Wyniki wyszukiwania dla: POLYMERASE CHAIN REACTION-RESTRICTION FRAGMENT LENGTH POLYMORPHISM (PCR-RFLP)
-
Amplification of a single-locus variable-number direct-repeats with restriction fragment length polymorphism (DR-PCR/RFLP) for genetic typing of Acinetobacter baumannii strains
PublikacjaW celu poszukiwania nowych metod typowania genetycznego szczepów Acinetobacter baumannii przydatnych w szpitalnych badaniach epidemiologicznych przeprowadzono analizę dostępnych w banku genów sekwencji szczepów referencyjnych na obecność nowych motywów repetytywnych. Do badania polimorfizmu sekwencji repetytywnych w szczepach Acinetobacter sp. wykorzystano proste powtórzenia typu "direct repeat" (DR) zlokalizowane w jednym miejscu...
-
PCR-RFLP assays for species-specific identification of fungi belonging to Scopulariopsis and related genera
PublikacjaFungi of the Scopulariopsis genus, commonly found in the environment, are opportunistic pathogens that can cause various types of human infections. So far, no efficient molecular method has been developed for species differentiation among Scopulariopsis and related genera. In order to advance this field, we have evaluated performance of polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays, based...
-
Novel application of terminal restriction fragments length polymorphism method (t-RFLP) for identification of six clinically important Candida sp.
PublikacjaTerminal restriction fragments length polymorphism method (t-RFLP) is a modified classical RFLP method. The main difference is based on carrying out PCR using a fluorescently labeled primer or primers. The result of such modification is that after digestion of amplicons only terminal fragments of PCR product (containing fluorescent dye) are visualized. T-RFLP method has found application in environmental microbiology and epidemiology....
-
A new assay based on terminal restriction fragment length polymorphism of homocitrate synthase gene fragments for Candida species identification
PublikacjaCandida sp. have been responsible for an increasing number of infections, especially in patients with immunodeficiency. Species specific differentiation of the Candida sp. is difficult in routine diagnosis. This identification can have a highly significant association in therapy and prophylaxis. This work has shown a new application of terminal restriction fragment length polymorphism (t-RFLP) method in molecular identification...
-
A simple modification to improve the accuracy of methylation-sensitive restriction enzyme quantitative polymerase chain reaction
PublikacjaDNA 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...
-
New Approaches for Escherichia coli Genotyping
PublikacjaEasy-to-perform, fast, and inexpensive methods of differentiation of Escherichia coli strains beyond the species level are highly required. Herein two new, original tools for genotyping of E. coli isolates are proposed. The first of the developed method, a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) test uses a highly variable fliC gene, encoding the H antigen as a molecular target. The designing...
-
ALIS-FLP: amplified ligation selected fragment-length polymorphism method for microbial genotyping
PublikacjaA 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....
-
Molecular identification and genotyping of Staphylococci: genus, species, strains, colnes, lineages, and interspecies exchanges
PublikacjaStaphylococci 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....
-
Principles and applications of Ligation Mediated PCR methods for DNA-based typing of microbial organisms
PublikacjaA 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...
-
Fuzyjne polimerazy DNA – otrzymywanie, charakterystyka i zastosowanie
PublikacjaObecnie 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...
-
Identification and cloning of C. albicans SC5314 genes encoding L-methionine biosynthetic pathway enzymes.
Dane BadawczeEnzymes of fungal L-methionine biosynthetic pathway: homoserine O-acetyltransferase (Met2p), O-acetylhomoserine sulfhydrylase (Met15p) and cystathionine-γ-synthase (Str2p) could be exploited as molecular targets for antifungal chemotherapy. The goal of the study was to identify and clone genes encoding mentioned above enzymes. MET2, MET15 and STR2 genes...
-
Fungal Typin Methods
PublikacjaThe broad application of the molecular techniques in mycoses diagnosis is related to increase of infections caused by fungi in many countries. The oldest typing methods relaying on fenotypic observation, physiological and biochemical examination have had very limited importance from decades. Novadays, the molecular biology methods took their place. Most of the genotyping methods have been devoleped to be applied for typing of bacteria...
-
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
PublikacjaThis 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)...
-
Detection of Blastocystis hominis in unpreserved stool specimens byusing polymerase chain reaction
PublikacjaBlastocystis hominis jest pasożytem występującym na całym świecie. Patogenność tego pasożyta nie jest jeszcze w pełni udowodniona, aczkolwiek opisanych jest wiele przypadków, sugerujących, że B. hominis może wywoływać objawy żołądkowo-jelitowe. Wykrywanie tego pasożyta opiera się na bezpośredniej obserwacji mikroskopowej rozmazów i zagęszczonych próbek kału lub rozmazów utrwalonych, jednakże w pewnych przypadkach nie jest to możliwe.Celem...
-
Mechanism of reaction of RNA-dependent RNA polymerase from SARS-CoV-2
PublikacjaWe combine molecular dynamics, statistical mechanics, and hybrid quantum mechanics/molecular mechanics simulations to describe mechanistically the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp). Our study analyzes the binding mode of both natural triphosphate substrates as well as remdesivir triphosphate (the active form of drug), which is bound preferentially over ATP by RdRp while...
-
Fusion of DNA-binding domain of Pyrococcus furiosus ligase with TaqStoffel DNA polymerase as a useful tool in PCR with difficult targets
PublikacjaThe 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...
-
Różnicowanie C. albicans i C. dubliniensis w oparciu o polimorfizm genu syntazy homocytrynianowej z wykorzystaniem metody PCR RFLP
PublikacjaNajczęstszym czynnikiem etiologicznym zakażeń wywołanych przez drożdżaki z rodzaju Candida jest C. albicans. Gatunek ten posiada bardzo zbliżone cechy fenotypowe do C. dubliniensis co często wiąże się z błędną identyfikacją. Skutkuje to wtórnym zakażeniem wywołanym przez współwystepujący gatunek C. dubliniensis, który nabywa oporność na flikonazol stosowany w terapii C. albicans. Celem badań była konstrukcja układu diagnostycznego...
-
The suitability of DNA extracted from formalin-fixed, paraffin-embedded tissues for double differential polymerase chain reaction analysis
Publikacja -
Detection ofHelicobacterspecies in liver and stomach tissues of patients with chronic liver diseases using polymerase chain reaction-denaturing gradient gel electrophoresis and immunohistochemistry
Publikacja -
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
Publikacja