Quantitative Measurement How many of each taxa are present

1
Quantitative MeasurementHow many of each taxa
are present?

• Methods
• T-RFLP
• FisH
• Real-time PCR
• ARDRA and DGGE also give an idea of which
  organisms are most abundant.
• WARNING true quantitation is virtually
  impossible!
• PCR bias leads to unfair representation of
  quantity of each species present.

2
T-RFLPTerminal-Random Fragment Length
Polymorphisms

• PCR with fluorescently labelled primers, so all
  PCR products have fluorescent terminal.
• Digest PCR products with restriction enzymes.
• Run restriction digest on a gel and read with a
  laser.
• The brighter the signal the more copies of DNA.
• We can identify individual species by checking
  T-RFLP pattern of pure culture.

Brightness of Fluor.
Size of fragment
3
FisHFluorescence in situ Hybridisation

• Purpose To check whether a particular organism
  exists in a
• sample and in what proportion.
• 1) Find out the sequence of your organism (from
  database).
• 2) Make a probe (oligonucleotide) that is
  complementary to the RNA of the organism and
  label it with fluorescent probe.
• 3) Fix your sample on a microscope slide and wash
  with labelled probe.
• 4) Look down a fluorescence microscope and see in
  which cells the fluorescent probe has bound.

4
Real-time PCR

• Purpose to quantify the amount of template
  that
• is amplified in a PCR reaction.
• Can be used to quantify different species present
  in a community.
• Need Primers specific for the organisms you
  want to quantify.
• A damned expensive machine that
• measures the PCR product as it is
• amplified.

5
Systematics/Taxonomy

• Cladistics - discrete character data
• a series of discrete variables (characters) with
  multiple character states.
• Example in higher organisms - Character 1
  toes
• character states humans 5 toes
• lions 4 toes
• tigers 4 toes
• Character 2 whiskers
• character states humans absent
• lions present
• tigers present
• Character 3 ears
• character states humans 2 ears
• lions 2 ears
• tigers 2 ears

H L T 4 Toes - Whiskers - 2
Ears Tigers and Lions are more closely
related to each other than they are to humans
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Cladistics continued .

• Problem many characters have evolved more than
  once in separate lineages e.g. wings in bats and
  birds
• Molecular Cladistics (Discrete character data)
• examples of characters presence/absence of
  gene
• presence/absence of RFLP band
• A/G/C/T at a particular position
• Example of Discrete Character Method
• Maximum Parsimony.

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Phenetics

• Phenetics - distance data
• a single variable (e.g. DNA hybridisation data)
• Discrete character data can be converted in
  distance data, but distance data can not tell us
  about discrete characters.
• DNA Sequence Data - the percent of base
  differences between sequences can put into a
  distance matrix.
• A B C D
• A 0 10 20 30
• B 0 10 20
• C 0 10

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• A B C D
• A 0 10 20 30
• B 0 20 20
• C 0 20
• A AGGGCCTTAA
• B AGGGCCTTAG
• C AGGGCCTTCC
• D AGGGCCTACG

A B A C C D D B A D
B C
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A B C DA 0 10 20 30B 0 20 20C 0 20
A B AB CD BD AC 10 20 20 20
70 C D A C AC DB CB AD 20 20 20
30 90 D B A D AD BC DB AC 30
20 20 10 80 B C
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Rooted and Unrooted Trees
A B
A B A B
C D
C D C D
A B C D
A B C D
A B C D
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ROOTED TREE TO SCALE
A B C
D
A B C D A 10 20 30 B
20 20 C
20
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Maths

• Phylogeny is not really all that simple!
• A AGGGCCTTAA
• B AGGGCCTTAG
• We need mathematical algorithms to convert
  percentage DIFFERENCE to GENETIC DISTANCE. Also
  most trees have more than 4 branches. These
  algorithms can be computationally difficult. Get
  a computer to do it!!

How do we know that this is one base change? It
could have changed from A to C then C to G