- MeSH
- DNA analysis isolation & purification MeSH
- Genetic Code MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
OBJECTIVES: To characterize by restriction fragment length polymorphism (RFLP) patterns, the distribution of different Mycobacterium tuberculosis strains isolated consecutively from 75 tuberculosis patients who resided in Prague and had culture-confirmed cases during a 4-month period in 1995. METHODS: The insertion sequence IS6110-based RFLP analysis of M. tuberculosis isolates was carried out. RESULTS: There were a total of 75 patients with various forms of tuberculosis (54 males; 21 females). The sources of M. tuberculosis isolates were sputum (n = 64), pleura or lymph node drainage (n = 8), and urine (n = 3). Fifty-three of the patients (70.7%) had isolates with unique RFLP patterns, while 22 (29.3%) had isolates that belonged to seven clusters of related RFLP patterns. The seven clusters consisted of four groups of two patients, two groups of four patients, and one group of six patients. Most of the patients whose isolates fell within a clustered RFLP pattern lived in different quarters of the city and had no identifiable contacts with other patients whose isolates had the same pattern. CONCLUSIONS: The finding that isolates from most patients (70.7%) had unique rather than clustered RFLP patterns suggests that endogenous reactivation rather than exogenous transmission is the major determinant of most of the tuberculosis cases in Prague. The occurrence of seven distinct clusters comprising 29.3% of the isolates suggests that approximately one third of cases developed active tuberculosis from recent exogenous transmission.
- MeSH
- DNA, Bacterial * analysis MeSH
- DNA Fingerprinting MeSH
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Lymph Nodes microbiology MeSH
- Urine microbiology MeSH
- Molecular Epidemiology MeSH
- Mycobacterium tuberculosis * genetics isolation & purification MeSH
- Polymorphism, Restriction Fragment Length * MeSH
- Aged MeSH
- Sputum microbiology MeSH
- Tuberculosis * epidemiology microbiology MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Czech Republic MeSH
Several methods of molecular analysis of microbial diversity, including terminal restriction fragment length polymorphism (T-RFLP) analysis are based on measurement of the DNA fragment length. Significant variation between sequence-determined and measured length of restriction fragments (drift) has been observed, which can affect the efficiency of the identification of microorganisms in the analyzed communities. In the past, this variation has been attributed to varying fragment length and purine content. In this study, principal component analysis and multiple regression analysis were applied to find the contributions of those and several other fragment characteristics. We conclude that secondary structure melting point and G+C nucleotide content, besides the fragment length, contribute to the variation observed, whereas the contribution of purine content is less important. Incomplete denaturation of the sample at the start of electrophoretic separation of fragments has been excluded as a major cause of the variation observed. Our regression model explains the observed drift variation by approximately 56%, with standard deviation of the prediction equal to approximately 1.3 bp.
- MeSH
- Amplified Fragment Length Polymorphism Analysis methods standards MeSH
- DNA, Fungal chemistry genetics MeSH
- Fungi chemistry genetics isolation & purification MeSH
- Nucleic Acid Conformation MeSH
- Phalaris microbiology MeSH
- Polymorphism, Restriction Fragment Length MeSH
- Transition Temperature MeSH
- Base Composition MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- Archaea genetics metabolism MeSH
- Bacteria genetics metabolism MeSH
- Chlorine metabolism MeSH
- Fungi genetics metabolism MeSH
- Microbial Consortia physiology MeSH
- Polymorphism, Restriction Fragment Length MeSH
- Soil analysis MeSH
- Soil Microbiology MeSH
- RNA, Ribosomal, 16S analysis MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Vyšetření chimerismu se stalo nedílnou součástí sledování pacientů po alogenní transplantaci krvetvornýchbuněk. Předkládaná publikace nejprve seznamuje s různými metodami jeho stanovení.Ve druhé části je pak detailně uvedena charakteristika metody kapilární elektroforézy s fluorescenčnídetekcí (fragmentační analýzy) produktů amplifikace vysoce polymorfních mikro- a mini- satelitníchoblastí genomové DNA, metody, která je v současnosti považována za „gold standard“ metodu stanovení.
Examination of chimerism has become an integral element of the monitoring of patients after allogenichematopoietic cell transplantation. Presented publication initially describes different methods ofchimerism evaluation. In the second part, there is detailed characterization of the capillary electrophoresiswith fluorescence detection (fragment analysis) of amplification products of high polymorficmicro- and mini- satellite DNA regions, technique, which become the gold standard for quantitativeevaluation.
- MeSH
- Biomarkers MeSH
- Electrophoresis, Capillary methods utilization MeSH
- Fluorescence MeSH
- Polymerase Chain Reaction methods utilization MeSH
- Polymorphism, Restriction Fragment Length MeSH
- DNA, Satellite analysis diagnostic use MeSH
- Transplantation Chimera MeSH
- Publication type
- Comparative Study MeSH
The hotspot c.7541_7542delCT NOTCH1 mutation has been proven to have a negative clinical impact in chronic lymphocytic leukemia (CLL). However, an optimal method for its detection has not yet been specified. The aim of our study was to examine the presence of the NOTCH1 mutation in CLL using three commonly used molecular methods. Sanger sequencing, fragment analysis and allele-specific PCR were compared in the detection of the c.7541_7542delCT NOTCH1 mutation in 201 CLL patients. In 7 patients with inconclusive mutational analysis results, the presence of the NOTCH1 mutation was also confirmed using ultra-deep next generation sequencing. The NOTCH1 mutation was detected in 15% (30/201) of examined patients. Only fragment analysis was able to identify all 30 NOTCH1-mutated patients. Sanger sequencing and allele-specific PCR showed a lower detection efficiency, determining 93% (28/30) and 80% (24/30) of the present NOTCH1 mutations, respectively. Considering these three most commonly used methodologies for c.7541_7542delCT NOTCH1 mutation screening in CLL, we defined fragment analysis as the most suitable approach for detecting the hotspot NOTCH1 mutation.
- MeSH
- Leukemia, Lymphocytic, Chronic, B-Cell genetics MeSH
- Humans MeSH
- Mutation * MeSH
- DNA Mutational Analysis methods MeSH
- Peptide Fragments analysis MeSH
- Receptor, Notch1 genetics MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
