Comparing COX1 Protein Sequences from Different Species
AMANDA BARCLAY, SOLOMON LARSEN, AND ALEX YOUNG
NUCLEOTIDE DATABASES AT THE NATIONAL CENTER OF BIOTECHNOLOGY INFORMATION
ABSTRACT
Cytochrome C Oxidase, also known as COX1, is a mitochondrial
gene that codes for protein that functions in the electron transport chain
within the inner mitochondrial genome. This gene is conserved over a
broad range of eukaryotic organisms, and its sequence is often used in
species identification. Two primers are needed to amplify a DNA segment
by PCR. Our goal was to identify the range of species for which a specific
set of PCR primers can be used to amplify the COX1 gene. We started
with the known COX1 gene sequence from a cricket. We searched the
nucleotide databases at the National Center of Biotechnology Information
webpage using the BLAST search engine tool and the cricket sequence as
a query. We searched a variety of different plant, animal, and fungi
species for COX1 gene sequences and determined where the two primers
are expected to anneal. We then compared the primer sequences to the
gene sequences and estimated the annealing temperature for each primer.
By doing this we have determined that the primers can be used to amplify
the COX1 gene from a variety of animal and fungal species, but not from
plants.
OBJECTIVES
- Determine where the two primers are expected to anneal on a gene
sequence and estimated the annealing temperature for each primer.
- Identify the range of species for which a specific set of PCR primers can be
used to amplify the COX1 gene.
BACKGROUND & OVERVIEW
• Classification of organisms is an ongoing process. New insights are being
gained as DNA sequences are being compared between species.
• Cytochrome C Oxidase (COX1) is a mitochondrial gene that codes for a
protein used in the electron transport chain. A variety of species are known
to carry this sequence in their genome.
• The National Center of Biotechnology Information webpage is used to
obtain information about the genomes of known organisms. There is a
variety of different search tools within the site that allow for biological data
and information to be found and compared between many different species.
• A PCR product can be expected to form if both primers have an annealing
temperature greater than 54 Co, which allows us to determine if a sequence
will amplify or not.
METHODS & MATERIALS RESULTS CONT.
Insect Species with COX1:
Materials:
• Orthetrum glaucum, which is an Asian dragonfly.
• Sequence of two primers used in the PCR amplification.
• Cosmoscarta dorsimacula, which is a froghopper.
Forward primer: 5’-TCTCAACCAACCACAAAGACATTGG-3’
• Peirates turpis, also known as the “Assassin bug.”
Reverse primer: 5’-TAGACTTCTGGGTGGCCAAAGAATCA-3’
• Drosphilia simulans, which is a fruit fly.
• Sequence of the complete cricket COX 1 gene.
• Anopheles argyritarsis, which is a mosquito. Miyakon.com. (n.d.). Retrieved November 27,
• The book “The Tree of Life” Edited by Pablo Vargas and Rafael Zardoya, Sinauer 2022, from
https://www.miyakon.com/kamemusi/sasigame.ht
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Associates, Inc., 2014.
Animal species with COX1:
• The formula to calculate the melting temperature:
Tm = 81.5 + 0.41(%GC)-(675/N)
• Dixiphia pipra voucher, which Is a type of bird.
• The formula to calculate the annealing temperature:
• Polioptila caerulea, which is another kind of bird.
Ta = Tm – 5
• Lasiopodomys brandtii, also known as Brandt’s Vole.
• The nucleotide databases at the National Center of Biotechnology Information
• Piabina argentea, also known as Bony Fish.
webpage using the BLAST search engine tool with the cricket sequence as a query.
• Darevskia valentine, which is a kind of lizard.
Yahoo! (2022, November 27). Static photos on Flickr.
Flickr. Retrieved November 27, 2022, from
https://www.flickr.com/photos/tags/static/
Methods:
Beginning with the complete COX1 gene sequence of a cricket, we conducted CONCLUSIONS
BLAST searches on the National Center of Biotechnology information webpage for a
variety of different plant, animal, and fungi species. We chose species that showed the
highest Percent Identity to the COX1 gene from the cricket. Next, we compared the Species that DO NOT have the COX1 protein sequence:
sequences between positions 16 and 40 and the other between 699 and 724. These are
the regions where the primers should bind if they are similar enough. Using this
• Plants – No species of plants have the COX1 sequence.
information, we calculated the melting temperature and the annealing temperature to
determine if the sequence was similar enough to allow a primer to bind.
Species that DO have the COX1 protein sequence:
Example: • Insects - A wide variety of insect species have the COX1 protein
sequence.
• Animals – A variety of animal species, including reptiles,
chordates, and mammals, have the COX1 protein sequence.
• Fungi - Only two known fungal species have the COX1 protein
sequence.
REFERENCES & ACKNOWLEDGMENTS
RESULTS • U.S. National Library of Medicine. (n.d.). Blast: Basic local alignment search tool. National
Center for Biotechnology Information. Retrieved November 27, 2022, from
We determined that the primers can be used to amplify the COX1 gene https://blast.ncbi.nlm.nih.gov/Blast.cgi
from animal and fungal species, but not from plants. • U.S. National Library of Medicine. (n.d.). Blast: Basic local alignment search tool. National
Center for Biotechnology Information. Retrieved November 27, 2022, from
https://blast.ncbi.nlm.nih.gov/Blast.cgi
Fungal Species with COX1: • U.S. National Library of Medicine. (n.d.). Taxonomy browser (pseudopipra pipra). National
Center for Biotechnology Information. Retrieved November 27, 2022, from
• Mucor piriformus, which causes fruit to mold. https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=415032
&lvl=3&keep=1&srchmode=1&unlock&mod=1&log_op=mod
• Peniophora lycii, which is similar to a lichen. ifier_toggle
Seiya Saito | Plant pathologist | ph.D | united states department of ...
(n.d.). Retrieved November 27, 2022, from
https://www.researchgate.net/profile/Seiya-Saito