Title: Studying Microbial Biodiversity Step 1: Qualitative Identification What do they look like What do th
1Studying 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
2The Martian View of Cats and Dogs
- CATS DOGS
- 4 legs
- long tail
- two ears
- often live with
- 2-legged things
- prey lives inside tin
3Molecular 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
4Molecular 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
5DNA 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
6Fragment 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
7PCR
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
81. 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
9Cloning 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
10Choosing 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
11Which 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,
12rRNA 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)
13DGGEDenaturing 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
14SSCPsingle-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.
15ARDRAAmplified 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
16DNA 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.
17 Sequencing