Soluciones Irrigantes Y Medicamentos Intraconducto
Páginas: 51 (12533 palabras)
Publicado: 22 de noviembre de 2012
FEMS Microbiology Reviews 21 (1997) 213^229
Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis
Friedrich v. Wintzingerode , Ulf B. Gobel Y *, Erko Stackebrandt ë
è Institut fur Mikrobiologie und Hygiene, Universitatsklinikum Charite, Dorotheenstr. 96, 10117 Berlin, Germany ë ë Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany
Received 20 June 1997 ; accepted 22 September 1997
Abstract After nearly 10 years of PCR-based analysis of prokaryotic small-subunit ribosomal RNAs for ecological studies it seems necessary to summarize reported pitfalls of this approach which will most likely lead to an erroneous description on the microbial diversity of a givenhabitat. The following article will cover specific aspects of sample collection, cell lysis, nucleic acid extraction, PCR amplification, separation of amplified DNA, application of nucleic probes and data analysis.
Keywords : Microbial diversity ; Nucleic acid; 16S rRNA gene; Polymerase chain reaction ; Phylogenetic analysis
Contents
Molecular rRNA-based strategies to determine microbial diversity. . Initial considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell lysis and extraction of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell lysis and extraction of RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . PCR ampli¢cation . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Inhibition of PCR ampli¢cation . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Di¡erential PCR ampli¢cation . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Formation of PCR artefacts . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.1. Formation of chimeric molecules . . . . . . . . . . . . . . . . . . . 6.3.2.Formation of deletion mutants . . . . . . . . . . . . . . . . . . . . 6.3.3. Formation of point mutants . . . . . . . . . . . . . . . . . . . . . . 6.4. Contaminating DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. 16S rRNA sequence variations due to rrn operon heterogeneity . 7. Separation of ampli¢ed 16S rRNA genes . . . . . . . . . . . . . . . . . . . . . 8. Analysis of16S rRNA sequence data . . . . . . . . . . . . . . . . . . . . . . . . 9. Cross-checking the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 2. 3. 4. 5. 6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 215 215 215 216 217 217 217 219 219 220 220 221 221 222 223 224
* Corresponding author. Tel.: +49 (30) 20934715; Fax: +49 (30) 2292741; E-mail: goebel@rz.charite.hu-berlin.de
0168-6445 / 97 / $32.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 1 6 8 - 6 4 4 5 ( 9 7...
Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis
Friedrich v. Wintzingerode , Ulf B. Gobel Y *, Erko Stackebrandt ë
è Institut fur Mikrobiologie und Hygiene, Universitatsklinikum Charite, Dorotheenstr. 96, 10117 Berlin, Germany ë ë Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany
Received 20 June 1997 ; accepted 22 September 1997
Abstract After nearly 10 years of PCR-based analysis of prokaryotic small-subunit ribosomal RNAs for ecological studies it seems necessary to summarize reported pitfalls of this approach which will most likely lead to an erroneous description on the microbial diversity of a givenhabitat. The following article will cover specific aspects of sample collection, cell lysis, nucleic acid extraction, PCR amplification, separation of amplified DNA, application of nucleic probes and data analysis.
Keywords : Microbial diversity ; Nucleic acid; 16S rRNA gene; Polymerase chain reaction ; Phylogenetic analysis
Contents
Molecular rRNA-based strategies to determine microbial diversity. . Initial considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell lysis and extraction of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell lysis and extraction of RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . PCR ampli¢cation . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Inhibition of PCR ampli¢cation . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Di¡erential PCR ampli¢cation . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Formation of PCR artefacts . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.1. Formation of chimeric molecules . . . . . . . . . . . . . . . . . . . 6.3.2.Formation of deletion mutants . . . . . . . . . . . . . . . . . . . . 6.3.3. Formation of point mutants . . . . . . . . . . . . . . . . . . . . . . 6.4. Contaminating DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. 16S rRNA sequence variations due to rrn operon heterogeneity . 7. Separation of ampli¢ed 16S rRNA genes . . . . . . . . . . . . . . . . . . . . . 8. Analysis of16S rRNA sequence data . . . . . . . . . . . . . . . . . . . . . . . . 9. Cross-checking the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 2. 3. 4. 5. 6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 215 215 215 216 217 217 217 219 219 220 220 221 221 222 223 224
* Corresponding author. Tel.: +49 (30) 20934715; Fax: +49 (30) 2292741; E-mail: goebel@rz.charite.hu-berlin.de
0168-6445 / 97 / $32.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 1 6 8 - 6 4 4 5 ( 9 7...
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