BIOL 3000 DNA Barcoding

Linnaean Taxonomy System

Based on morphological characteristics of individuals. Classical taxonomy falls short in cataloging biodiversity.

Kingdom Phylum Class Order Family Genus Species

DNA Barcode

Unique pattern of DNA sequence that identifies each living thing

Dr. Paul Hebert

“Father of DNA Barcode”

1. Help with “Hard-to-Identify” specimens

2. A new tool allowing non-experts to identify specimens

Limitations of Traditional Taxonomy Approach

• Phenotypic plasticity employed for species level identification can lead to incorrect IDS

• Morphologically cryptic taxa can be overlooked

• Morphological keys are often effective only in certain life stages

  • Expertise is required for sample analysis and identification

What are we looking for in a DNA Barcode?

Universal, differentiating, and size

Universal

It has to be easily identifiable in all species

Differentiating

Variable enough to provide a unique sequence for each species

Size

Long enough to provide differential sequence but short enough to sequence quickly, efficiently and cheaply

How many base positions?

600-700 base pairs, so 15 positions and there are 4 alternate characters for each position

Which gene?

Mitochondrial genome

Mitochondrial genome

• Plentiful mitochondria in the cell

• Lacks introns

• Limited recombination because haploid

• Mix of highly variable and conserved regions

Mitochondrial genes

Only codes for 13 individual genes, energy production (ETC). Can only use any gene of the ETC, but settled on the Cytochrome C Oxidase (COX)

Cytochrome C Oxidase (COX)

• Robust primers

• Increased phylogenetic signal

How does DNA Barcoding work?

1. Sample collection

2. DNA amplification

3. Sequencing and Analysis

Sample collection

Effective DNA barcoding depends on the quality of the biological material

Animals: freeze whole specimens at -20C, fixation in 95% pure Ethanol

Plants: Silica gel

Ancient DNA: Depends on age of samples and source used for DNA extraction

DNA Amplification

Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR)

Molecular biology technique used to amplify a small amount of DNA several orders of magnitude

What is used in PCR?

Template DNA, specific primers (sense and antisense), DNA polymerase (Taq polymerase), Di-nucleotides (dNTPs of A, T, C, and G), buffer system, and cations (Mg+2, K+2)

Taq polymerase

Isolated from bacteria that lives in hot environments

Steps of PCR

1. Denaturation (94-96°C): Hydrogen bonds tend to break first

2. Annealing (65-68°C)

3. Elongation (72-74°C): Turns one strand into two. Exponential amplification of DNA fragment of interest

Sequencing and Analysis

Sanger Sequencing

Sanger Sequencing

Using purified DNA template, specific primer, Taq polymerase, Di-nucleotides (dNTPs of A, T, C, and G), labeled di-deoxynucleotides (ddNTPs of A, T, C, and G), buffer system, and cations (Mg+2, K+2)

Di-deoxyribonucleotides

Cannot add anything to the 3’ carbon

Steps of Sanger Sequencing

Denaturation (94-96°C)

Annealing (65-68°C)

Elongation (72-74°C): Adds every base at line until at random it will grab a di-deoxyribose which will stop elongation.

Why barcoding?

1. Works with DNA fragments

2. Works with all stages of life

3. Allows look-a-likes and mimics to be identified

4. Reduces ambiguities in identification

5. Makes expertise go further, doesn’t require much

6. Speeds up determination of the Encyclopedia of Life

How is DNA Barcoding used?

1. Species Identification

2. Consumer Protection

3. Health related issues

4. Identify ancient specimen