodę genotypowania szczepów bakterii z gatunku Mycobacterium ulcerans. Polimorfizm genetyczny 32 badanych izolatów Mycobacterium ulcerans odzwierciedla ich geograficzne pochodzenie z rejonów Afryki Centralnej, Ameryki Południowej i Łacińskiej, Azji Południowo-Wschodniej, Malezji oraz Australii.
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Modyfikacje polimeraz DNA jako rozwiązania problemów w reakcjach PCR
PublicationReakcja PCR już od wielu lat jest podstawowym narzędziem stosowanym w biotechnologii molekularnej. Nadal napotykamy jednak liczne problemy podczas jej przeprowadzania związane z trudnymi matrycami, obecnością inhibitorów itp. Optymalizacja reakcji PCR jest więc niezbędna w celu poprawy wyników. Jednym z rozwiązań tych problemów może być modyfikowanie polimeraz DNA. W powyższym artykule opisano problemy i rozwiązania zależne polimerazy...
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A novel method of Mycobacterium tuberculosis complex strain differentiation using polymorphic GC-rich gene sequences.
PublicationTuberculosis is one of the leading infectious diseases. In this work, a new genotyping method of Mycobacterium tuberculosis (Mtb) complex strain is presented. 27 Mtb genomes were analyzed for the presence of length polymorphism within polymorphic GC-rich gene sequences. Four genes, Rv3345c, Rv3507, Rv0747 and Rv3511, showing variation in length depending on the Mtb strain were selected for designing primer sequences flanking variable...
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Principles and applications of Ligation Mediated PCR methods for DNA-based typing of microbial organisms
PublicationA significant number of DNA-based techniques has been introduced into the field of microorganisms’ characterization and taxonomy. These genomic fingerprinting methods were developed to detect DNA sequence polymorphisms by using general principles, such as restriction endonuclease analysis, molecular hybridization, and PCR amplification. In recent years, some alternative techniques based on ligation of oligonucleotide adapters before...
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PCR-Based Genotyping of Mycobacterium tuberculosis with New GC-Rich Repeated Sequences and IS6110 Inverted Repeats Used as Primers.
PublicationW pracy przedstawiono nową metodę genotypowania Mycobacterium tuberculosis. Zastosowanie oligonukleotydu Mtb1 5'-CCG-GCG-GGG-CCG-GCG-G lub Mtb2 5'-CGG-CGG-CAA-CGG-CGG-C wraz z oligonukleotydami specyficznymi do terminalnych sekwencji elementu insercyjnego IS6110 pozwala na różnicowanie szczepów Mycobacterium tuberculosis w metodzie PCR. Opracowana metoda może być wykorzystana w badaniach epidemiologicznych gruźlicy.
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Diagnostyka zakażeń Candida spp. oparta o amplifikację DNA techniką PCR
PublicationZakażenia wywoływane przez oportunistyczne grzyby z rodzaju Candida są poważnym problemem klinicznym. Niezbędne jest poszukiwanie nowych szybkich, czułych i pewnych metod do identyfikacji gatunkowej tych drobnoustrojów. W artykule przedstawiono opisane w literaturze metody diagnostyki molekularnej drożdżaków z rodzaju Candida oparte o amplifikację DNA techniką PCR.
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DNA Methylation Changes Induced by Redox-Active Compounds—Choosing the Right PCR-Based Method
PublicationThe impact of catechins on the expression profile of redox-related genes in HT29 cell line has been studied recently by our group using Oxidative Stress RT2 Profiler PCR Array. Within the examined panel of 84 genes, the down-regulation of SRXN1 gene was unique among other up-regulated genes. We hypothesized that the observed down-regulation resulted from DNA methylation and have exploited this observation to choose the proper strategy...
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A New Double Digestion Ligation Mediated Suppression PCR Method for Simultaneous Bacteria DNA-Typing and Confirmation of Species: An Acinetobacter sp. Model
PublicationWe have designed a new ddLMS PCR (double digestion Ligation Mediated Suppression PCR) method based on restriction site polymorphism upstream from the specific target sequence for the simultaneous identification and differentiation of bacterial strains. The ddLMS PCR combines a simple PCR used for species or genus identification and the LM PCR strategy for strain differentiation. The bacterial identification is confirmed in the...
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Usefulness of the PCR technique for bacterial DNA detection in blood of the patients after ''oppened heart'' operations.
PublicationOperacje kardiochirurgiczne w krążeniu pozaustrojowym (KPU) poprzez ogromny uraz, powodowanie występowania zaburzeń odporności oraz uszkodzenie barier anatomicznych stwarzają sytuację wybitnie sprzyjającą rozwojowi powikłań infekcyjnych u tych chorych. Prowadzenie terapii antybiotykowej profilaktycznej i leczniczej, a także selektywna dekontaminacja przewodu pokarmowego może utrudniać i przedłużać czas rozpoznania obecności bakterii...
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Characterization of DNA Polymerase from Thermus thermophilus MAT72 Phage Tt72
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The suitability of DNA extracted from formalin-fixed, paraffin-embedded tissues for double differential polymerase chain reaction analysis
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Quantitative fluorescent determination of DNA – Ochratoxin a interactions supported by nitrogen-vacancy rich nanodiamonds
PublicationOchratoxin A (OTA) is a hazardous contaminant of a large variety of plant and animal originated food. Herein, we report an interaction of OTA with calf thymus DNA (ct DNA) on the nanodiamond surface. We employed multispectroscopic techniques to elucidate the binding mechanism of OTA with ct DNA. The fluorescence and UV–Vis spectroscopy results show that OTA binds to ds ct DNA and forms complexes. We obtained the binding constants...
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Detection of Helicobacter rodentium-like DNA in the liver tissue of patients with chronic liver diseases by polymerase chain reaction–denaturing gradient gel electrophoresis and DNA sequence analysis
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Endogenous oxidative DNA base modifications analysed with repair enzymes and GC/MS technique
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Molecular Characterization of a DNA Polymerase from Thermus thermophilus MAT72 Phage vB_Tt72: A Novel Type-A Family Enzyme with Strong Proofreading Activity
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One-tube cell lysis and DNA extraction procedure for PCR based detection of Mycobacterium ulcerans in aquatic insects, molluscs and fish.
PublicationMycobacterium ulcerans jest bakterią wywołującą choroby skóry u ludzi i zwierząt. W trakcie prowadzonych badań opracowano prostą w wykonaniu metodę ekstrakcji DNA Mycobacterium ulcerans z próbek środowiskowych oraz klinicznych. Opracowana metoda izolacji DNA może być przydatna w badaniu źródeł infekcji oraz dróg szerzenia się Mycobacterium ulcerans.
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Induction of unique structural changes in guanine-rich DNA regions by the triazoloacridone C-1305, a topoisomerase II inhibitor with antitumor activities.
PublicationCelem pracy było scharakteryzowanie odziaływania związku C-1305 z DNA w porównaniu do innych inhibitorów topoizomerazy II. Badania pokazują, że C-1305 odziaływuje preferencyjnie z parami CG, interkaluje do DNA i zaburza sąsiednie otoczenie w bardzo charakterystyczny sposób, który nie został zaobserwowany u pozostałych 22 badanych związków.
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Discovery and characterization of RecA protein of thermophilic bacterium Thermus thermophilus MAT72 phage Tt72 that increases specificity of a PCR-based DNA amplification
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PCR-ELISA: inexpensive alternative to quantitative PCR
PublicationPCR-ELISA (polymerase chain reaction-enzyme linked immunosorbent assay), a combination of PCR and ELISA methods, has been used since late 1980s. The technique is based on specially labelled DNA fragments which are captured by specific DNA sequences and detected by antibodies. The whole procedure of PCR-ELISA is divided into three steps: DNA extraction, PCR reaction and detection by ELISA. The method has been found as very specific...
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Real-Time PCR: molecular technique of many applications
PublicationReal-Time PCR is a sensitive DNA amplification technique initially applied in genetics and molecular biology. It enables in vivo copying of the selected DNA fragment (flanked by two primers) by the thermostable polymerase (in the presence of magnesium ions and deoxynucleotide triphosphates) and simultaneous measurement of the fluorescence. For one or more specific sequences in a DNA sample, real-time PCR enables both detection...
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The New LM-PCR/Shifter Method for the Genotyping of Microorganisms Based on the Use of a Class IIS Restriction Enzyme and Ligation Mediated PCR
PublicationThis study details and examines a novel Ligation-Mediated - Polymerase Chain Reaction (LM-PCR) method. Named the LM-PCR/Shifter, it relies on the use of a Class IIS restriction enzyme giving restriction fragments with different 4 base, 5' overhangs, this being the Shifter, and the ligation of appropriate oligonucleotide adapters. A sequence of 4-base, 5' overhangs of the adapter and a 4-base sequence of the 3' end of the primer(s)...
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Techniki biologii molekularnej w analityce produktów mięsnych
PublicationW pracy przedstawiono możliwości wykorzystania reakcji PCR (Polymerase Chain Reaction) do identyfikacji patogennych drobnoustrojów a także do badania składu surowcowego w mięsnych produktach żywnościowych.
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Real-time isothermal DNA amplification monitoring in picoliter volumes using an optical fiber sensor
PublicationRolling circle amplification (RCA) of DNA can be considered as a great alternative to the gold standard polymerase chain reaction (PCR), especially during this pandemic period, where rapid, sensitive, and reliable test results for hundreds of thousands of samples are required daily. This work presents the first research to date on direct, real-time and label-free isothermal DNA amplification monitoring using a microcavity in-line...
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DDLMS PCR double digestion Ligation Mediated Suppression PCR - a new technique for bacterial specific differentiation
PublicationA new diagnostic kit for K. oxytoca specific differentiation based on ddLMS PCR (ang. double digest ligation Mediated PCR) technique is shown. As a species-specific DNA fragment pehX gene, encoding the enzyme polygalactouronase, was chosen. The genome sequence of K. oxytoca is digested with two endonucleases: AclI and BclI which cut DNA before and after pehX gene. The polymorphic DNA fragments are ligated with AclI-end-specific...
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Interaction of the conserved region 4.2 of sigma(E) with the RseA anti-sigma factor
PublicationEo-E RNA polymerase transcribes a regulon of folding factors for the bacterial envelope and is induced by physical and chemical stresses. The RseA anti-sigma factor inhibits the activity of Esigma(E) RNA, polymerase. It is shown here that the N-terminal portion of sigma(E), residues 1-153, binds core RNA polymerase. RseA interacts with residues 154-191 of sigma(E), a site that is homologous to region 4, the sigma factor binding...
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A simple modification of PCR thermal profile applied to evade persisting contamination
PublicationThe polymerase chain reaction (PCR), one of the most commonly applied methods of diagnostics and molecular biology has a frustrating downside known as the false positive signal or contamination. Several solutions to avoid and to eliminate PCR contaminations have been worked out to date but the implementation of these solutions to laboratory practice may be laborious and time consuming. A simple approach to circumvent the problem...
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Molecular identification and genotyping of Staphylococci: genus, species, strains, colnes, lineages, and interspecies exchanges
PublicationStaphylococci are increasingly recognized as etiological agents of many opportunistic human and animal infections, emphasizing the need for a rapid and accurate identification, even to a genotypical level of these bacteria. In the recent years, there has been a significant progress in typing and phylogenetic study of Staphylococcus species. Here, we describe molecular methods used in taxonomy as well as staphylococci characterization....
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Specific detection of Alternaria alternata by PCR and real-time PCR
PublicationFungi of Alternaria genus are cosmopolitan organisms, which spores can be found in the air, soil, water, clothing and food. They commonly occur as saprotrophs on the plant remains, contributing to the decomposition of organic matter. Additionally, they are components of the normal human and animal skin flora. Alternaria spp. are also known human allergens, causing hay fever and allergic reactions that can lead to the development...
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Loop-mediated isothermal amplification (LAMP) as a diagnostic tool in detection of infectious diseases
PublicationLoop-mediated isothermal amplification (LAMP) is a gene amplification method which amplifies DNA with high specificity and efficiency under isothermal conditions. Because of its rapidity and simplicity, it is a valuable diagnostic tool in the early detection and identification of infectious diseases. LAMP method is based on the use of a set of four to six specially designed primers spanning six to eight distinct sequences on the...
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Recombinant !ermostable AP Exonuclease from Thermoanaerobacter tengcongensis: Cloning, Expression, Purification, Properties and PCR Application
PublicationApurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity, especially at high temperatures. !e gene encoding a homologue of AP exonuclease was cloned from the thermophilic anaerobic bacterium Thermoanaerobacter tengcongensis and transformed into Escherichia coli. The protein product showed high identity (80%) to human Ape1 nuclease, whereas to E. coli exonuclease...
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PCR and real-time PCR approaches to the identification of Arthroderma otae species Microsporum canis and Microsporum audouinii/Microsporum ferrugineum
PublicationObjectives The identification of species in the Arthroderma otae complex is essential to determine the origin of infection and to eliminate the risk of transmission. Microsporum canis is a zoophilic species, whereas Microsporum audouinii and Microsporum ferrugineum are anthropophilic species. In this paper, we propose alternative methods that permit species-specific identification of both anthropophilic and zoophilic members of...
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Versatile method employing basic techniques of genetic engineering to study the ability of low molecular weight compounds to bind covalently with DNA in cell free systems.
PublicationMechanizm działania większości leków przeciwnowotworowych oraz związków rakotwórczych polega na ich kowalencyjnym wiązaniu z DNA. Poziom tego wiązania jest zazwyczaj bardzo niski, co powoduje, że potrzebne są niezwykle czułe metody aby kowalencyjna modyfikacja mogła być w ogóle wykryta. My podjęliśmy próbę zastosowania w tym celu prostych, szybkich i sprawdzonych technik inżynierii genetycznej: metody PCR do uzyskania fragmentu...
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The New LM-PCR/Shifter Method for the Genotyping of Microorganism
PublicationTechniques relies on the ligation of appropriates adapters (LM-PCR) as AFLP, PCR MP and ADSRRS are successfully used for epidemiological studies for prokaryotic and eukaryotic microorganisms. In this study we propose a new method, called the LM-PCR/Shifter, based on the use of a Class IIS restriction enzyme giving restriction fragments with different 4 base 5' overhangs (Shifter) and the ligation of appropriate oligonucleotide...
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Diagnostic PCR tests for Microsporum audouinii, M. canis and Trichophyton infections
PublicationSince traditional diagnosis of dermatophyte infections is slow, we present a rapid new pcr test for detection of trichophyton spp., microsporum canis and m. audouinii infections. the performance of the test was evaluated with: 58 dermatophyte isolates; 10 yeast, mould and human dna control samples; 25 routine specimens from patients suspected of having dermatophytosis; 10 hair specimens from guinea pigs experimentally infected...
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PCR and real-time PCR assays to detect fungi of Alternaria alternata species
PublicationFungi of the Alternaria genus are mostly associated with allergic diseases. However, with a growing number of immunocompromised patients, these fungi, with A. alternata being the most prevalent one, are increasingly recognized as etiological agents of infections (phaeohyphomycoses) in humans. Nowadays, identification of Alternaria spp. requires their pure culture and is solely based on morphological criteria. Clinically, Alternaria...
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Application of SSB-like protein from Thermus aquaticus in multiplex PCR of human Y-STR markers identification
PublicationW tej publikacji prezentujemy zastosowanie białka SSB-podobnego Thermus aquaticus (TaqSSB) w reakcji multiplex PCR w celu identyfikacji markerów ludzkiego Y-STR. Wykorzystanie termostabilnego białka TaqSSB chroni lub redukuje tworzenie przez startery struktur dimerycznych, które powodują hamowanie hybrydyzacji starterów do badanego DNA i redukują ilość starterów dostępnych w reakcji PCR.
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New PCR test for detection of Candida glabrata based on the molecular target chosen by the RAPD technique
PublicationRapid, reliable diagnosis is a necessary condition for the successful treatment of infections. Such diagnostic assays are continually being developed. #e paper presents a method for selecting the molecular target for PCR-based diagnostics based on the comparison of RAPD patterns. A sequence encoding Candida glabrata CBS138 hypothetical protein was selected. The limit of detection for PCR and real-time PCR reactions with DNA extracted...
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Characterization of exceptionally thermostable single-stranded DNA-binding proteins from Thermotoga maritima and Thermotoga neapolitana
PublicationBACKGROUND: In recent years, there has been an increasing interest in SSBs because they find numerous applications in diverse molecular biology and analytical methods.RESULTS: We report the characterization of single-stranded DNA binding proteins (SSBs) from the thermophilic bacteria Thermotoga maritima (TmaSSB) and Thermotoga neapolitana (TneSSB). They are the smallest known bacterial SSB proteins, consisting of 141 and 142 amino...
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MutL protein as a constituent of vsp, ner and mmr repair systems
PublicationMutS and MutL proteins are renowned mostly for their functions in well-characterized, post-DNA replication mis- match repair system (MMR). However, there is growing evidence that MMR system is not the only field of action for these pro- teins. Moreover, the participation in MMR does not even have to be their primary function. There are some reports indicat- ing involvement of MutL in BER, NER and VSP (very short patch repair)....
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MUTL PROTEIN AS A COMMON CONSTITUENT OF VSP, BER, NER AND MMR REPAIR SYSTEMS
PublicationMutS and MutL proteins are renowned mostly for their functions in well-characterized, post-DNA replication mismatch repair system (MMR). However, there is growing evidence that MMR system is not the only field of action of these proteins. Moreover, involvement in MMR does not even have to be their primary function. There are some reports indicating involvement of MutL in BER, NER and VSP (very short patch repair). MutL protein...
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A universal method for the identification of genes encoding amatoxins and phallotoxins in poisonous mushrooms
PublicationABSTRACT Background. As the currently known diagnostic DNA targets amplified in the PCR assays for detection of poisonous mushrooms have their counterparts in edible species, there is a need to design PCR primers specific to the genes encoding amanitins and phallotoxins, which occur only in poisonous mushrooms. Objective. The aim of the study was testing of PCR-based method for detection of all genes encoding hepatotoxic cyclic...
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Zakażenia bakteryjne w przewlekłym zapaleniu ucha środkowego - ocena przydatności diagnostyki molekularnej opartej o technikę PCR. Bacterial infection in chronic otitis media - assessment of usefulness of molecular diagnostic based in PCR method.
PublicationIdentyfikacja zakażeń w przewlekłym zapaleniu ucha środkowego jest trudna, o czym świadczą wysokie odsetki ujemnych wyników badań bakteriologicznych. Przyczyną tego jest trudność w uzyskiwaniu wiarygodnego materiału do badań oraz stosunkowo niska czułość klasycznych metod diagnostycznych. Amplifikacja bakteryjnego DNA techniką PCR jest badaniem, które może przyczynić się do zwiększenia efektywności i czułości badań bakteriologicznych....
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ALIS-FLP: amplified ligation selected fragment-length polymorphism method for microbial genotyping
PublicationA DNA fingerprinting method known as ALIS-FLP (amplified ligation selected fragment-length polymorphism) has been developed for selective and specific amplification of restriction fragments from TspRI restriction endonuclease digested genomic DNA. The method is similar to AFLP, but differs in that only one specific restriction enzyme (TspRI) is used. The cohesive ends of the DNA fragments are ligated with two types of oligonucleotide....