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DNA-DNA hybridisationDNA-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.
RAPD, AFLP, MinisatellitesRAPD, 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
PCR-RFLP / CAP 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)
CAPSCAPS
580 bp
290 bp
180 bp
580 bp 180 bp
180 bp290 bp290 bp
PCR products of the trnS-trnM region (cpDNA) restricted by TaqI
PCR-RFLP / CAP 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).
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
Restricted by DraI (TTTAAA)
DNA SequencingDNA 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.
DNA SequencingDNA 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
Amino Acid SequencingAmino 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).
Single-Letter Amino Acid CodeSingle-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 SequencingAmino 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.