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Molecular Techniques in
Molecular Systematics
DNA-DNA hybridisation
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Measures the degree of genetic similarity
between pools of DNA sequences.
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Normally used to determine the genetic
distance between two species.
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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.
RAPD, AFLP, Minisatellites
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Data is scored as presence (1) or absence (0) of
a DNA band/fragment.
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Suitable for studies involving closely related
taxa because the variation detected by these
markers is high.
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Homology problem may arise due to comigration of DNA bands/fragments.
RADP data
PCR-RFLP / CAP
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Data scored as presence (1) or absence (0) of a
restriction site.
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4-base and 6-base cutters are used to generate
data.
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Data scoring becomes difficult if the variations
involve length mutations (deletions or
insertions / indels)
CAPS
PCR products of the trnS-trnM region (cpDNA) restricted by TaqI
580 bp
290 bp
180 bp
580 bp
290 bp
180 bp
290 bp
180 bp
PCR-RFLP / CAP
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The probability of restriction site loss is higher
than restriction site gain.
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A site loss could be due to various characterstates (conditions).
Restricted by DraI (TTTAAA)
Taxon
Species
Species
Species
Species
Species
Sequence
A
B
C
D
E
CGTATTTAAACCGCTC
CGTATTTAAACCGCTC
CGTATTCAAACCGCTC
CGTATTGAAACCGCTC
CGTATTTCAACCGCTC
Restriction
Cut
Cut
No Cut
No Cut
No Cut
DNA Sequencing
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DNA data is multiple state data. It normally exist
in 4 different bases (A, T, C and G).
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DNA data must be aligned (multiple sequence
alignment) in order to be scored.
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CLUSTAL software is used to align DNA
sequences.
DNA Sequencing
Multiple Sequence Alignment using CLUSTAL
DNA Sequencing
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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.)
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For protein coding region, the mutation rate for
synonymous base substitution is higher than
non-synonymous base substitution.
Taxon
DNA Sequence
Amino acid sequece
Species A
Species B
Species C
TTT GTT TGG
TTT GTA TGG
TTT TTT TCG
Phe-Val-Trp
Phe-Val-Trp
Phe-Phe-Trp
Amino Acid Sequencing
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Amino acid sequencing was practiced before the
establishment of DNA sequencing methods.
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Amino acid data is a multiple state data. It consists
of 20 states (20 different amino acids).
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)
Amino Acid Sequencing
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Amino acid sequencing is time consuming. It is done
by HPLC approach.
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At present the amino acid sequences are generated
mainly from the inference of protein coding DNA
sequences.
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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.