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The Evolution of Direct Amplification: From Sample to Result in 2 Hours*
Nicola Oldroyd Forensic Applications Consultant Human Identification
* Typical workflow for 24 samples
5/10/2012 | © Life Technologies™ 2
Traditional Single Source Sample Workflow
Time to Result (hrs/per 24 samples)
~1-3.5 ~0-2 ~4 ~1.5 ~1
Total Time to Result = ~7-12 hrs
Collection Sample
Preparation Quantitation/ Normalization
Amplification Detection Analysis
5/10/2012 | © Life Technologies™ 4
Evaluation of Direct Amp Capability of the Identifiler® Kit
Control DNA 007
25 μL Reaction Volume
1.2 mm Blood on FTA® Disc
No full profiles under direct amp conditions
5/10/2012 | © Life Technologies™ 5
Identifiler® Kit Direct Amp Performance Evaluation
Possible explanations for poor performance
− Insufficient DNA?
− Excess PCR inhibitors?
0.75 mm disc ~ 22.08 ng DNA
0.5 mm disc ~ 9.81 ng DNA
1.2 mm disc ~ 56.52 ng DNA Height = 1mm
1 μL blood ~ 50 ng DNA
Sufficient DNA still available
Inhibitor concentration reduced by >5-fold
0.5 mm disc ~ 9.81 ng DNA
5/10/2012 | © Life Technologies™ 6
Effect of Reducing Disc Size on Identifiler® Kit Direct Amp Performance
0.5 mm blood on FTA® disc
1.2 mm blood on FTA® disc
Control DNA 007 (1 ng)
0.75 mm blood on FTA® disc
But: − No automated
0.5 mm punch head options
− 0.5 mm disc size too small for buccal samples
− 0.5 mm discs very hard to handle
5/10/2012 | © Life Technologies™ 7
Optimisation of Reaction Buffer for 1.2 mm Discs
Component 1
Response Component 2
Use of Design of Experiments (DOE) Approach
5/10/2012 | © Life Technologies™ 8
Optimisation of Identifiler® Direct Kit Mastermix Buccal sample on 1.2 mm FTA® disc before DOE
5/10/2012 | © Life Technologies™ 9
Optimisation of Identifiler® Direct Kit Mastermix Buccal sample on 1.2 mm FTA® disc after DOE
5/10/2012 | © Life Technologies™ 10
− 3 x liquid blood samples
− 80 μL of blood onto FTA® Classic (passive diffusion)
− Sample 1.2 mm discs from the center to the edge of the blood stain
− Perform replicates for each position
Effect of Punch Position on Sample Peak Heights
1 3
2 4 5
5/10/2012 | © Life Technologies™ 11
FTA® Technology by Whatman
FTA® Classic Card
EasiCollect ® System
Identifiler® Direct Kit
Identifiler® Kit
Exam
ple
1
Exam
ple
2
Identifiler®Direct Kit
Identifiler® Kit
Blood on FTA® samples
5/10/2012 | © Life Technologies™ 12
Identifiler® Direct Kit Validation: External Test Site Results
Sample Type
PCR Success Rate Interpretation Success Rate Number of
Samples Tested Range of Success rates
Mean Success Rate
Range of Success rates
Mean Success Rate
VTS Stu
dy
Blood 99.4 % 99.4 % 95.7 – 98.8% 97.3 % 414
Buccal 91.8 – 99.4% 97.1 % 84.2 – 95.5% 90.9 % 653
CTS Stu
dy
Blood 100% 100 % 98.8 – 100% 99.8 % 437
Buccal 98.7 – 100% 99.0 % 91.7 – 100% 94.7 % 703
1st Pass Success Rate Definition
− All profile peaks higher than specified threshold
− Off scale peaks produce no artifacts which interfere with profile interpretation
> OL-labelled pull-up peaks <20% of highest peak of the marker
> No split, double called peaks
> Stutter peaks < 20% of marker or < marker stutter cut off, whichever is higher
5/10/2012 | © Life Technologies™ 13
hello
NUCLEICard™ System by Copan
NUCLEICard™ System
Chemically-treated paper-based
substrate to:
Lyse cell membranes and denature
proteins upon contact
Prevent the growth of bacteria and
other microorganisms
Maintein the integrity of nucleic acids
Allow direct PCR amplification from a
card punch
Certified human DNA, DNAse, RNAse
free and EtO treated
5/10/2012 | © Life Technologies™ 14
NUCLEICard™ Performance Study
PCR Success Rate Interpretation Success Rate
Substrate N Number of
Full Profiles
First Pass
Success Rate
Number of
Full Profiles
First Pass
Success Rate
FTA® Card 39 39/39 100% 39/39 100%
NUCLEICard™ Lot 1 40 40/40 100% 40/40 100%
NUCLEICard™ Lot 2 40 40/40 100% 39/40 97.5%
NUCLEICard™ Lot 3 40 40/40 100% 40/40 100%
Blood samples on FTA® Paper and NUCLEICard™ substrates
All data run on 3500 series platforms
5/10/2012 | © Life Technologies™ 15
Blood on FTA® Card Blood on NUCLEICard™
Profile Comparison: NUCLEICard vs. FTA® Paper
Identifiler® Direct Kit Amplifications
5/10/2012 | © Life Technologies™ 16
Expansion of the Direct Amplification Workflow to Non-FTA Substrates
Untreated Paper
5/10/2012 | © Life Technologies™ 17
Identifiler® Direct Kit development and optimisation performed on FTA® substrates only
Direct amplification workflow proven to deliver time and cost savings for database sample processing
Many laboratories in the US and international jurisdictions want to take advantage of direct amplification but use alternative substrates and/or alternative marker sets
Expansion of the Direct Amplification Workflow to Non-FTA® Substrates
Goal: Enable direct amplification of non-FTA® buccal samples
Prep-n-Go™ Buffer
5/10/2012 | © Life Technologies™ 18
Minimal additional workflow steps
Efficient cell lysis without the requirement for heat incubation
Good sample peak heights
High quality, well-balanced STR profiles
No introduction of artifacts
Optimized for Bode Buccal DNA Collector™ and Identifiler® Direct
Tested on buccal swabs and other STR chemistries
Prep-n-Go™ Buffer: Development Requirements & Considerations
5/10/2012 | © Life Technologies™ 19
Direct Amp Workflow: Untreated Paper Substrates
Collect Samples on Untreated Paper
One New Step
Electrophorese
Amplify
Punch 1.2 mm disc
Add PCR reagents
Add Prep-n-Go™ Buffer
5/10/2012 | © Life Technologies™ 20
Bode Buccal DNA Collector™ Samples Amplified with the Prep-n-Go™ Buffer and the Identifiler® Direct kit
2 different individuals amplified for 26 PCR cycles Well-balanced profiles within each dye colour
5/10/2012 | © Life Technologies™ 21
Performance Study Results Summary
Test Site Number of
Samples Tested
PCR Cycle
Number
CE Platform
PCR Success Rate Interpretation Success Rate
Number of Full Profiles
First Pass Success Rate
Number of Full Profiles
First Pass Success Rate
1 82* 27 3130xl 81/82 98.8% 78/82 95.1%
2 80 26 3130xl 78/80 97.5% 78/80 97.5%
3 84* 26 3130xl 83/84 98.8% 79/84 94.0%
4 84* 26 3130xl 80/81 98.8% 76/81 93.8%
5 84* 26 3730 83/84 98.8% 83/84 98.8%
6 84 26 3500 82/84 97.6% 74/84 88.1%
Life Technologies
84 26 3130xl 82/84 97.6% 79/84 94.0%
Total 582 569/582 97.8% 547/582 94.0%
* Used real offender samples
5/10/2012 | © Life Technologies™ 22
hello
Buccal DNA Collector™ Performance Study
Peak Heights Intracolor Balance
5/10/2012 | © Life Technologies™ 23
Identifiler® Direct Kit w/ Prep-n-Go™ Buffer Identifiler® Direct Kit w/Other Buffer
1200 1200
Lysis Buffer Performance Comparison
No allelic drop-out using Prep-n-Go™ Buffer
5/10/2012 | © Life Technologies™ 24
Expansion of the Direct Amplification Workflow to Non-FTA Substrates
Buccal Swabs
5/10/2012 | © Life Technologies™ 25
Pros
− Easy to use
− Inexpensive
Cons
− High performance variability among swab types
− Limited opportunities for automation increasing labour requirements and the risk of contamination
− Result quality dependent on collection process and storage conditions
Buccal Swabs
5/10/2012 | © Life Technologies™ 26
hello
Total recovery of DNA from FLOQSwabs™
Maximum efficiency in collection capacity and sample release
Available with different anatomical and ergonomic design
Human DNA, DNASE and RNASE free certified
ETO treated (Ethylene Oxide)
• Patented worldwide: Copan WO 2004/086979
Pre-scored breaking point
Fibre v Floq swab
5/10/2012 | © Life Technologies™ 27
Direct Amp Workflow: Buccal Swabs
Collect Samples on Buccal Swab
Lyse swab in Prep-n-Go™ Buffer
Add PCR Reagents
Transfer Lysate
New steps
Electrophorese
Amplify
Stand @ RT for 20 minutes
5/10/2012 | © Life Technologies™ 28
Copan 4N6 Swab Puritan® Cotton Swab Whatman® Omni Swab
Buccal Swab Performance Comparison
Identifiler® Direct Kit Amplifications
5/10/2012 | © Life Technologies™ 29
Expansion of the Direct Amplification Workflow to Other STR Marker Sets
5/10/2012 | © Life Technologies™ 30
Direct Amplification: The NGM™ & NGM SElect™ Kits
NGM SElect™ Kit NGM™ Kit
Amplification of buccal samples on Bode Buccal Collector™ treated with Prep-n-Go™ Buffer
5/10/2012 | © Life Technologies™ 31
Utilises the same primer sequences as the NGM™ and NGM SElect™ Kits
Includes a new mastermix optimised specifically to support direct amplification of swab and treated/untreated paper substrates
Delivers rapid cycling times through the introduction of a new fast-capable enzyme (< 1 hr)
May be amplified using the Veriti® 96-Well or 9700 (silver or gold-plated silver block) thermal cyclers
− Veriti® 96-Well Thermal Cycler (standard) now supported for use with all existing AmpFℓSTR® kits
Direct Amplification for the Expanded ESSL
AmpFℓSTR® NGM SElect™ Express Kit
5/10/2012 | © Life Technologies™ 32
AmpFℓSTR® NGM SElect™ Express Kit Configuration & Cycling Conditions
Components Master Mix Primer Set
Control DNA 007 Allelic ladder
PCR Conditions 95°C/ 1 min 94°C/ 3 sec 59°C/ 16 sec 65°C/ 29 sec 60°C/ 5 min
25-28 x
*Cycle number requires optimisation by each laboratory for each sample type/substrate, additional cycles will increase the cycling time slightly
Cycling Time: ~45* min!
5/10/2012 | © Life Technologies™ 35
AmpFℓSTR® NGM SElect™ Express Kit Example Profiles: Treated Paper
Blood on FTA® Classic Card
Buccal on FTA® Indicating Card
5/10/2012 | © Life Technologies™ 36
AmpFℓSTR® NGM SElect™ Express Kit Example Profiles: Untreated Paper
Blood on 903 Paper
Buccal on Bode Buccal Collector™
5/10/2012 | © Life Technologies™ 37
AmpFℓSTR® NGM SElect™ Express Kit Example Profiles: Swabs
Buccal on Copan Flocked Swab
Buccal on Whatman Omniswab
5/10/2012 | © Life Technologies™ 38
AmpFℓSTR® NGM SElect™ Express Kit Instrument Compatibility Study
25 cycles
Buccal samples on Omniswab
5/10/2012 | © Life Technologies™ 39
AmpFℓSTR® NGM SElect™ Express Kit Species Specificity Study
Dog
Cat
Horse
Sheep
Cow
Pig
Rat
Rabbit
10 ng input DNA, 28 cycles
5/10/2012 | © Life Technologies™ 40
AmpFℓSTR® NGM SElect™ Express Kit Negative Control Reaction
Low level peaks in the NED dye channel occasionally observed at ~ 70 bp
No artifacts within the read region
5/10/2012 | © Life Technologies™ 41
Test Site Results
Reproducibility Study
− Either 10 buccal swabs or 10 buccal samples on treated paper amplified in replicates of 6
Test Site Sample
Type Cycle #
CE Platform
PCR Success Rate Interpretation Success Rate
Number of
Full Profiles
First Pass
Success Rate
Number of
Full Profiles
First Pass
Success Rate
1
FTA® Paper
26 3130xl 60/60 100% 60 100%
2 28 3130xl 60/60 100% 60 100%
3 27 3500xL 60/60 98% 57 98%
4
Copan 4N6 FLOQ Swabs
25 3500xL 57/60 95% 57 95%
5 26 3130xl 60/60 100% 60 100%
Life Technologies
25 3130xl 60/60 100% 60 100%
5/10/2012 | © Life Technologies™ 42
A Complete Set of Direct Amplification Workflows
Collect Samples
Electrophorese
Amplify
Punch 1.2 mm Disc
Add PCR Reagents
Treated Paper
Add Prep-n-Go™ Buffer
Punch 1.2 mm Disc
Add PCR Reagents
Untreated Paper
Lyse Swab in Prep-n-Go™ Buffer
Add PCR Reagents
Transfer Lysate
Buccal Swabs
5/10/2012 | © Life Technologies™ 44
Factors Influencing Direct Amplification Results
Choice of Kit
Choice of Sample Type/Substrate Sample Transfer Efficiency
Punch Size and Position
Cycle Number
Data Analysis Parameters
5/10/2012 | © Life Technologies™ 45
Optimising Data Analysis Parameters for Single-Source Samples
Use of a global cut-off filter can reduce significantly the amount of editing required for single-source sample data
Peak Quality settings may also be adjusted to better reflect the characteristics of single-source samples
5/10/2012 | © Life Technologies™ 46
Optimised Parameters = More Efficient Analysis
Un
op
tim
ised
Op
timised
5/10/2012 | © Life Technologies™ 47
Time to Result (hrs/per 24 samples)
~1-3.5 ~0-2 ~4 ~1.5 ~1
Maximized Efficiency for Single Source Sample Processing
Time to Result (hrs/per 24 samples)
0 0 0.75 0.55 0.1
Total Time to Result = ~7-12 hrs
Collection Extraction Quantitation/ Normalization
Amplification Detection Analysis
5/10/2012 | © Life Technologies™ 48
Time to Result (hrs/per 24 samples)
~1-3.5 ~0-2 ~4 ~1.5 ~1
Maximized Efficiency for Single Source Sample Processing
Time to Result (hrs/per 24 samples)
0 0 0.75 0.55 0.1
Total Time to Result = ~7-12 hrs
Collection Amplification Detection Analysis
Total Time to Result = ~2 hrs
5/10/2012 | © Life Technologies™ 53
Thank You
© 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. EasiCollect and FTA are registered trademarks of Whatman Limited. Buccal DNA collector is a trademark of Bode Technology. FLOQswabs is a trademark of Copan Italia S.P.A.
Refer to product page on the Life Technologies website for Limited Use License.
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