Generate A DNA Barcode and Identify Fish Species

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
2 Biotechnology Explorer™ | explorer.bio-rad.com

Workshop Timeline
 Introduction
 Fish DNA extraction
 Gel electrophoresis
 DNA visualization with UViewTM
 Bioinformatics and species identification
 Inquiry Questions

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)

Visual Classification
 Some distinct species are not easy to differentiate by eye…
vs
or

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

Barcode Of Life (BOL)
 iBOL
– 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!

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)

“Sushigate”
 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

“Sushigate”
 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)

Food Fraud in the News

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? Mitochondrial
– Mitochondrial genome lacks introns DNA
– 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!

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?

Fish DNA Barcoding Kit Start to Finish
DNA BARCODE

DNA Barcoding Kit Workflow
PCR amplification
Fish sample Extract genomic DNA
Sequencing,
Sequence Analysis
Gel electrophoresis

Barcoding Overview

Barcoding Overview

Barcoding Overview

DNA Extraction Overview
+ Resuspension + Lysis Incubate

Buffer Buffer 10 min
at 55oC
Bind DNA + Neutralization

to column Buffer
(Matrix Solution)
Wash column Elute DNA

with Wash buffer

Quick Guide – Fish Prep
Label tubes “1” for fish sample

1 1, “2” for fish sample 2. Also
label with your initials.
2
1
Cut a piece of fish

approximately the size of a
pencil eraser-head, from your
2 first fish sample. Slice it until
finely minced. Transfer the
sample into microcentrifuge
tube 1. 1

Quick Guide – Fish Prep
Using a new cutting implement, cut a

piece of fish approximately the size of a
pencil eraser-head, from your second fish
3 sample. Slice it until finely minced.
Transfer the sample into microcentrifuge
tube 2. 2
Add 200 ml of Resuspension to

your two tubes and flick several
4 times to ensure full submersion of
the fish in the resuspension solution.

Quick Guide – DNA Extraction
Add 250 µl of Lysis to each tube and

5 mix gently by inverting tubes 10 times
to mix contents.
Incubate samples at 55oC for 10

6 min. The samples do not need to be
shaken during incubation.

What is happening during DNA extraction?
Where is the DNA at each step?
mito
 Resuspension DNA
– buffered solution with chelating
agents to destabilize cell membranes nuclear
– DNA: pellet (fish) or supernatant? DNA
 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?

What is happening during DNA extraction?
Where is the DNA at each step?
 Matrix
– silica based suspension that binds mito
DNA
DNA but not RNA or proteins
pelleted proteins,
– DNA: column or flow through? 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?

Add 250 ml of Neutralization to

each tube and mix gently by
7
inverting tubes 10 times to mix. A
visible cloudy precipitate may form.
Centrifuge the tubes for 5 min at top

speed (14,000 x g) in the
8 microcentrifuge. A compact pellet will
form along the side of the tube. The
supernatant contains the DNA.

Snap (do not twist!) the bottoms

off of the spin columns and insert
9 each column into a capless 2 ml 1 2
microcentrifuge tube. Label
columns 1 and 2 + your initials.
Transfer the entire supernatant

10 (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 1 2
because they will clog the column
and prevent you from continuing.

Thoroughly mix the tube labeled
Matrix to make sure particulates
are completely resuspended
before use. 1 2
Add 200 ml of thoroughly
11 resuspended Matrix to the first
column and pipet up and down to
mix.
Using a new pipet tip, add 200 ml of

12 thoroughly resuspended Matrix to
the second column and pipet up and
down to mix. 1 2
Centrifuge the columns for
30 sec at full speed.
13
Remove flow through to
waste.

Add 500 µl of Wash and wash the

14 samples by centrifugation for 30 sec. 1 2
Remove flow through to waste.
Wash
15 Repeat wash step and

centrifugation as shown above.

Centrifuge columns for a full 2 min to 1 2

16 remove residual traces of Wash and dry
out the samples
Remove the spin columns and

discard the 2 ml microcentrifuge
17 wash tubes. Place the spin column 1
for each sample into a new capless 2
ml tube.
new capless
tube

Using a fresh pipet tip for each

sample, add 100 µl of distilled
18 water to each spin column, being 1 2
careful not to touch the resin. Elute
the DNA by centrifuging for 1 min.
Label two clean 2 ml microcentrifuge

19 tubes (with caps) Fish 1 and Fish 2
and your initials. Transfer the eluted 1 2
DNA into the appropriately labeled
tube.

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
 Assemble reactions of
– Template DNA
– Primers Mitochondrial
– Nucleotides DNA
– Taq polymerase
– Magnesium chloride
 Multiple rounds of thermal cycling to

amplify DNA

PCR – Degenerate primers
 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
– Pike: A-C-T-G-G-C-T-T-A-G-C
– Carp: A-C-T-G-G-A-T-T-A-G-C
– Tuna: A-C-T-G-G-G-T-T-A-A-C
– Bass: A-C-T-G-G-T-T-T-A-G-C
– Hake: A-C-T-G-G-A-T-T-T-A-C
– CONS.: 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!

Fish Barcoding PCR Primers

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

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 Sample 200 V

1 EMPTY 20 min
2 EMPTY 0.25x TAE
3 20 µl MWR
4 12 µl (+) E
5 12 µl (–) E
6 12 µl 1 E
7 12 µl 2 E
8 EMPTY

Visualizing DNA after electrophoresis
UViewTM Ethidium Bromide Fast BlastTM
Nontoxic Toxic, Mutagen Nontoxic

Loading dye + Stain Stain Stain
Instant Instant Requires wait time
View with UV View with UV View by eye
Very sensitive Most sensitive Less sensitive

Use UV transilluminator to visualize UView or
Ethidium Bromide
1. MW ruler
UViewTM
2. (+) control
3. (-) control
4. Fish 1
5. Fish 2 Ethidium
bromide
1 2 3 4 5

Sequencing of PCR products

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

Run
.....
What is the longest run of tails I should

expect for 100 tosses?
R = log1/0.5 (100) = 6.64
….so if I get more than 6.64 tails in a
row when tossing 100 times, I might
wonder if something besides
Paul Erdos randomness is going on
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

DNA is not a 4 sided Coin

- Account for probability of bases being switched out for
each other by a scoring matrix
Match
Transversion Transition
Change of base type – Purine for Pyrimidine Same base type – Purine for Purine

G A A T T C A G T T A
G 1 1 1 1 1 1 1 1 1 1 1
G 1 1 1 1 1 1 1 2 2 2 2
A 1 2 2 2 2 2 2 2 2 2 2
T 1 2 2 3 3 3 3 3 3 3 3
C 1 2 2 3 3 4 4 4 4 4 4
G 1 2 2 3 3 4 4 5 5 5 5
A 1 2 3 3 3 4 5 5 5 5 6

G A A T T C A G T T A
G 1 1 1 1 1 1 1 1 1 1 1
G 1 1 1 1 1 1 1 2 2 2 2
A 1 2 2 2 2 2 2 2 2 2 2
T 1 2 2 3 3 3 3 3 3 3 3
C 1 2 2 3 3 4 4 4 4 4 4
G 1 2 2 3 3 4 4 5 5 5
A 1 2 3 3 3 4 5 5 5 5 6

Bioinformatics – BLAST tool
Query Database
ATTCGCAT
ATT 1) Break into words

TTC
2) Find Matches and let
TCG
go of all the other
CGC
database words that
GCA
don’t match
CAT
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
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

Bioinformatics – Linking DNA Barcoding and
Protein Profiler
+
Compare light chain of myosin sizes Compare E-value for CO1 gene
Stronger Evidence for Evolutionary Relationship

=
Bioinformatics using BOLD-SDP
BOLD-SDP = Barcode Of Life Data systems – Student Data Portal
Quick Start tutorial available online

Create an Instructor Account

Fill Out Required Information

Receive Two Important Emails – Login and
Registration Keys
Registration keys

Log In and Register a New Course

Fill in Course Info,List Students, Receive Class
Login

Students Log In and Enter Specimen Info

Enter Specimen Information

View Class Specimen List

Upload Sequencing Data Files

Select PCR and Sequencing Primers Used

Class List Updates with Specimen Records
Uploaded

View Data

Uploaded trace files receive data quality
assessment

Trace files viewable

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)

Generate contig (“sequence”) from trace files

Examine base calls in contig
Contig
sequence

Contig generated,
trim primer sequences

Run contaminant check and submit contig
Contig 573 bp with no ambiguous nucleotides

Data summary
barcode
generated
from data
contig
sequence
translation

Search full database for genetic match

Search species database for genetic match

Species match!

Education and DNA Barcoding - Resources
 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

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!

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

Student Inquiry - Teacher Considerations

Generate A DNA Barcode and Identify Fish Species

Enviado por

Dados do documento

Título original

Direitos autorais

Formatos disponíveis

Compartilhar este documento

Compartilhar ou incorporar documento

Opções de compartilhamento

Você considera este documento útil?

Este conteúdo é inapropriado?

Direitos autorais:

Formatos disponíveis

Generate A DNA Barcode and Identify Fish Species

Enviado por

Direitos autorais:

Formatos disponíveis

Generate a DNA Barcode and Identify Species

Is there something fishy about what you’re eating?

Sherri Andrews, Ph.D.

Leigh Brown, M.A.

2 Biotechnology Explorer™ | explorer.bio-rad.com

 Fish DNA extraction

 DNA visualization with UViewTM

 Bioinformatics and species identification

3 Biotechnology Explorer™ | explorer.bio-rad.com

 Only about 1.7 million species have been formally identified

 Current limitation to studies of biological diversity - humans are

 How do we complete the task of identifying the remaining

4 Biotechnology Explorer™ | explorer.bio-rad.com

 Some distinct species are not easy to differentiate by eye…

5 Biotechnology Explorer™ | explorer.bio-rad.com

 A worldwide effort (International Barcode of Life, iBOL) exists to “barcode” or

6 Biotechnology Explorer™ | explorer.bio-rad.com

– Opportunity to contribute to the global initiative to barcode life on Earth!

7 Biotechnology Explorer™ | explorer.bio-rad.com

 Participate in the largest biodiversity cataloging project

 Design a market study to look at local food supply,

Axolotl / Mexican salamander (critically endangered)

8 Biotechnology Explorer™ | explorer.bio-rad.com

9 Biotechnology Explorer™ | explorer.bio-rad.com

10 Biotechnology Explorer™ | explorer.bio-rad.com

11 Biotechnology Explorer™ | explorer.bio-rad.com

12 Biotechnology Explorer™ | explorer.bio-rad.com

What did I eat

13 Biotechnology Explorer™ | explorer.bio-rad.com

14 Biotechnology Explorer™ | explorer.bio-rad.com

15 Biotechnology Explorer™ | explorer.bio-rad.com

16 Biotechnology Explorer™ | explorer.bio-rad.com

17 Biotechnology Explorer™ | explorer.bio-rad.com

18 Biotechnology Explorer™ | explorer.bio-rad.com

+ Resuspension + Lysis Incubate

Bind DNA + Neutralization

Wash column Elute DNA

19 Biotechnology Explorer™ | explorer.bio-rad.com

Label tubes “1” for fish sample

Cut a piece of fish

20 Biotechnology Explorer™ | explorer.bio-rad.com

Using a new cutting implement, cut a

Add 200 ml of Resuspension to

21 Biotechnology Explorer™ | explorer.bio-rad.com

Add 250 µl of Lysis to each tube and

Incubate samples at 55oC for 10

22 Biotechnology Explorer™ | explorer.bio-rad.com

23 Biotechnology Explorer™ | explorer.bio-rad.com

24 Biotechnology Explorer™ | explorer.bio-rad.com

Add 250 ml of Neutralization to

Centrifuge the tubes for 5 min at top

25 Biotechnology Explorer™ | explorer.bio-rad.com

Snap (do not twist!) the bottoms

Transfer the entire supernatant

26 Biotechnology Explorer™ | explorer.bio-rad.com

Using a new pipet tip, add 200 ml of

27 Biotechnology Explorer™ | explorer.bio-rad.com

Add 500 µl of Wash and wash the

15 Repeat wash step and

28 Biotechnology Explorer™ | explorer.bio-rad.com

Centrifuge columns for a full 2 min to 1 2

Remove the spin columns and

29 Biotechnology Explorer™ | explorer.bio-rad.com