Documente Academic
Documente Profesional
Documente Cultură
Damon Tighe,
damon_tighe@bio-rad.com
Introduction
Gel electrophoresis
Inquiry Questions
vs
or
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
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
What did I
catch
yesterday?
What is the
genetic
signature
of this rare
species?
DNA BARCODE
PCR amplification
Fish sample Extract genomic DNA
Sequencing,
Sequence Analysis
Gel electrophoresis
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?
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?
Wash
new capless
tube
Assemble reactions of
– Template DNA
– Primers Mitochondrial
– Nucleotides DNA
– Taq polymerase
– Magnesium chloride
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!
Repeat 35 cycles
1. MW ruler
UViewTM
2. (+) control
3. (-) control
4. Fish 1
5. Fish 2 Ethidium
bromide
1 2 3 4 5
Run
.....
Run
.....
AATCGTACTG
AACCATTCAG
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
Query Database
ATTCGCAT
E-value
45 Biotechnology Explorer™ | explorer.bio-rad.com
Bioinformatics – BLAST tool
E-value
Theoretically, we could trust any result with an E-value ≤ 1
+
Compare light chain of myosin sizes Compare E-value for CO1 gene
Registration keys
Contig
sequence
barcode
generated
from data
contig
sequence
translation
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!