DNA Barcoding with Cytochrome c Oxidase Subunit I (COI)

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DNA Barcoding with Cytochrome c Oxidase Subunit I (COI)

Introduction to DNA Barcoding

DNA barcoding is a molecular technique that uses short, standardized genetic markers to identify species. The concept, formalized by Hebert et al. (2003), relies on the principle that genetic variation between species exceeds variation within species for specific marker genes.

Why COI for Animals?

The mitochondrial cytochrome c oxidase subunit I (COI) gene has become the standard barcode for animals because:

1. Universal across animals: Present in all eukaryotic organisms with mitochondria

2. Sufficient variability: Shows enough sequence divergence between species while being conserved within species

3. Low recombination: Mitochondrial genome is maternally inherited and rarely recombines

4. High copy number: Mitochondrial DNA is abundant in cells (hundreds to thousands of copies per cell)

5. Conserved flanking regions: Allows design of "universal" primers that work across many taxa

Key Reference:

> Hebert, P. D. N., Cywinska, A., Ball, S. L., & deWaard, J. R. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society of London. Series B: Biological Sciences, 270(1512), 313-321. https://doi.org/10.1098/rspb.2002.2218

COI Barcoding in Mosquitoes

The Challenge with Folmer Primers

The original "universal" COI primers designed by Folmer et al. (1994) work well for many invertebrates but have poor success rates with mosquitoes. Studies show only 16.7% amplification success in mosquitoes using standard Folmer primers (LCO1490/HCO2198).

Folmer Primers:

LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3'
HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'
Amplicon size: ~710 bp

Key Reference:

> Folmer, O., Black, M., Hoeh, W., Lutz, R., & Vrijenhoek, R. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3(5), 294-299.

The AU-COI Primers: A Better Solution

Hoque et al. (2022) designed modified primers specifically optimized for mosquitoes. These primers incorporate degenerate bases at positions where mosquito mitochondrial genomes show variation.

AU-COI Primers:

AU-COI-F: 5'-TATTTTCWACAAATCATAARGATATTGGWAC-3'
AU-COI-R: 5'-TAWACTTCWGGRTGWCCRAARAATCA-3'
Amplicon size: ~712 bp
Success rate: 67.5% in mosquitoes (4× better than Folmer)

Degenerate Base Codes:

W = A or T
R = A or G

Why These Work Better:

1. Account for sequence variation at wobble positions in mosquito COI genes

2. Bind to more conserved regions flanking the barcode region

3. Optimized annealing temperature for mosquito templates

4. Tested on 40 mosquito species across multiple genera

Key Reference:

> Hoque, M. M., Arafat, Y., Ahmed, T., Hasan, M., & Ullah, M. S. (2022). Development of species-specific primers for DNA barcoding of mosquitoes (Diptera: Culicidae) in Bangladesh. PLoS ONE, 17(7), e0270030. https://doi.org/10.1371/journal.pone.0270030

Target Species in ENTM201L

In this lab, we are working with mosquitoes from the genus Aedes, which are important vectors of arboviruses:

Aedes aegypti (Yellow Fever Mosquito)

Native range: Africa
Current distribution: Pantropical (worldwide in tropics and subtropics)
Medical importance: Primary vector of dengue, Zika, chikungunya, yellow fever
Morphology: White lyre-shaped markings on thorax, banded legs
COI barcode: GenBank accession KC920138 (648 bp)

Aedes albopictus (Asian Tiger Mosquito)

Native range: Southeast Asia
Current distribution: Invasive worldwide, established in California since 2001
Medical importance: Secondary vector of dengue, chikungunya, Zika
Morphology: White stripe down center of thorax and head, banded legs
COI barcode: GenBank accession MN736318 (658 bp)

References:

Kraemer, M. U., et al. (2015). The global distribution of the arbovirus vectors Aedes aegypti and Ae. albopictus. eLife, 4, e08460. https://doi.org/10.7554/eLife.08460
Gloria-Soria, A., et al. (2016). Global genetic diversity of Aedes aegypti. Molecular Ecology, 25(21), 5377-5395. https://doi.org/10.1111/mec.13866

PCR Amplification Strategy

Enzyme Selection: Q5 High-Fidelity DNA Polymerase

We use Q5® High-Fidelity DNA Polymerase (New England Biolabs) for COI amplification:

Advantages:

1. Proofreading ability: 3'→5' exonuclease activity corrects errors (100× more accurate than Taq)

2. High fidelity: Error rate of 1 in 10^6 bases (compared to 1 in 10^4 for Taq)

3. Robust amplification: Works with difficult templates (GC-rich, repetitive)

4. High yield: Produces sufficient product for Sanger sequencing

5. Generates blunt ends: Easier for downstream cloning applications

Why Fidelity Matters for Barcoding:

Single nucleotide differences distinguish closely related species
PCR errors could create false variation that confuses species identification
Sequencing authentic DNA sequence is critical for database submission

Reference:

> New England Biolabs (2023). Q5® High-Fidelity DNA Polymerase. https://www.neb.com/products/m0491-q5-high-fidelity-dna-polymerase

PCR Reaction Setup

Master Mix Composition (25 μL reaction):

Component	Volume (μL)	Final Concentration
Q5® 2X Master Mix	12.5	1×
10 μM AU-COI-F Primer	1.25	0.5 μM
10 μM AU-COI-R Primer	1.25	0.5 μM
Nuclease-free H₂O	8.0	—
Master Mix Subtotal	23.0	—
Template DNA (from extraction)	1-2	~10-50 ng
Nuclease-free H₂O (if needed)	0-1	—
Total Reaction	25.0	—

Q5® 2X Master Mix Contents (Pre-formulated):

Q5 High-Fidelity DNA Polymerase (0.02 U/μL final)
dNTPs (200 μM each - dATP, dGTP, dCTP, dTTP)
Q5 Reaction Buffer (1X - includes Mg²⁺)

Thermocycler Program

Standard COI PCR Protocol for Folmer/AU-COI Primers:

Step	Temperature	Time	Cycles
Initial Denaturation	94°C	2 minutes	1×
Cycling:
Denaturation	94°C	30 seconds	35×
Annealing	48°C	30 seconds	35×
Extension	72°C	1 minute	35×
Final Extension	72°C	7 minutes	1×
Hold	4°C	∞	—

Notes on Thermocycler Program:

94°C denaturation: Standard temperature for COI gene amplification; 2-minute initial denaturation ensures complete separation of template DNA strands
30 sec denaturation cycles: Provides adequate time to fully denature DNA at 94°C without excessive primer denaturation
48°C annealing: Standard for COI Folmer/AU-COI primers (LCO1490/HCO2198); this lower Tm (~48-50°C) accommodates degenerate bases in AU-COI primers and sequence variation in mosquito COI genes
30 sec annealing time: Standard timing for primer binding; provides sufficient time for primer-template hybridization and accounts for degenerate base pairing in AU-COI primers
72°C extension: Standard temperature for polymerase extension; 1 minute accommodates 712 bp product amplification (~1 kb/min for most polymerases)
35 cycles: Standard cycle number for COI barcoding; optimal for mitochondrial COI amplification without excessive template depletion
7 min final extension: Extended final extension ensures all PCR products are completely synthesized and 3' polished, critical for downstream Sanger sequencing

Expected Results

Successful Amplification:

Single band at ~712 bp on agarose gel
Band intensity indicates sufficient DNA for Sanger sequencing (>20 ng/μL)
No primer dimers (~60 bp) or non-specific products

Troubleshooting:

No band: Insufficient template, primer degradation, incorrect thermocycler program
Multiple bands: Non-specific amplification, contamination, primer design issues
Primer dimers: Excess primers, low annealing temperature, insufficient extension time

Sanger Sequencing and Analysis

Sequencing Preparation

After successful PCR amplification:

1. Gel electrophoresis: Verify product size and purity

2. PCR cleanup: Remove primers and dNTPs (ExoSAP-IT or column purification)

3. Quantification: Measure DNA concentration (NanoDrop, Qubit, or gel band intensity)

4. Sequencing submission: Submit 20-50 ng/μL purified PCR product with primers

Sequencing Primers:

Use the same AU-COI-F and AU-COI-R primers for sequencing
Sequence both strands (forward and reverse) for quality control
Typical read length: 800-900 bp (covers entire 712 bp amplicon)

Bioinformatics Pipeline

Step 1: Quality Control and Trimming

View chromatograms in SnapGene, Geneious, or ApE
Trim low-quality ends (Phred score < 20)
Reverse complement the reverse read

Step 2: Consensus Sequence Assembly

Align forward and reverse reads
Generate consensus sequence
Resolve any ambiguities by referring to chromatograms

Step 3: BLAST Search

Submit consensus to NCBI BLAST (https://blast.ncbi.nlm.nih.gov/)
Use Nucleotide BLAST (blastn) against "Nucleotide collection (nr/nt)"
Filter for "Culicidae" (mosquito family) to avoid false hits

Step 4: Species Identification

Top hit with >97% identity typically indicates species-level match
95-97% identity may indicate subspecies or geographic variation
<95% identity suggests potentially novel species or barcode gap

Reference Sequences for Comparison:

Aedes aegypti COI: GenBank KC920138
Aedes albopictus COI: GenBank MN736318

Phylogenetic Analysis (Module 10)

Once all students have sequenced their samples, we will:

1. Multiple sequence alignment: MAFFT or MUSCLE

2. Phylogenetic tree inference: IQ-TREE2 (maximum likelihood)

3. Visualization: iTOL or FigTree

4. Interpretation: Do samples cluster by species? Any unexpected results?

Medical and Ecological Importance

Why Species Identification Matters

Disease Transmission:

Aedes aegypti is the primary vector of dengue, with higher vectorial capacity
Aedes albopictus is a secondary vector but more cold-tolerant (range expanding)
Correct species ID informs vector control strategies and risk assessment

Invasive Species Monitoring:

Aedes albopictus is invasive in California (first detected 2001, now widespread)
DNA barcoding enables rapid identification of new invasions
Morphological ID requires taxonomic expertise; DNA barcoding is more accessible

Insecticide Resistance:

Different populations show different resistance alleles
Genetic monitoring (including barcoding) tracks resistance spread
COI is neutral marker, but linked to other mitochondrial genes under selection

Climate Change:

Both species expanding northward as climate warms
Monitoring species distributions requires accurate identification
DNA barcoding creates permanent genetic records for long-term studies

References:

Powell, J. R., & Tabachnick, W. J. (2013). History of domestication and spread of Aedes aegypti—A Review. Memórias do Instituto Oswaldo Cruz, 108(suppl 1), 11-17. https://doi.org/10.1590/0074-0276130395
Bonizzoni, M., et al. (2013). The invasive mosquito species Aedes albopictus: current knowledge and future perspectives. Trends in Parasitology, 29(9), 460-468. https://doi.org/10.1016/j.pt.2013.07.003

Learning Objectives

By the end of this lab, you should be able to:

1. Explain the principle of DNA barcoding and why COI is used for animals

2. Describe the advantages of AU-COI primers over Folmer primers for mosquitoes

3. Set up a PCR reaction using Q5 High-Fidelity DNA Polymerase

4. Interpret gel electrophoresis results to assess PCR success

5. Use BLAST to identify species from COI sequences

6. Discuss the medical importance of Aedes species identification

7. Appreciate how molecular tools complement traditional morphological taxonomy

Pre-Lab Quiz Questions

Test your understanding before lab:

1. What is the approximate size of the PCR product amplified by AU-COI primers?

2. Why is Q5 polymerase preferred over Taq polymerase for DNA barcoding?

3. What percentage of mosquito species were successfully amplified by AU-COI primers in Hoque et al. (2022)?

4. What does a BLAST identity of 98% typically indicate?

5. Name two diseases transmitted by Aedes aegypti.

6. Why is Aedes albopictus considered more invasive than Aedes aegypti?

7. What temperature is used for annealing in the Q5 PCR protocol?

8. What is a "degenerate base" and why are they used in the AU-COI primers?

Additional Resources

Databases:

BOLD Systems: http://www.boldsystems.org/ (Barcode of Life Database)
NCBI GenBank: https://www.ncbi.nlm.nih.gov/genbank/
VectorBase: https://vectorbase.org/ (Invertebrate vectors of disease)

Tools:

NCBI BLAST: https://blast.ncbi.nlm.nih.gov/
Primer-BLAST: https://www.ncbi.nlm.nih.gov/tools/primer-blast/
IQ-TREE: http://www.iqtree.org/
MAFFT: https://mafft.cbrc.jp/alignment/server/

Mosquito Identification:

CDC Mosquito ID: https://www.cdc.gov/mosquitoes/about/identify.html
Walter Reed Biosystematics Unit: https://www.wrbu.org/

Next Steps:

1. Review this theoretical background before lab

2. Complete pre-lab quiz on Canvas

3. Bring lab notebook and safety goggles to lab

4. Review PCR setup protocols and thermocycler operation

Document prepared for ENTM201L - Molecular Entomology: DNA Barcoding Laboratory UC Riverside, Department of Entomology Fall 2025

Scientific Literature References

Folmer et al. (1994) - Universal COI Primers

Citation: Folmer, O., Black, M., Hoeh, W., Lutz, R., & Vrijenhoek, R. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3(5), 294-299.

PMID: 7881515

Primers: LCO1490 / HCO2198 (~710 bp fragment)

The foundational "Folmer primers" designed from conserved regions across metazoan invertebrates. These universal primers have become the primary tool for metazoan DNA barcoding, generating nearly 3 million COI sequences since 1994.

Hebert et al. (2004) - Lepidoptera-Specific Primers

Citation: Hebert, P. D. N., Penton, E. H., Burns, J. M., Janzen, D. H., & Hallwachs, W. (2004). Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the National Academy of Sciences USA, 101(41), 14812-14817.

DOI: 10.1073/pnas.0406166101 | PMID: 15465915

Primers: LepF1 / LepR1 (~658 bp fragment)

Designed specifically for Lepidoptera to improve amplification success over universal Folmer primers. Landmark paper demonstrating that DNA barcoding could reveal cryptic species complexes - finding 10 distinct species within what was thought to be a single butterfly species.

Miller et al. (2016) - Modified Lepidoptera Primers

Citation: Miller, S. E., Hausmann, A., Hallwachs, W., & Janzen, D. H. (2016). Advancing taxonomy and bioinventories with DNA barcodes. Philosophical Transactions of the Royal Society B, 371(1702), 20150339.

DOI: 10.1098/rstb.2015.0339

Primers: MLepF1 / MLepR1

Modified versions of LepF1/LepR1 designed to improve amplification success in Lepidoptera, especially for degraded DNA samples and museum specimens.

Hebert et al. (2003) - DNA Barcoding Proposal

Citation: Hebert, P. D. N., Cywinska, A., Ball, S. L., & deWaard, J. R. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences, 270(1512), 313-321.

DOI: 10.1098/rspb.2002.2218

The seminal paper that proposed using a standardized 658 bp fragment of the mitochondrial COI gene as a universal barcode for animal species identification. This laid the groundwork for the global DNA barcoding initiative and the Barcode of Life Database (BOLD).