Studying Microbial Biodiversity Step 1: Qualitative Identification What do they look like What do th

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Studying Microbial BiodiversityStep 1
Qualitative IdentificationWhat do they look
like? What do they do? What do they eat?

• Naked Eye Colony - shape, colour.
• Microscopy Cell - shape, colour, size
• Biochemistry Staining
• Assimilation Studies
• These techniques usually require culturing
  in-vitro.
• Over 99 of bacteria are unculturable
• Molecular Level

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The Martian View of Cats and Dogs

• CATS DOGS
• 4 legs
• long tail
• two ears
• often live with
• 2-legged things
• prey lives inside tin

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Molecular Methods for Identification of
Genotypes Basic Concept

• The number of nucleic acid or amino acid
  differences between two organisms is proportional
  to the time since they diverged from a common
  ancestor.

1 2 3
1 AAGGCTA 2 AAGGGTA 3 AAGGATG Example
Rate of Evolution 1bp per 100 years
MOLECULAR DIFFERENCES
100years
200 years
TIME
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Molecular Methodsmany ways to determine
difference in molecular structure

• Proteins
• amino acid sequencing
• allozyme electrophoresis
• Nucleic Acids
• Hybridisation
• Fragment Polymorphisms
• DNA sequencing

need culture
Genomic DNA (need culture)
Amplified/Cloned fragments
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DNA Hybridisation low level resolutionHomo and
Heteroduplexes - Jackets and Jeans - zips

• Principle
• ds DNA is held together by H-bonds. When heated
  the strands disassociate. As they cool they
  re-anneal.
• The annealing temperature is dependent on the
  level of mismatch between the two strands.
  Strands of DNA from the same organism will
  re-associate more easily than strands of DNA from
  distantly related organisms

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Fragment Polymorphismsmedium level resolution

• Principle
• DNA fragments can be separated electrophoretically
  according to their size, number or conformation.
• Fragment Analysis Methods
• Restriction Site Analysis/ARDRA
• SSCP/DGGE
• RAPDs
• VNTRs
• Microsatellites

Mainly used for analysing populations of higher
organisms
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PCR
As a preliminary step to amplify DNA for REA,
DGGE or sequencing
As a stand-alone diagnostic tool
PRIMERS
Universal or specific to a certain group of
organisms
Specific ONLY to one organism or group of
organisms
APPLICATIONS
Identifying novel taxa and community analysis
Identifying the presence of known organisms in
the environment
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1. Amplify genomic DNA template with
ssu-rRNA primers.2. Observe on agarose gel.
Diagnostic Test using Specific Primers
Identifying a new organism from a pure culture
Amplifying from a community or mixed culture
Sequence the PCR product
Clone into TA vector
DGGE/SSCP Analysis
Presence of a PCR product indicates that the
organism is present in the sample
ARDRA Analysis
Sequence
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Cloning into TA Vectors

• PCR product contains DNA from several organisms.
• Each copy of the vector only receives one DNA
  fragment (from one organism)
• Each E.coli cell receives PCR product from JUST
  one organism.
• Each E.coli cell can be grown up into colonies -
  amplifying PCR product from each organism.

A
PCR product
A
LIGATE
T
T
vector
TRANSFORM INTO E. coli
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Choosing Markers

• All DNA does NOT evolve at the same rate
• General Rule
• genes coding for essential functions evolve
  slowly
• non-coding DNA usually evolves faster
• Need to choose gene (or marker) carefully

difference
Need 1-20 differences
No. of base differences
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Which marker for which purpose?

• How long ago did organism A and organism B last
  have a common ancestor?
• Very recently - RAPDs/VNTRs/Microsatellites/
  resistance genes
• 10,000s - 100,000s yrs - RNA- ITS, various
  protein- coding genes
• 100,000s - 1,000,000s yrs - ssu rRNA, HSPs,

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rRNA genes - the ideal markers for microbial
identification

• Small subunit - highest order differences
  (domains)
• Large subunit - medium order differences
• ITS - low order differences (species/strains?)
• Small Sub-Unit rRNA (16S)
• ubiquitous
• 1.6 - 2.0kb
• good molecular chronometer.
• some areas conserved (for priming/alignment)
• some areas variable (for resolving differences)

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DGGEDenaturing Gradient Gel Electrophoresis

• Purpose to electrophoretically separate PCR
  fragments that differ by a few bases.
• PCR hypervariable fragment of gene.
• Make an agarose gel that has a gradient of
  denaturant
• Run PCR products on gel.
• Separation depends on motility in denaturing
  environment

Normal agarose gel
Denaturing gel
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SSCPsingle-strand conformation polymorphism

• Theory the conformation of single-stranded DNA
  is dependent on its primary sequence
• Purpose to electrophoretically separate PCR
  fragments that differ by a few bases.
• PCR hypervariable fragment of gene.
• Denature into single-strands by heating.
• Run on a polyacrylamide gel
• PCR products with different sequences will run a
  different speeds on the gel.

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ARDRAAmplified Ribosomal DNA Restriction Analysis

• rRNA gene is PCRd
• Cloned into E. coli
• rRNA gene is amplified out of E.coli
• DNA is digested with restriction enzymes
• Restriction digest is run out on an agarose gel.
• Restriction patterns are identified.
• Can identify ARDRA types by sequencing or just
  make measure of diversity by numbers of patterns

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DNA Sequencing high resolution analysis

• Purpose to identify the DNA sequence. This is
  the ultimate analysis - but is expensive.
• Partial Sequencing - sequencing a gene fragment
  to check its identity against other organisms in
  the database. If you use an invariable gene or
  region two species may share the same sequence.

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Sequencing