1. 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

2. Instructors - Bio-Rad Curriculum and Training Specialists
Damon Tighe,
Sherri Andrews, Ph.D.
Leigh Brown, M.A.

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

4. Diversity of Life
It is estimated that there are 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)

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

6. 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

7. 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!

8. 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)

9. “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

10. “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)

11. Food Fraud in the News

12. 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!
Mitochondrial
DNA

13. Applications of DNA Barcoding
What did I eat
last night (and
is it what they
said it was)?
What is the
genetic
signature
of this rare
species?
What did I
catch
yesterday?

14. Fish DNA Barcoding Kit Start to Finish
DNA BARCODE

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

16. Barcoding Overview

17. Barcoding Overview

18. Barcoding Overview

19. DNA Extraction Overview
Incubate
10 min
at 55oC
+ Resuspension Lysis
Buffer Buffer
Bind DNA
to column
(Matrix Solution)
+ Neutralization
Buffer
Wash column
with Wash buffer
Elute DNA

20. Quick Guide – Fish Prep
Label tubes “1” for fish sample 1, “2” for fish sample 2. Also label with your initials. 1 2 1
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. 2 1

21. 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. 3 2
Add 200 ml of Resuspension to your two tubes and flick several times to ensure full submersion of the fish in the resuspension solution. 4

22. Quick Guide – DNA Extraction
Add 250 µl of Lysis to each tube and mix gently by inverting tubes 10 times to mix contents. 5
Incubate samples at 55oC for 10 min. The samples do not need to be shaken during incubation. 6

23. What is happening during DNA extraction? Where is the DNA at each step?
mito
DNA
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
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
nuclear
DNA

24. 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
Elute
low ionic strength buffer or water
releases DNA from the silica
mito
DNA
pelleted proteins,
membranes, etc

25. Quick Guide – DNA Extraction
Add 250 ml of Neutralization to each tube and mix gently by inverting tubes 10 times to mix. A visible cloudy precipitate may form. 7
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. 8

26. Quick Guide – DNA Extraction
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. 9 1 2
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. 10 1 2

27. Quick Guide – DNA Extraction
Thoroughly mix the tube labeled Matrix to make sure particulates are completely resuspended before use.
Add 200 ml of thoroughly resuspended Matrix to the first column and pipet up and down to mix.
Using a new pipet tip, add 200 ml of thoroughly resuspended Matrix to the second column and pipet up and down to mix. 1 2 11 1 2 12
Centrifuge the columns for 30 sec at full speed.
Remove flow through to waste. 13

28. Quick Guide – DNA Extraction
Wash 1 2
Add 500 µl of Wash and wash the samples by centrifugation for 30 sec.
Remove flow through to waste. 14 15
Repeat wash step and
centrifugation as shown above.

29. Quick Guide – DNA Extraction1 2
Centrifuge columns for a full 2 min to remove residual traces of Wash and dry out the samples 16
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. 17 1
new capless
tube

30. Quick Guide – DNA Extraction
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. 18 1 2
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. 1 2 19

31. 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 to
amplify DNA
Mitochondrial
DNA

32. 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!

33. Fish Barcoding PCR Primers

34. 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

35. 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 V
20 min
0.25x TAE

36. 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

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

38. Sequencing of PCR products

39. Bioinformatics – Alignment
Run
What is the longest run of tails I should expect for 100 tosses?
R = log1/p (n)
WARNING: THIS is a BIOLOGIST hand waiving of this more complex mathematical model….but we need to at least know at some level what the computer scientist are doing, so we can communicate and solve bigger problems. When we align DNA sequences we are in general asking how many times do the sequences have the same bases in a run (streak). To talk about the statistics of runs its easier to start with the classic coin toss problem – How many runs of the same (heads/tails) would I expect if I flipped a coin 100 times? Paul Erdos - Mathematical bad ass and most prolific mathematician ever….he lived in your lifetime!!!, do you know your Erdos number? Paul and his house host Alfred Renyi went after this question and they found a mathematical model that could work to predict runs for coin tosses in There is a great radio lab piece on this.
Paul Erdos‐Alfréd Rényi law
R = longest run
p = probability (for “fair” coins its 0.5)
n = number of tosses
Paul Erdos

40. Bioinformatics – Alignment
Run
What is the longest run of tails I should expect for 100 tosses?
R = log1/0.5 (100) = 6.64
Erods and Renyi find out that if you toss a coin 100 times you should expect to see runs of almost 7 heads or tails. Its no opening scene to Rosencrantz and Guildenstern, but its more then most people would guess and gives us a quantitative way to talk about the appearance of runs.
….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
Paul Erdos

41. 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?
Biologists found that if you were comparing sequences of DNA you could equate places in a sequence that were the same with the idea of a run, so in the sequence the first two positions are the same (run of 2). because you are comparing two strings (sequences) you have to multiply them together to get the number of “tosses”.
Also you have to change the model a little bit now to deal with the new probability of having 4 things your space could be occupied instead of just two. This model gives us a good baseline, but needs further refinement because nucleotides don’t switch out perfectly….so we have to adjust how we calculate the probability
R = log1/0.25 (100) = 3.32
Sequence lengths multiplied
¼ chance for getting same base

42. 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
Transition
Change of base type – Purine for Pyrimidine
Same base type – Purine for Purine

43. Bioinformatics – Alignment
DNA is not a 4 sided Coin 6 5 4 3 2 1 A G C T

44. Bioinformatics – Alignment
DNA is not a 4 sided Coin 6 5 4 3 2 1 A G C T

45. 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

46. 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

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

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

49. Create an Instructor Account

50. Fill Out Required Information

51. Receive Two Important Emails – Login and Registration Keys

52. Log In and Register a New Course

53. Fill in Course Info,List Students, Receive Class Login

54. Students Log In and Enter Specimen Info

55. Enter Specimen Information

56. View Class Specimen List

57. Upload Sequencing Data Files

58. Select PCR and Sequencing Primers Used

59. Class List Updates with Specimen Records Uploaded

60. View Data

61. Uploaded trace files receive data quality assessment

62. Trace files viewable

63. 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)

64. Generate contig (“sequence”) from trace files

65. Examine base calls in contig
Contig sequence

66. Contig generated, trim primer sequences

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

68. Data summary
barcode
generated
from data
contig
sequence
translation

69. Search full database for genetic match

70. Search species database for genetic match

71. Species match!

72. Education and DNA Barcoding - Resources
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

73. 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!

74. 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

75. Student Inquiry - Teacher Considerations