natalia v. ivanova paul d.n. hebert express barcodes: racing from bugs to identifications
Post on 14-Dec-2015
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Conventional DNA barcoding at CCDB
Automation:
• 3 Biomek robots (Beckman Coulter )
• 2 3730xl DNA sequencers (ABI)
Methods: • Automated DNA
extraction
• Pre-made frozen PCR and sequencing plates
• PCR with Platinum Taq
• E-gel PCR check
• No PCR cleanup
• Sequencing with 1/24 BigDye v. 3.1
• Automated Agencourt sequencing cleanup
0.5M specimens1M sequences per year
Outline of the analytical chain
ExtractDNA
PCR Amplify SequenceSpecimen Tissue Sample
ExtractDNA
PCR Amplify Sequence
Specimen
Tissue Sample Photograph
Collection Data Web-Accessible Data and DNA Barcode
Time required for conventional DNA barcoding
Total: ~ 8 hours
ExtractDNA
PCR Amplify SequenceSpecimen Tissue Sample
DNA Extraction PCR Sequencing Sequencing run
PCR check
Sequencing cleanup
Why do we need express DNA barcoding?
Express Identification System:
• Monitoring of invasive species• Identification of all life stages• Simple• Fast• Portable
Keeping everything simple
Merging Technology with a Simple Approach
• Basic equipment
• Fast DNA extraction
• Pre-made frozen or lyophilized reagents for PCR and sequencing
• Size-exclusion sequencing cleanup
Express DNA extraction – 5 min
Alkaline Lysis (Whatman)
• Boil water
• Take a small bug fragment
• Place in a tube with alkaline buffer (0.1 N NaOH, 0.3 mM EDTA, pH 13.0)
• Incubate for 2 min at 95°C
• Add neutralization solution (0.1 M Tris-HCl pH 7.0)
PCR amplification
TaKaRa Z-Taq
QIAGEN Fast Cycling Kit
Platinum Taq (control)
Fresh moth DNA, ~300 bp:
5 min – 95 ºC
40 cycles: 5 sec – 98 ºC, 5 sec – 51 ºC, 10 sec – 68 ºC1 min – 72 ºC
Z-Taq PCR optimization – how fast can it work?
PCR optimization on moth legs extracted with alkaline lysis
5 sec – 98 ºC, 5 sec – 51 ºC, 10 sec – 68 ºC
Z-Taq Optimization
0
10
20
30
40
50
60
70
80
90
100
40 cycles25 min
35 cycles22 min
30 cycles19 min
20 cycles16 min
% P
CR
su
cces
s
Undiluted
2x
4x
10x
TemplateDilution
Cycle sequencing optimization
Cycle sequencing parameters to get ~300 bp:
2 min – 96 ºC 30 cycles: 5 sec – 96 ºC, 5 sec – 55 ºC, 20 sec – 60 ºC
26 minutes
Sequencing cleanup using size-exclusion
Size-exclusion SequencingCleanup using Nanosep (PALL)
• Dilute sample with 0.5 mM EDTA
• Apply onto membrane, centrifuge
• Wash with 0.5 mM EDTA, centrifuge
• Add 0.5 mM EDTA , collect from the top and transfer to collection plate
10 min
Making lab procedures fast and simple
< 2 hours
~ 8 hours
European chafer Rhizotrogus (Amphimallon) majalis
DNA Extraction PCR Sequencing Sequencing run
PCR check
Sequencing cleanup
Portable PCR and sequencing devices available today
PNAS | January 22, 2002 | vol. 99 | no. 2 | 574-579
Microchip sequencing combined with on-chip cleanup could be completed in 20-25 min
A place for a hand-held barcoder?
Merging Technology with Express-barcoding Methods:
• Lyophilized reagents
• Quick on-a-chip alkaline lysis
• PCR on a microchip
• Sequencing on a microchip
• Connection to BOLD
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