The use of polynucleotide RNA probes for detection, identification and cell sorting of microorganisms [Elektronische Ressource] / Katrin Zwirglmaier

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2003
• Nombre de lectures: 47
• Langue: Deutsch
• Poids de l'ouvrage: 32 Mo

Extrait

Lehrstuhl für Mikrobiologie
der Technischen Universität München






The Use of Polynucleotide RNA Probes for
Detection, Identification and Cell Sorting of
Microorganisms





Katrin Zwirglmaier




Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan
für Ernährung, Landnutzung und Umwelt
der Technischen Universität München
zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation







Vorsitzender: Univ.-Prof. Dr. W. Höll

Prüfer der Dissertation: 1. Univ.-Prof. Dr. K.-H. Schleifer
2. Univ.-Prof. Dr. R. F. Vogel
3. Univ.-Prof. Dr. H. Brunner, Universität Stuttgart




Die Dissertation wurde am 19.02.2003 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 21.05.2003 angenommen.
Table of contents

A. Introduction 1

B. Material and methods

B.1. Organisms 5
B.2. Cultivation of organisms 7
B.3. Primers 8
B.4. Probes 10
B.5. Isolation of nucleic acids 12
B.5.1. genomic DNA
B.5.2. of plasmid 13
B.6. Photometric measurement of nucleic acid solutions 14
B.7. Agarose gel electrophoresis 15
B.8. Polymerase chain reaction (PCR) 16
B.9. Labelling of nucleic acids with biotin, digoxigenin or fluorescent dyes 18
B.9.1. Oligonucleotide probes 18
B.9.2. Polynucleotide RNA probes – in vitro transcription 18
B.10. Cell fixation 20
B.10.1. Cell with PFA 20
B.10.2. ethanol
B.10.3. Cell fixation with formaldehyde / acetic acid 21
B.10.4. Cell fixation with methanol 21
B.10.5. Postfixational treatments 22
B.10.5.1. Heat 22
B.10.5.2. Lysozyme 22
B.10.5.3. Proteinase K 23
B.11. Hybridisation techniques 23
B.11.1. Evaluation of hybridisation conditions 23
B.11.2. Hybridisation with oligonucleotide probes 25
B.11.3. Hybridisation with RNA polynucleotide probes 26
B.11.3.1. Probes targeting rRNA 26
B.11.3.2. plasmids 27
B.11.3.3. genomic DNA 27
B.12. Cell sorting 28
B.12.1. Magnetic cell sorting (MACS) 28
B.12.2. Cell in microplates 28
B.12.2.1. Streptavidin coated mi
B.12.2.2. DNA coated mi 29
B.12.2.2.1. Coating of microplates with DNA 29
B.12.2.2.2. Cell sorting (Hybridisation in coated microplates – HYCOMP) 30
B.13. Detection of cells after hybridisation 31
B.13.1. Detection in microplates 31
B.13.2. for microscope analysis 32
B.14. Quantification of cell sorting efficiency 32
B.14.1. Semiquantitative PCR 32
B.14.2. Manual cell counts 33
B.14.3. Computer based quantification 34
B.15. Software 34


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C. Results

Part I – Fluorescence In situ Hybridisation (FISH) with polynucleotide transcript probes

C.1. Groups of organisms targeted with polynucleotide probes 36
C.1.1. Gram-negative bacteria 38
C.1.2. Gram-positive 40
C.1.3. Yeasts 44
C.1.4. Mammalian cells 50

C.2. Classes of target molecules for polynucleotide probes 52
C.2.1. Polynucleotide probes targeting rRNA 52
C.2.1.1. Standard polynucleotide probes 52
C.2.1.2. Evaluation of probe length 52
C.2.1.3. Influence of secondary structure on hybridisation signal 54
C.2.2. Polynucleotide probes targeting plasmids 57
C.2.3. Polynucleotide probes targeting chromosomal DNA 65
C.2.3.1. Glycerol aldehyde 3-phosphate dehydrogenase (GAPDH) 65
C.2.3.2. Xanthomonas campestris pathovars 68



Part II – Cell sorting with polynucleotide probes

C.3. Cell sorting methods 70

C.4. Optimisation of the reaction parameters 72
C.4.1. Sample preparation 72
stC.4.2. Parameters for 1 hybridisation 73
C.4.2.1. Stringency 73
C.4.2.2. Hybridisation time 74
C.4.2.3. Amount of probe
ndC.4.3. Parameters for 2 hybridisation 76
C.4.3.1. Stringency 76
C.4.3.2. Hybridisation time 76

C.5. Cell sorting efficiency 77
C.5.1. Evaluation via semiquantitative PCR 77
C.5.2. Quantification via manual cell counts 78
C.5.3. Computer based quantification 78

C.6. Applicating the method to environmental samples 80





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D.Discussion
Part I – Fluorescence In situ Hybridisation (FISH) with polynucleotide transcript probes

D.1. Target organisms 83
D.2. molecules 85
D.2.1. rRNA 85
D.2.2. DNA : Plasmids and chromosomal DNA – RING-FISH 86
D.3. Network hypothesis 93
D.4. Probe design 99
D.5. Possible applications 100

Part II – Cell sorting with polynucleotide probes

D.6. Comparison of cell sorting methods 101
D.7. Cell sorting in DNA coated microplates 102
D.7.1. Cell fixation 102
D.7.2. Hybridisation parameters 102
D.7.3. sorting efficiency 103
D.8. Environmental samples
D.9. Possible applications 104


E. Summary

Summary (English) 105
Zusammenfassung (German) 106

F. Appendix 107


G. References 118

















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Abbreviations

A adenine
abs. absolute
Ac acetate
AP alkaline phosphatase
amp ampicillin
APS moniumperoxodisulfate
BIO biotin
bp base pairs
°C degree centigrade
C cytosine
CIA chloroform : isoamylalcohol = 24:1 (v/v)
cf. confer
cm centimeter
d day(s)
DAPI 4', 6'-Diamino-2-phenylindol-dihydrochloride
dATP deoxyadenosinetriphosphate
dCTP deoxycytidinetriphosphate
DIG digoxigenin
DNA deoxyribonucleic acid
DNase deoxyribonuclease
ddNTP 2’,3’ dideoxy-nucleoside-5’-triphosphate
dGTP deoxyguanosinetriphosphate
dNTP 2’deoxy-nucleoside-5’-triphosphate
ds double stranded
DSMZ Deutsche Sammlung für Mikroorganismen und Zellkulturen,
Braunschweig, Germany
dTTP deoxythymidinetriphosphate
E extinction
EDTA ethylenediamintetraacetate
e.g. exemplum gratiae
et al. et alteri
etc. cetera
EtBr ethidiumbromide
EtOH 100% ethanol abs.
FA formamide
FISH fluorescence in situ hybridiszation
FLUOS 5,(6)-carboxyfluorescein-N-hydroxysuccinimidester
g gram
G guanine
GC mol % guanine + cytosine
H O ultra pure water (Millipore-system) 2 MQ
h hour(s)
HD yeast-dextrose medium
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HEPES 2-[4-(2-hydroxyethyl)-piperazinyl-(1)]-ethansulfonic acid C H N O S 8 18 2 4
i.e. id est
IPTG isopropyl-β-D-thiogalactoside
kb kilo bases
kDa kilodalton
l liter
LB Luria-Bertani medium
LMG Laboratorium voor Microbiologie Universiteit Gent, Belgien
M molar
-3m eter; milli(10 )
min inutes
mRNA messenger RNA
-6µ micro (10 )
-9n nano (10 )
n.a. not applicable
NaAc sodium acetate
n.d. no data, not determined
nt nucleotides
OD optical density
o/n overnight
-12p pico (10 )
Pa pascal
p.a. pro analysi
PBS phosphate buffered saline
PCR polymerase chain reaction
PFA paraformaldehyde
pH negative decadic logarithm of the proton concentration
pv. pathovar
q.v. quod vide
resp. respectively
RNA ribonucleic acid
RNase ribonuclease
rDNA ribosomal DNA
rpm revolutions per minute
rRNA al RNA
RT room temperature
SDS sodiumdodecylsulfate
sec seconds
SSC standard saline citrate
sp. species
ss single stranded
T thymine
TAE tris-acetate-EDTA
TE tris-EDTA
T melting temperature of nucleic acids m
v
Tab. table
Taq Thermus aquaticus
TE tris-EDTA
TEMED N,N,N’,N’-tetramethylethylendiamine
Tris tris-(hydroxmethyl-)aminomethane
U Unit or uracil
UV ultra violet
v. vide
V volt
v/v volume/volume
W watt
w/v weight/volume
WS Weihenstephan, bakteriologisches Institut des Forschungszentrums
für Milch und Lebensmittel der TUM, Freising, Germany
X-Gal 5-chlor-4-brom-3-indolyl-β-D-galactoside

































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