A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis
| Autor: | Carlos E. Salas, Miriam T. P. Lopes, I. F. Veloso, E. C. Moreira |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2000 |
| Předmět: |
DNA
Bacterial Microbiology (medical) Tris lcsh:Arctic medicine. Tropical medicine lcsh:RC955-962 polymerase chain reaction lcsh:QR1-502 Biology lcsh:Microbiology law.invention Silver stain chemistry.chemical_compound leptospira law Leptospira Endopeptidases Polyacrylamide gel electrophoresis DNA extraction Polymerase chain reaction Phenol Plants Proteinase K biology.organism_classification Molecular biology chemistry biology.protein Electrophoresis Polyacrylamide Gel Chloroform Endopeptidase K DNA |
| Zdroj: | Memórias do Instituto Oswaldo Cruz., Vol 95, Iss 3, Pp 339-343 (2000) Memórias do Instituto Oswaldo Cruz, Volume: 95, Issue: 3, Pages: 339-343, Published: JUN 2000 |
| ISSN: | 1678-8060 0074-0276 |
| Popis: | Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100 degrees C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2) leptospira, using the silver stain procedure. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|