generate a dna barcode and identify species is there something fishy about what you’re eating?

Generate a DNA Barcode and Identify SpeciesIs there something fishy about what you’re eating?

Bio-Rad Biotechnology Explorer Fish DNA Barcoding Kit and DNA Barcoding Sequencing Module

Biotechnology Explorer™ | explorer.bio-rad.com2

Instructors - Bio-Rad Curriculum and Training Specialists

Damon Tighe, [email protected]

Sherri Andrews, [email protected]

Leigh Brown, M.A. [email protected]


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.

CCCTCCTA

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


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 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!

MitochondrialDNA


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

signatureof this rare

species?


Fish DNA Barcoding Kit Start to Finish

DNA BARCODE


DNA Barcoding Kit Workflow

Fish sample Extract genomic DNA

Gel electrophoresis

Sequencing,Sequence Analysis

PCR amplification


Barcoding Overview


DNA Extraction Overview

+ Resuspension + Lysis Buffer Buffer

Incubate10 minat 55oC

Bind DNAto column

(Matrix Solution)

+ Neutralization Buffer

Wash columnwith Wash buffer

Elute DNA


Quick Guide – Fish Prep

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.

Label tubes “1” for fish sample 1, “2” for fish sample 2. Also label with your initials. 1

2

1

1

2


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 sample. Slice it until finely minced. Transfer the sample into microcentrifuge tube 2. 2

Add 200 l of Resuspension to your two tubes and flick several times to ensure full submersion of the fish in the resuspension solution.

3

4


Quick Guide – DNA Extraction

Add 250 µl of Lysis to each tube and mix gently by inverting tubes 10 times to mix contents.

Incubate samples at 55oC for 10 min. The samples do not need to be shaken during incubation.

5

6


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?

mitoDNA

nuclearDNA


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?

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?

mitoDNA

pelleted proteins,membranes, etc



Add 250 l of Neutralization to each tube and mix gently by 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 microcentrifuge. A compact pellet will form along the side of the tube. The supernatant contains the DNA.

7

8



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.

9

10

1 2

1 2



Thoroughly mix the tube labeled Matrix to make sure particulates are completely resuspended before use.

Add 200 l of thoroughly resuspended Matrix to the first column and pipet up and down to mix.

Using a new pipet tip, add 200 l of thoroughly resuspended Matrix to the second column and pipet up and down to mix.

Centrifuge the columns for 30 sec at full speed.Remove flow through to waste.

11

12

13

1 2

1 2



Repeat wash step andcentrifugation as shown above.

Add 500 µl of Wash and wash the samples by centrifugation for 30 sec.Remove flow through to waste.

14

15

Wash

1 2



Centrifuge columns for a full 2 min to remove residual traces of Wash and dry out the samples

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.

16

17

new caplesstube

1 2

1



1 2

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.

18

19

1 2


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– Nucleotides– Taq polymerase– Magnesium chloride

Multiple rounds of thermal cycling toamplify DNA

MitochondrialDNA


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

200 V20 min

0.25x TAE


Visualizing DNA after electrophoresis

UViewTM Ethidium Bromide Fast BlastTM

Nontoxic Toxic, Mutagen NontoxicLoading dye + Stain Stain StainInstant Instant Requires wait timeView with UV View with UV View by eyeVery sensitive Most sensitive Less sensitive


Use UV transilluminator to visualize UView or Ethidium Bromide

UViewTM

Ethidium bromide

1 2 3 4 5

1. MW ruler2. (+) control3. (-) control4. Fish 15. Fish 2


Sequencing of PCR products


Bioinformatics – Alignment

What is the longest run of tails I should expect for 100 tosses?

. . . . .

Run

R = log1/p (n)

R = longest runp = probability (for “fair” coins its 0.5)n = number of tosses

Paul Erdos

Paul Erdos‐Alfréd Rényi law

http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=a-ERxSZLOydmSM&tbnid=h6fjew2V0u66pM:&ved=0CAUQjRw&url=http%3A%2F%2Fmembers.iif.hu%2Fvisontay%2Fponticulus%2Fbritannicus%2Fpgom.html&ei=tGJiUYjvAcnAhAfjj4HgDg&bvm=bv.44770516,d.ZG4&psig=AFQjCNFWz_FDI0iawLJK0cx-nS8fWnvFPA&ust=1365488654606701

http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=85YEomT6thEk3M&tbnid=CbWFujsvFd-j_M:&ved=0CAUQjRw&url=http%3A%2F%2Fgentilesteven.blogspot.com%2F2013%2F02%2Frs-4-would-you-let-coin-toss-determine.html&ei=aWNiUaGoItGIhQfEyYDQCg&bvm=bv.44770516,d.ZG4&psig=AFQjCNGPgAdab_lSxqn2B4bK8tiCIl_o9Q&ust=1365488816044423

http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=bk5IL5F3NN-CyM&tbnid=GL3Sf4biSa4EjM:&ved=0CAUQjRw&url=http%3A%2F%2Fwww.taxfreegold.co.uk%2F2003twopoundsdnagold.php&ei=RmViUY_sM4i5hAeShIC4DA&bvm=bv.44770516,d.ZG4&psig=AFQjCNED5zdtH6irJrpUWzGE1AqfsaGLfg&ust=1365489239185738

http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=WuLodKS0P3AoUM&tbnid=G1kbez8L0XvvUM:&ved=0CAUQjRw&url=http%3A%2F%2Fwww.taxfreegold.co.uk%2Fweselltwopounds.php&ei=ZGViUc2PCcOVhQedkoGIBA&bvm=bv.44770516,d.ZG4&psig=AFQjCNED5zdtH6irJrpUWzGE1AqfsaGLfg&ust=1365489239185738





What is the longest run of tails I should expect for 100 tosses?

. . . . .

Run

R = log1/0.5 (100) = 6.64

Paul Erdos

….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

http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=a-ERxSZLOydmSM&tbnid=h6fjew2V0u66pM:&ved=0CAUQjRw&url=http%3A%2F%2Fmembers.iif.hu%2Fvisontay%2Fponticulus%2Fbritannicus%2Fpgom.html&ei=tGJiUYjvAcnAhAfjj4HgDg&bvm=bv.44770516,d.ZG4&psig=AFQjCNFWz_FDI0iawLJK0cx-nS8fWnvFPA&ust=1365488654606701

http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=85YEomT6thEk3M&tbnid=CbWFujsvFd-j_M:&ved=0CAUQjRw&url=http%3A%2F%2Fgentilesteven.blogspot.com%2F2013%2F02%2Frs-4-would-you-let-coin-toss-determine.html&ei=aWNiUaGoItGIhQfEyYDQCg&bvm=bv.44770516,d.ZG4&psig=AFQjCNGPgAdab_lSxqn2B4bK8tiCIl_o9Q&ust=1365488816044423







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

AATCGTACTGAACCATTCAG

¼ chance for getting same base

Sequence lengths multiplied

http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=M3qrN0rCinhZ9M&tbnid=wSyQwEyJqvq2nM:&ved=0CAUQjRw&url=http%3A%2F%2Fblog.arjournals.com%2F2011%2F04%2Fwomen-in-science-negative-results.html&ei=MQZjUZKMJbDXiALskoCgBw&bvm=bv.44770516,d.cGE&psig=AFQjCNFHuvC3z3Td9PAxPmoaZJC0D3ql1Q&ust=1365530529586319






DNA is not a 4 sided Coin- Account for probability of bases being switched out for each other by a scoring matrix

Match

TransitionTransversionSame base type – Purine for Purine Change of base type – Purine for Pyrimidine



DNA is not a 4 sided Coin

65555433321A55554433221G44444433221C33333333221T22222222221A

22221111111G11111111111GATTGACTTAAG



DNA is not a 4 sided Coin

65555433321A5554433221G44444433221C33333333221T22222222221A

22221111111G11111111111GATTGACTTAAG



Bioinformatics – BLAST tool

ATTCGCAT

Query Database

1) Break into words

2) Find Matches and let go of all the other database words that don’t match

ATTTTCTCGCGCGCACAT

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-valueTheoretically, 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 (similardomains, maybe non-homologous).• E-values between 10-2 and 1 do not indicate a good homology


Bioinformatics – Linking DNA Barcoding and Protein Profiler

+

Stronger Evidence for Evolutionary Relationship

Compare light chain of myosin sizes Compare E-value for CO1 gene

=


Bioinformatics using BOLD-SDP

Quick Start tutorial available online

BOLD-SDP = Barcode Of Life Data systems – Student Data Portal


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 valuesExamine peaks

Low quality valueExamine 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

contig sequence

translation

barcode generatedfrom data


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


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


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


Student Inquiry - Teacher Considerations

generate a dna barcode and identify species is there something fishy about what you’re eating?

Documents

speciesbiotechnology

eyevsorbiotechnology

species of organisms

remaining species

designated dna sequence

substantial sequence

sushigate79 samples

genetic registry of