Generate a DNA Barcode and Identify Species
Is there something fishy about what you’re eating?
Bio-Rad Biotechnology Explorer
Fish DNA Barcoding Kit and DNA Barcoding Sequencing Module
Instructors - Bio-Rad Curriculum and Training
Specialists
Damon Tighe,
damon_tighe@bio-rad.com
Sherri Andrews, Ph.D.
sherri_andrews@bio-rad.com
Leigh Brown, M.A.
leigh_brown@bio-rad.com
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Workshop Timeline
 Introduction
 Fish DNA extraction
 Gel electrophoresis
 DNA visualization with UViewTM
 Bioinformatics and species identification
 Inquiry Questions
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Diversity of Life
 It is estimated that there are 10 -100 million species of
organisms on Earth
 Only about 1.7 million species have been formally identified
 Current limitation to studies of biological diversity - humans are
limited in their ability to recognize and recall morphological
variation
 Few taxonomists can even reliably identify a collection of ~1000
species
 How do we complete the task of identifying the remaining
species, let alone recognizing them once they are identified?
 Solution – Create a genetic based identification system (DNA
barcode)
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Visual Classification
 Some distinct species are not easy to differentiate by eye…
vs
or
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What is DNA Barcoding?

A worldwide effort (International Barcode of Life, iBOL) exists to “barcode” or
generate standard genetic sequence identification of all species on Earth.

What is a barcode?
– UPC (Universal Product Code) Symbol – 11 variable positions
with 10 possible numbers
– Ability to assign a unique identifier to over 100 billion items

What is a DNA barcode?
– Use of a designated DNA sequence to serve as a unique species identifier
– Ideal sequence is constrained by overall conservation (preserve gene function), but
still has substantial sequence variation which differentiates species
CCCTCCTA
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Barcode Of Life (BOL)
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iBOL
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International Barcode of Life Project
Hub is at Biodiversity Institute of Ontario (BIO) at U Guelph
Goal to generate 5 million barcodes representing 500,000 species
Currently: 2 million barcodes representing 300,000 species in the database
– Opportunity to contribute to the global initiative to barcode life on Earth!
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Be a citizen scientist!

Participate in the largest biodiversity cataloging project
ever undertaken and help build a genetic registry of life

Design a market study to look at local food supply,
or local flora and fauna
Axolotl / Mexican salamander (critically endangered)
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“Sushigate”
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2008, two 11th graders in New York did a market substitution study
Surveyed 60 samples collected from 4 restaurants and 10 grocery stores
Of the 60 samples, 54 could be genetically identified
13 of the 54 were mislabeled (23%)!
2/4 restaurants and 6/10 grocery stores had sold mislabeled fish
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“Sushigate”
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7/9 samples listed as Red Snapper were mislabeled, and included
substitutions: Acadian redfish from North Atlantic, Pinjalo from SE Asia, Lavender
jobfish from So. Pacific, Nile perch from Africa, and Atlantic Cod.
Spotted Goatfish (restricted to the Caribbean) sold as Mediterranean Red Mullet
White Bass (farmed freshwater fish) sold as Sea Bass
Smelt Roe sold as Flying Fish Roe
White (albacore) tuna sushi was Mozambique tilapia (commonly farmed)
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Food Fraud in the News
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DNA Barcode Region Defined
Genes Designated as Barcode Regions:

Fungi
– ITS – nuclear ribosomal internal transcribed spacer region

Plants – 2 genes required
– rbcL – chloroplast ribulose-1,5-bisphosphate carboxylate
– matK – chloroplast maturase K

Animals
– COI – mitochondrial cytochrome C oxidase subunit I

Why COI?
–
–
–
–
–
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Mitochondrial genome lacks introns
Limited exposure to recombination
Haploid mode of inheritance
Universal primers are robust
Hundreds to thousands of mitochondria/cell – this
means many more copies of the COI gene in your sample!
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Mitochondrial
DNA
Applications of DNA Barcoding
What did I eat
last night (and
is it what they
said it was)?
What did I
catch
yesterday?
What is the
genetic
signature
of this rare
species?
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Fish DNA Barcoding Kit Start to Finish
DNA BARCODE
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DNA Barcoding Kit Workflow
PCR amplification
Fish sample
Extract genomic DNA
Sequencing,
Sequence Analysis
Gel electrophoresis
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Barcoding Overview
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Barcoding Overview
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Barcoding Overview
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DNA Extraction Overview
+ Resuspension + Lysis
Buffer
Buffer
Bind DNA
to column
(Matrix Solution)
Wash column
with Wash buffer
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Incubate
10 min
at 55oC
+ Neutralization
Buffer
Elute DNA
Quick Guide – Fish Prep
1
2
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Label tubes “1” for fish sample
1, “2” for fish sample 2. Also
label with your initials.
Cut a piece of fish
approximately the size of a
pencil eraser-head, from your
first fish sample. Slice it until
finely minced. Transfer the
sample into microcentrifuge
tube 1.
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2
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Quick Guide – Fish Prep
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Using a new cutting implement, cut a
piece of fish approximately the size of a
pencil eraser-head, from your second fish
sample. Slice it until finely minced.
Transfer the sample into microcentrifuge
tube 2.
4
Add 200 ml of Resuspension to
your two tubes and flick several
times to ensure full submersion of
the fish in the resuspension solution.
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Quick Guide – DNA Extraction
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Add 250 µl of Lysis to each tube and
mix gently by inverting tubes 10 times
to mix contents.
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Incubate samples at 55oC for 10
min. The samples do not need to be
shaken during incubation.
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What is happening during DNA extraction?
Where is the DNA at each step?
 Resuspension
– buffered solution with chelating
agents to destabilize cell membranes
– DNA: pellet (fish) or supernatant?
 Lysis
– alkaline solution that disrupts
membranes, releases DNA,
denatures DNA
– DNA: pellet (fish) or supernatant?
 Heating
– helps to break down tissue to
recover more DNA
 Neutralization
– solution that counteracts the effects of
alkalinity, renatures smaller pieces of DNA,
helps precipitate DNA and remove detergents
– DNA: pellet (fish) or supernatant?
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mito
DNA
nuclear
DNA
What is happening during DNA extraction?
Where is the DNA at each step?
 Matrix
– silica based suspension that binds
DNA but not RNA or proteins
– DNA: column or flow through?
pelleted proteins,
membranes, etc
 Wash
– removes other small particles in the
prep that are nonspecifically bound
to the Matrix
– DNA: column or flow through?
 Elute
– low ionic strength buffer or water
releases DNA from the silica
– DNA: column or flow through?
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mito
DNA
Quick Guide – DNA Extraction
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Add 250 ml of Neutralization to
each tube and mix gently by
inverting tubes 10 times to mix. A
visible cloudy precipitate may form.
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Centrifuge the tubes for 5 min at top
speed (14,000 x g) in the
microcentrifuge. A compact pellet will
form along the side of the tube. The
supernatant contains the DNA.
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Quick Guide – DNA Extraction
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10
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Snap (do not twist!) the bottoms
off of the spin columns and insert
each column into a capless 2 ml
microcentrifuge tube. Label
columns 1 and 2 + your initials.
Transfer the entire supernatant
(500–550 µl) of each fish sample into
the appropriately labeled spin
column. Try not to get any of the
particulates into the spin column
because they will clog the column
and prevent you from continuing.
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1
2
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Quick Guide – DNA Extraction
Thoroughly mix the tube labeled
Matrix to make sure particulates
are completely resuspended
before use.
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Add 200 ml of thoroughly
resuspended Matrix to the first
column and pipet up and down to
mix.
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Using a new pipet tip, add 200 ml of
thoroughly resuspended Matrix to
the second column and pipet up and
down to mix.
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Centrifuge the columns for
30 sec at full speed.
Remove flow through to
waste.
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1
2
2
Quick Guide – DNA Extraction
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Add 500 µl of Wash and wash the
samples by centrifugation for 30 sec.
Remove flow through to waste.
1
Wash
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Repeat wash step and
centrifugation as shown above.
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Quick Guide – DNA Extraction
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Centrifuge columns for a full 2 min to
remove residual traces of Wash and dry
out the samples
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Remove the spin columns and
discard the 2 ml microcentrifuge
wash tubes. Place the spin column
for each sample into a new capless 2
ml tube.
1
2
1
new capless
tube
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Quick Guide – DNA Extraction
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Using a fresh pipet tip for each
sample, add 100 µl of distilled
water to each spin column, being
careful not to touch the resin. Elute
the DNA by centrifuging for 1 min.
Label two clean 2 ml microcentrifuge
tubes (with caps) Fish 1 and Fish 2
and your initials. Transfer the eluted
DNA into the appropriately labeled
tube.
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1
2
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PCR amplification of COI gene

Fish DNA has been extracted

Next step is to amplify a portion of the
mitochondrial COI gene
– Generate enough DNA to visualize on a gel
– Generate enough DNA to send for sequencing
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Assemble reactions of
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Template DNA
Primers
Nucleotides
Taq polymerase
Magnesium chloride

Multiple rounds of thermal cycling to
amplify DNA
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Mitochondrial
DNA
PCR – Degenerate primers
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When trying to amplify DNA from a wide variety of samples (many different fish)
using the same primer set, creating degenerate primers is a useful approach
Determine a consensus sequence derived from several species
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Pike:
Carp:
Tuna:
Bass:
Hake:
CONS.:
A-C-T-G-G-C-T-T-A-G-C
A-C-T-G-G-A-T-T-A-G-C
A-C-T-G-G-G-T-T-A-A-C
A-C-T-G-G-T-T-T-A-G-C
A-C-T-G-G-A-T-T-T-A-C
A-C-T-G-G-N-T-T-A-R-C
The consensus/degenerate primers bind to DNA from all of these fish, whereas
regular primers would only bind to one
The primers used in our Fish DNA barcoding kit contain degenerate positions to
amplify DNA from as many different fish as possible!
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Fish Barcoding PCR Primers
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PCR – Overview
Heat (94oC) to denature DNA strands
Cool (55oC) to anneal primers to template
Warm (72oC) to activate Taq polymerase, which
extends primers and replicates DNA
Repeat 35 cycles
Your PCR products will be given to you now for electrophoresis
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Quick Guide - Electrophoresis
Add 2 µl of UView 6x loading dye to
each sample, using a new pipet tip
each time. Mix samples well.
Load the agarose gel in the following
lane order and volumes, using a new
pipet tip each time:
Lane
1
2
3
4
5
6
7
8
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Sample
EMPTY
EMPTY
20 µl MWR
12 µl (+) E
12 µl (–) E
12 µl 1 E
12 µl 2 E
EMPTY
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200 V
20 min
0.25x TAE
Visualizing DNA after electrophoresis
UViewTM
Nontoxic
Loading dye + Stain
Instant
View with UV
Very sensitive
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Ethidium Bromide
Toxic, Mutagen
Stain
Instant
View with UV
Most sensitive
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Fast BlastTM
Nontoxic
Stain
Requires wait time
View by eye
Less sensitive
Use UV transilluminator to visualize UView or
Ethidium Bromide
1.
2.
3.
4.
5.
MW ruler
(+) control
(-) control
Fish 1
Fish 2
UViewTM
Ethidium
bromide
1
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2
3
4
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Sequencing of PCR products
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Bioinformatics – Alignment
Run
.....
What is the longest run of tails I should
expect for 100 tosses?
R = log1/p (n)
Paul Erdos‐Alfréd Rényi law
R = longest run
p = probability (for “fair” coins its 0.5)
n = number of tosses
Paul Erdos
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Bioinformatics – Alignment
Run
.....
What is the longest run of tails I should
expect for 100 tosses?
R = log1/0.5 (100) = 6.64
Paul Erdos
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….so if I get more than 6.64 tails in a
row when tossing 100 times, I might
wonder if something besides
randomness is going on
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Bioinformatics – Alignment
AATCGTACTG
AACCATTCAG
If I call alignments tails. What is the longest
run of tails I should expect for comparing
two 10 bp sequences?
R = log1/0.25 (100) = 3.32
Sequence lengths multiplied
¼ chance for getting same base
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Bioinformatics – Alignment
DNA is not a 4 sided Coin
- Account for probability of bases being switched out for
each other by a scoring matrix
Match
Transversion
Change of base type – Purine for Pyrimidine
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Transition
Same base type – Purine for Purine
Bioinformatics – Alignment
DNA is not a 4 sided Coin
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G
A
A
T
T
C
A
G
T
T
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Bioinformatics – Alignment
DNA is not a 4 sided Coin
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G
A
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T
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A
G
T
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G
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G
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Bioinformatics – BLAST tool
Query
Database
ATTCGCAT
ATT
TTC
TCG
CGC
GCA
CAT
1) Break into words
2) Find Matches and let
go of all the other
database words that
don’t match
3) Extend from match 1 base at a time until score falls off
4) Use two anchors to define and alignment, compare, score
E-value
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Bioinformatics – BLAST tool
E-value
Theoretically, we could trust any result with an E-value ≤ 1
In practice – BLAST uses estimations.
• E-values of 10-4 and lower indicate a significant homology.
• E-values between 10-4 and 10-2 should be checked (similar
domains, maybe non-homologous).
• E-values between 10-2 and 1 do not indicate a good homology
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Bioinformatics – Linking DNA Barcoding and
Protein Profiler
+
Compare light chain of myosin sizes
Compare E-value for CO1 gene
=
Stronger Evidence for Evolutionary Relationship
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Bioinformatics using BOLD-SDP
BOLD-SDP = Barcode Of Life Data systems – Student Data Portal
Quick Start tutorial available online
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Create an Instructor Account
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Fill Out Required Information
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Receive Two Important Emails – Login and
Registration Keys
Registration keys
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Log In and Register a New Course
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Fill in Course Info,List Students, Receive Class
Login
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Students Log In and Enter Specimen Info
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Enter Specimen Information
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View Class Specimen List
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Upload Sequencing Data Files
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Select PCR and Sequencing Primers Used
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Class List Updates with Specimen Records
Uploaded
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View Data
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Uploaded trace files receive data quality
assessment
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Trace files viewable
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Quality Scores
Light blue bars in background represent assigned quality value for each nucleotide
(scale on right axis)
High quality values
Examine peaks
Low quality value
Examine peaks (mixed call – overlap)
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Generate contig (“sequence”) from trace files
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Examine base calls in contig
Contig
sequence
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Contig generated,
trim primer sequences
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Run contaminant check and submit contig
Contig 573 bp with no ambiguous nucleotides
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Data summary
barcode
generated
from data
contig
sequence
translation
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Search full database for genetic match
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Search species database for genetic match
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Species match!
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Education and DNA Barcoding - Resources
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www.educationandbarcoding.org
Links to content to aid in classroom presentations
Check out ongoing student barcoding campaigns
Register your own barcoding campaign!
Link to BOLD-SDP workbench (Student Data Portal)
Engage in citizen science
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Student Inquiry
Questions to consider:
– How important is each step in the lab protocol?
– What part of the protocol can I manipulate to see a change
in the results?
– Possible variables / questions:
• How will results be affected by the use of different fish sources
(fresh, frozen, dried, canned)?
• Will different fish tissue yield better results (muscle vs fin, gills,
or scales)?
• Cleanliness and attention to detail during fish processing
– How do I ensure the changes I make are what actually
affects the outcome (importance of controls).
– Write the protocol. After approval – do it!
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Student Inquiry - Teacher Considerations
 What materials and equipment do I have on hand,
and what will I need to order?
– Extra gels, different organisms?
– Other supplies depending on student questions
– Consider buying extras in bulk or as refills – many have 1
year + shelf life.
 What additional prep work will I need?
– Order supplies
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Student Inquiry - Teacher Considerations
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