Development of microsystem technology suitable for bacterial identification and gene expression monitoring [Elektronische Ressource] / von Andriy Ruryk

Dietzel

Sujets

• Biologie

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2005
• Nombre de lectures: 27
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

DEVELOPMENT OF MICROSYSTEM TECHNOLOGY
SUITABLE FOR BACTERIAL IDENTIFICATION AND
GENE EXPRESSION MONITORING
DISSERTATION


ZUR ERLANGUNG DES AKADEMISCHEN GRADES
doctor rerum naturalium






vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät der
Friedrich-Schiller-Universität Jena






von

Andriy Ruryk
geboren am 08. März 1971 in Lviv/Ukraine


Jena, im August 2004



















Referees
1. _______________________
2. _______________________
3. _______________________
Day of the open defence: _______________________
IIIContent
CONTENT ..............................................................................................................................IV
INTRODUCTION....................................................................................................................1
THE PROSPECTIVE MICROBIOLOGY TASKS AND THE BIOLOGY OF ANTIBIOTIC PRODUCTION BY
ACTINOMYCETES. .................................................................................................................... 1
CURRENT TAXONOMY ISSUES ON ACTINOMYCETE BACTERIA .................................................. 5
DNA MICROARRAYS: NEW FRONTIERS IN PROSPECTIVE MICROBIOLOGY ................................ 8
AIMS ....................................................................................................................................... 15
16S RRNA SEQUENCE INTERROGATION................................................................................ 15
EXPRESSION MONITORING..................................................................................................... 15
MATERIALS AND METHODS........................................................................................... 16
MATERIALS 16
• Bacterial strains ....................................................................................................... 16
• Nucleic acids ............................................................................................................ 16
• Media........................................................................................................................ 16
• Compounds............................................................................................................... 18
• Buffers ...................................................................................................................... 19
• Microscopic glass slides........................................................................................... 20
• Plastic consumables ................................................................................................. 20
• Enzymes kits ............................................................................................................. 20
• DNA and RNA molecular labels and stains ............................................................. 20
• Nucleic acid purification columns............................................................................ 21
• Nucleic acid labelling and purification kits 21
• Hybridization chambers, spotting pins..................................................................... 21
• Software.................................................................................................................... 21
• Additional devices .................................................................................................... 21
METHODS.............................................................................................................................. 21
• Morphological, physiological and chemotaxonomical characterization of freshly
isolated eubacterial strains .............................................................................................. 21
• Genomic DNA isolation from Actinomycetales........................................................ 22
• Total RNA isolation.................................................................................................. 22
• Nucleic acid sequence analysis................................................................................ 23
• Polymerase chain reactions ..................................................................................... 24
• Transformation of E. coli cells................................................................................. 25
• Plasmid DNA minipreps from E.coli cells ............................................................... 25
• Cloning of PCR fragments ....................................................................................... 25
• Sequencing of PCR fragments of 16S rDNA ............................................................ 25
• Direct chemical labelling of total RNA.................................................................... 25
• RNA fragmentation................................................................................................... 26
• Primer design for cDNA labelling ........................................................................... 26
• RNA labelling ........................................................................................................... 26
• Quantification of nucleic acids and labelling efficiencies ....................................... 26
• Activation of solid support surface for DNA coupling............................................. 26
• DNA sampling for array fabrications ...................................................................... 27
• Array fabrication...................................................................................................... 27
IV• Array hybridization .................................................................................................. 29
• Hybridization data retrieval..................................................................................... 29
• Data analysis............................................................................................................ 30
RESULTS................................................................................................................................ 31
1.1 TAXONOMICAL ASSIGNMENT OF NEWLY ISOLATED STRAINS ..................................... 31
1.2 PRIMERS DESIGNED FOR PCR AMPLIFICATION OF 16S-RRNA GENE FRAGMENTS ..... 32
OLIGONUCLEOTIDE PROBES INTERROGATING THE 16S-RRNA GENE SEQUENCES.................. 32
SOLID SUPPORTS.................................................................................................................... 36
ACTIVATION OF THE SOLID-SUPPORT SURFACE...................................................................... 36
• Activation by gelatine-carrying linkers.................................................................... 36
• Polylysine coating .................................................................................................... 36
• Silanization of microscopic slides by alkylaminosilanes (Corning’s gamma-
aminopropyl silane slides). .............................................................................................. 38
1.2.1 Aldehyde coupling............................................................................................ 39
PREPARATION OF IMMOBILIZATION PROBES .......................................................................... 43
1.2.2 PCR fragments ................................................................................................. 43
1.2.3 Oligonucleotide probes .................................................................................... 43
IMMOBILIZATION OF DNA PROBES ....................................................................................... 45
LABELLING EFFICIENCY FOR HYBRIDIZATION TARGET MOLECULES....................................... 49
ARRAY HYBRIDIZATION ........................................................................................................ 50
1.2.4 Final hit selection and reliability..................................................................... 63
DISCUSSION ......................................................................................................................... 68
OLIGONUCLEOTIDES TO INVESTIGATE THE 16S-RRNA GENE SEQUENCE. ............................. 68
ACTIVATION OF SURFACES .................................................................................................... 70
PRIMARY AMINOGROUPS....................................................................................................... 70
ALDEHYDE GROUPS............................................................................................................... 71
IMMOBILIZATION OF PROBE MOLECULES ............................................................................... 73
TARGET MOLECULES ............................................................................................................. 74
HYBRIDIZATION FACTORS ..................................................................................................... 76
Buffer................................................................................................................................ 76
Temperature and probe positioning effects...................................................................... 77
Hybridiza