Transcript DNA Barcoding

DNA Barcoding
From DNA to ID
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What is DNA Barcoding?
ACGAGTCGGTAGCTGCCCTCTGACTGCA
TCGAATTGCTCCCCTACTACGTGCTATA
TGCGCTTACGATCGTACGAAGATTTAT
AGAATGCTGCTAGCTGCTCCCTTATTCG
ATAACTAGCTCGATTATAGCTACGATG
?
=
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What DNA Barcoding Works On
CHLOROPLAST
rbcL
MITOCHONDRION
matK
CO1
Why DNA Barcoding Works
Win: Sequence shows ~70% conservation, good for PCR, good as barcode
Fail: Sequence is completely conserved, good for PCR, but uninformative as barcode
Fail: Sequence shows no conservation, impossible for PCR, but good as barcode
Why DNA Barcoding Works
CHLOROPLAST
rbcL
MITOCHONDRION
matK
CO1
How DNA Barcoding Works
1. Sample Organism
2. Extract DNA
3. Amplify “Barcode” DNA
ACGAGTCGGTAGCTGCCCTCTGACTGCA
TCGAATTGCTCCCCTACTACGTGCTATA
TGCGCTTACGATCGTACGAAGATTTAT
AGAATGCTGCTAGCTGCTCCCTTATTCG
ATAACTAGCTCGATTATAGCTACGATG
4. Sequence DNA
5. Compare sequence against database
Why is DNA Barcoding Important?
ACGAGTCGGTAGCTGCCCTCTGACTGCA
TCGAATTGCTCCCCTACTACGTGCTATA
TGCGCTTACGATCGTACGAAGATTTAT
AGAATGCTGCTAGCTGCTCCCTTATTCG
ATAACTAGCTCGATTATAGCTACGATG
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Issue #1: No one knows how many species there are.
How Many Species Can You Name?
How many Animals did you name?
How many mammals?
How many plants?
How many insects?
“Dog”
Canis lupus familiaris
“Cat”
Felis catus
“Shark”
Ginglymostoma cirratum
“Oak Tree”
Quercus alba
“Beetle”
Popillia
japonica
Vertebrates
Species
Mammals
5,490
Birds
9,998
Reptiles
9,084
Amphibians
6,433
Fishes
31,300
Total
62,305
Invertebrate
s
Species
Plants
Species
Angiosperms
281,821
Insects
1,000,000
Gymnosperms
1,021
Mollusks
85,00
47,000
Ferns and
Allies
12,000
Crustaceans
Corals
2,175
Mosses
16,236
Arachnids
102,248
Algae
10,134
Total
(+others)
1,305,250
Total
321,212
• Currently between 1.5 and 2 million species are described/known
• This number may represent as little as half of the true number of species
• Perhaps more than 1/3 of all species are threatened
(IUCN Red list version 2010.1)
Issue #2: There is a lack of agreement of what “species” means.
Defining “species” is complex and
depends on many factors:
• Interbreeding capabilities
Canis lupus
Canis lupus (familiaris)
• Morphological variation
• Ecological context
• Genetic similarities
Anas platyrhynchos
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Issue #3: Traditional taxonomic identification methods may be inadequate
and too slow to capture vanishing biodiversity
Classical taxonomy is difficult for non-experts to understand
Leaves alternate proximally, opposite and ultimately
decussate distally, 6–16 × 4–13 cm; petiole ca. as long as
blade, winged, base clasping, basal lobes stipulate,
growing as extensions of wings, less than 1 mm wide; blade
5–7-veined, ovate, glabrous, base typically sagittate,
margins entire, apex acute to acuminate. Staminate
inflorescences axillary, 1–2 per axil, paniculate,
fasciculate; panicles bearing flowers singly,bracteolate,
in a zigzag pattern along rachis, internodes less than 2
mm; rachis to 25 cm, secondary axes 1–3(–6), fasciculate,
less than 3 cm, each subtended by deltate-ovate bracteole
shorter than 1 mm. Pistillate inflorescences solitary, 4–
8(–20)-flowered, 6–35 cm, internodes ca. 1 cm.
Adding to the complexity: immature, damaged or incomplete specimen
may make identification impossible.
Issue #4: DNA Barcoding provides opportunities to investigate things
DNA Barcoding Engages and Transforms Curiosity
Into Practical Inquiry
Kate Stoeckle
August 23, 2008
What research questions could you ask?
Research questions can be about any living thing or about
non-living things (foods or other products) that have DNA.
Examples:
• Are there invasive (non-native) plants in my local park?
• What are the most popular types of flowers in my city?
• Do the teas I buy at my supermarket really contain the
ingredients on the package?
• How many different living organisms can I find in an office building?
• Whose droppings are these?
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Materials & Equipment for DNA Barcoding
• DNA extraction Kit
• PCR machine and reagents
• DNA sequencing (Genewiz, other)
• Bioinformatic tools (analysis of DNA sequence)
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DNA Barcoding Analysis
• Sequence
• Prepare
• Compare
• Identify
• Share
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Contributing to big science
BOLD
GenBank
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How DNA Barcoding Works
1. Sample Organism
2. Extract DNA
3. Amplify “Barcode” DNA
ACGAGTCGGTAGCTGCCCTCTGACTGCA
TCGAATTGCTCCCCTACTACGTGCTATA
TGCGCTTACGATCGTACGAAGATTTAT
AGAATGCTGCTAGCTGCTCCCTTATTCG
ATAACTAGCTCGATTATAGCTACGATG
4. Sequence DNA
5. Compare sequence against database
Assignments
• Find NCBI’s MapViewer; identify difference between human
and chimpanzee genome; check situation in all 4 other primate
genomes provided
• Make sea shell phylogenetic tree, work through
hhmi.org/biointeractive/activities/shells/online/index.html
• Review users.ugent.be/~avierstr/principles/phylogeny.html*
• Review
http://www.hiv.lanl.gov/content/sequence/TUTORIALS/TREE
_TUTORIAL/Tree-tutorial.html*
• Review Constructing an Evolutionary Tree (Binder)
• Review Stockle paper on DNA, Taxonomy & Barcode of Life
(pre-Readings)
* Links worked on 2012 05 31; if they fail, find the 2 sites through Google
Questions?
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