Natalia V. Ivanova Paul D.N. Hebert

EXPRESS BARCODES:

Racing from Bugs to Identifications

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

Rick TurnerAlex Borisenko

Acknowledgements

Jeremy deWaard Janet Topan

and CCDB