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Identification of bacteria with

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Title: Identification of bacteria with


1
2003-09-29
Identification of bacteria with 16S rRNA
sequencing
2
2003-09-29
Identification of bacteria
Patient sample (blood, urin, faeces)
Primary culture
- Selective growth media - Antibiotics
Secondary culture
Identification?
Biochemical typing
API strip
Identification
3
2003-09-29
Why sequencing?
Cultivation of bacteria fails - cultivation
methods are lacking - the bacteria are to few
to be cultivated - the bacteria are
inactive Biochemical identification fails -
methods are missing - the bacteria do not
perform biochemically correct
4
2003-09-29
Identification Flowchart
Patient sample (blood, urin, faeces)
Primary culture fails
Sequencing
Secondary culture
Identification?
Biochemical typing
Identification fails
Sequencing
5
2003-09-29
The Molecular Clock
Criteria -Randomly occuring mutations at
neutral sites -Constant mutation rate The
degree of sequence similarity reflects
phylogenetic distance, i.e. the more similar the
sequences- the more closely related the
species Ribosomal genes - contain regions
that are conserved among all self replicating
organisms, together with highly variable
regions -Are large enough to contain
adequate amount of information
6
2003-09-29
Evolutionary tree of the domain Bacteria
7
2003-09-29
The bacterial 16S rRNA molecule
Part of the ribosomal small subunit together with
a set of ribosomal proteins 16S the
sedimentation coefficient in ultra
centrifugation, measured in Svedberg rRNA
ribosomal RNA 1500 bases long RNA molecule
Conserved regions primer design Variable
regions identification
8
2003-09-29
Historical highlights of the bacterial 16S rRNA
molecule
  • Woese Fox 1977 Archaea, Bacteria and
    Eucaryotes could be classified acording to their
    phylogeny
  • Classification of bacteria with phylogenetic
    trees
  • Detection of non-cultivatible bacteria. 99 can
    not be cultivated!
  • Wilson et al. 1991 Identification of the
    etiologic agent of Whipples disease

9
2003-09-29
Sequencing of the 16S rRNA gene
  • DNA extraction 1 h (more for gram positives)
  • PCR 2,5 h
  • Cycle sequencing 3 h
  • Sequence analysis 1,5 h
  • Editing of raw data 20 min
  • Search in database 10 min lt 9 h

10
2003-09-29
Conclusions
11
2003-09-29
Lab. Groups
12
2003-09-29
Wet-Lab content
  • DNA preparation
  • PCR with primers directed against 16S rDNA
  • Analysis of PCR product on agarose gel
  • Purification of PCR product
  • Cyclic Sequencing reaction (PCR)
  • Automated sequencing
  • Identification of organism by database searches

13
2003-09-29
PCR with primers directed against 16S rDNA
  • Each PCR reaction containing
  • Master Mix (dNTP, MgCl buffer)
  • TaqDNApolymerase
  • Forward Reverse Primers
  • DNA Template
  • Water to adjust the reaction volume

The samples are put in a Thermocycler 95C,
5min. Initial denaturation 95C,
45s. Denaturation of DNA 50C, 45s. Annealing of
Primers 72C, 1min. Elongation of new DNA 72C,
10min. Final elongation
X 35
14
2003-09-29
Purification of PCR product
  • Commercial kits (e.i QIAqiuck Spin) are used to
    remove the excess of primers and nucleotides from
    the PCR reaction
  • The principle
  • Binding of DNA to a silica filter at a suitable
    pH, adjusted with a buffer solution
  • Washing the DNA with a wash buffer
  • Elute the DNA by changing the pH with another
    buffer solution

15
2003-09-29
Cyclic Sequencing reaction (PCR)
16
2003-09-29
Identification of organism by database searches
BLAST www.ncbi.nlm.nih.gov
BLAST- search at the National Center for
Biotechnology Information (NCBI) site by using
the retrieved nucleotide sequence (or translated
amino acid sequence) in FASTA- format gtSeqname
Nucleotide sequence........
17
2003-09-29
Computer sessions
http//www.kvir.uu.se/molbiokurs/molbiokurs_uu.htm
l
18
2003-09-29
Demonstration of some useful techniques in
molecular biology
  • Real- time PCR
  • Automated Capillary Sequenator
  • Amersham Pharmacia Biotech
  • Gyros
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