Molecular Techniques in Molecular Systematics

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

Multiple Sequence Alignment using CLUSTAL

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.