Molecular Techniques in Molecular Systematics

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Molecular Techniques in Molecular Systematics
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DNA-DNA hybridisation

• Measures the degree of genetic similarity between
  pools of DNA sequences.
• Normally used to determine the genetic distance
  between two species.
• The method compares the melting of a labeled
  sample after it is hybridized to iteself vs its
  melting after hybridized to unlabeled DNA of
  another organism.

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RAPD, AFLP, Minisatellites

• Data is scored as presence (1) or absence (0) of
  a DNA band/fragment.
• Suitable for studies involving closely related
  taxa because the variation detected by these
  markers is high.
• Homology problem may arise due to co-migration of
  DNA bands/fragments.

RADP data
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PCR-RFLP / CAP

• Data scored as presence (1) or absence (0) of a
  restriction site.
• 4-base and 6-base cutters are used to generate
  data.
• Data scoring becomes difficult if the variations
  involve length mutations (deletions or insertions
  / indels)

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CAPS
PCR products of the trnS-trnM region (cpDNA)
restricted by TaqI
580 bp
290 bp
180 bp
580 bp
180 bp
180 bp
290 bp
290 bp
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PCR-RFLP / CAP

• The probability of restriction site loss is
  higher than restriction site gain.
• A site loss could be due to various
  character-states (conditions).

Restricted by DraI (TTTAAA)
Taxon Species A Species B Species C Species D
Species E
Sequence CGTATTTAAACCGCTC
CGTATTTAAACCGCTC CGTATTCAAACCGCTC
CGTATTGAAACCGCTC CGTATTTCAACCGCTC
Restriction Cut Cut No Cut No Cut
No Cut
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DNA Sequencing

• DNA data is multiple state data. It normally
  exist in 4 different bases (A, T, C and G).
• DNA data must be aligned (multiple sequence
  alignment) in order to be scored.
• CLUSTAL software is used to align DNA sequences.

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DNA Sequencing
Multiple Sequence Alignment using CLUSTAL
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DNA Sequencing

• The evolution of DNA sequences is various
  depending on DNA regions (protein coding region,
  non-protein coding region, intergenic spacer,
  intron, repetitive DNA region etc.)
• For protein coding region, the mutation rate for
  synonymous base substitution is higher than
  non-synonymous base substitution.

Taxon Species A Species B Species C
DNA Sequence TTT GTT TGG TTT GTA TGG TTT TTT
TCG
Amino acid sequece Phe-Val-Trp Phe-Val-Trp
Phe-Phe-Trp
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Amino Acid Sequencing

• Amino acid sequencing was practiced before the
  establishment of DNA sequencing methods.
• Amino acid data is a multiple state data. It
  consists of 20 states (20 different amino acids).

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Single-Letter Amino Acid Code
G - Glycine (Gly) P - Proline (Pro) A - Alanine
(Ala) V - Valine (Val) L - Leucine (Leu) I -
Isoleucine (Ile) M - Methionine (Met) C -
Cysteine (Cys) F - Phenylalanine (Phe) Y -
Tyrosine (Tyr)
W - Tryptophan (Trp) H - Histidine (His) K -
Lysine (Lys) R - Arginine (Arg) Q - Glutamine
(Gln) N - Asparagine (Asn) E - Glutamic Acid
(Glu) D - Aspartic Acid (Asp) S - Serine (Ser)
T - Threonine (Thr)
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Amino Acid Sequencing

• Amino acid sequencing is time consuming. It is
  done by HPLC approach.
• At present the amino acid sequences are generated
  mainly from the inference of protein coding DNA
  sequences.
• Amino acid of hemoglobin (in animals) and amino
  acid of large subunit ribulose-1,5-bisphophate
  carboxylase (rbcL in plants) are widely used for
  phylogenetic studies.