The Evolution of Direct Amplification: From Sample to Result in 2 Hours*
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1 The Evolution of Direct Amplification: From Sample to Result in 2 Hours* Nicola Oldroyd Forensic Applications Consultant Human Identification * Typical workflow for 24 samples
2 Traditional Single Source Sample Workflow Collection Sample Preparation Quantitation/ Normalization Amplification Detection Analysis Time to Result (hrs/per 24 samples) ~1-3.5 ~0-2 ~4 ~1.5 ~1 Total Time to Result = ~7-12 hrs 5/10/2012 Life Technologies 2
3 Development of Direct Amplification on Treated Paper Substrates 5/10/2012 Life Technologies 3
4 Evaluation of Direct Amp Capability of the Identifiler Kit Control DNA μl Reaction Volume 1.2 mm Blood on FTA Disc No full profiles under direct amp conditions 5/10/2012 Life Technologies 4
5 Identifiler Kit Direct Amp Performance Evaluation Possible explanations for poor performance Insufficient DNA? 1 μl blood ~ 50 ng DNA Height = 1mm 1.2 mm disc ~ ng DNA 0.75 mm disc ~ ng DNA 0.5 mm disc ~ 9.81 ng DNA Excess PCR inhibitors? 0.5 mm disc ~ 9.81 ng DNA Sufficient DNA still available Inhibitor concentration reduced by >5-fold 5/10/2012 Life Technologies 5
6 Effect of Reducing Disc Size on Identifiler Kit Direct Amp Performance Control DNA 007 (1 ng) 0.5 mm blood on FTA disc 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 1.2 mm blood on FTA disc 5/10/2012 Life Technologies 6
7 Optimisation of Reaction Buffer for 1.2 mm Discs Use of Design of Experiments (DOE) Approach Response Component 2 Component 1 5/10/2012 Life Technologies 7
8 Optimisation of Identifiler Direct Kit Mastermix Buccal sample on 1.2 mm FTA disc before DOE 5/10/2012 Life Technologies 8
9 Optimisation of Identifiler Direct Kit Mastermix Buccal sample on 1.2 mm FTA disc after DOE 5/10/2012 Life Technologies 9
10 Effect of Punch Position on Sample Peak Heights 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 /10/2012 Life Technologies 10
11 Example 2 Example 1 FTA Technology by Whatman Blood on FTA samples Identifiler Kit FTA Classic Card Identifiler Direct Kit Identifiler Kit EasiCollect System Identifiler Direct Kit 5/10/2012 Life Technologies 11
12 Identifiler Direct Kit Validation: External Test Site Results Sample Type PCR Success Rate Range of Success rates Mean Success Rate Interpretation Success Rate Range of Success rates Mean Success Rate Number of Samples Tested VTS Study Blood 99.4 % 99.4 % % 97.3 % 414 Buccal % 97.1 % % 90.9 % 653 CTS Study Blood 100% 100 % % 99.8 % 437 Buccal % 99.0 % % 94.7 % st 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 12
13 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 hello Life Technologies 13
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 /40 100% 40/40 100% NUCLEICard Lot /40 100% 39/ % NUCLEICard Lot /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 14
15 Profile Comparison: NUCLEICard vs. FTA Paper Blood on FTA Card Blood on NUCLEICard Identifiler Direct Kit Amplifications 5/10/2012 Life Technologies 15
16 Expansion of the Direct Amplification Workflow to Non-FTA Substrates Untreated Paper 5/10/2012 Life Technologies 16
17 Expansion of the Direct Amplification Workflow to Non-FTA Substrates 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 Goal: Enable direct amplification of non-fta buccal samples Prep-n-Go Buffer 5/10/2012 Life Technologies 17
18 Prep-n-Go Buffer: Development Requirements & Considerations 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 5/10/2012 Life Technologies 18
19 Direct Amp Workflow: Untreated Paper Substrates Collect Samples on Untreated Paper One New Step Add Prep-n-Go Buffer Punch 1.2 mm disc Add PCR reagents Amplify Electrophorese 5/10/2012 Life Technologies 19
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 20
21 Performance Study Results Summary Test Site Number of Samples Tested PCR Cycle Number CE Platform PCR Success Rate Number of Full Profiles First Pass Success Rate Interpretation Success Rate Number of Full Profiles First Pass Success Rate 1 82* xl 81/ % 78/ % xl 78/ % 78/ % 3 84* xl 83/ % 79/ % 4 84* xl 80/ % 76/ % 5 84* / % 83/ % / % 74/ % Life Technologies xl 82/ % 79/ % Total / % 547/ % * Used real offender samples 5/10/2012 Life Technologies 21
22 Buccal DNA Collector Performance Study Peak Heights Intracolor Balance 5/10/2012 hello Life Technologies 22
23 Lysis Buffer Performance Comparison Identifiler Direct Kit w/other Buffer Identifiler Direct Kit w/ Prep-n-Go Buffer No allelic drop-out using Prep-n-Go Buffer 5/10/2012 Life Technologies 23
24 Expansion of the Direct Amplification Workflow to Non-FTA Substrates Buccal Swabs 5/10/2012 Life Technologies 24
25 Buccal Swabs 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 5/10/2012 Life Technologies 25
26 Pre-scored breaking point Total recovery of DNA from FLOQSwabs Maximum efficiency in collection capacity and sample release Fibre v Floq swab Available with different anatomical and ergonomic design Human DNA, DNASE and RNASE free certified ETO treated (Ethylene Oxide) Patented worldwide: Copan WO 2004/ /10/2012 hello Life Technologies 26
27 Direct Amp Workflow: Buccal Swabs Collect Samples on Buccal Swab Lyse swab in Prep-n-Go Buffer RT for 20 minutes New steps Add PCR Reagents Transfer Lysate Amplify Electrophorese 5/10/2012 Life Technologies 27
28 Buccal Swab Performance Comparison Copan 4N6 Swab Puritan Cotton Swab Whatman Omni Swab Identifiler Direct Kit Amplifications 5/10/2012 Life Technologies 28
29 Expansion of the Direct Amplification Workflow to Other STR Marker Sets 5/10/2012 Life Technologies 29
30 Direct Amplification: The NGM & NGM SElect Kits NGM Kit NGM SElect Kit Amplification of buccal samples on Bode Buccal Collector treated with Prep-n-Go Buffer 5/10/2012 Life Technologies 30
31 Direct Amplification for the Expanded ESSL AmpFlSTR NGM SElect Express Kit 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 5/10/2012 Life Technologies 31
32 AmpFlSTR NGM SElect Express Kit Configuration & Cycling Conditions Components Master Mix Primer Set Control DNA 007 Allelic ladder Cycling Time: ~45* min! PCR Conditions 95 C/ 1 min 94 C/ 3 sec x 59 C/ 16 sec 65 C/ 29 sec 60 C/ 5 min *Cycle number requires optimisation by each laboratory for each sample type/substrate, additional cycles will increase the cycling time slightly 5/10/2012 Life Technologies 32
33 AmpFlSTR NGM SElect Express Kit Allelic Ladder 5/10/2012 Life Technologies 33
34 AmpFlSTR NGM SElect Express Kit Control DNA 007 5/10/2012 Life Technologies 34
35 AmpFlSTR NGM SElect Express Kit Example Profiles: Treated Paper Buccal on FTA Indicating Card Blood on FTA Classic Card 5/10/2012 Life Technologies 35
36 AmpFlSTR NGM SElect Express Kit Example Profiles: Untreated Paper Buccal on Bode Buccal Collector Blood on 903 Paper 5/10/2012 Life Technologies 36
37 AmpFlSTR NGM SElect Express Kit Example Profiles: Swabs Buccal on Whatman Omniswab Buccal on Copan Flocked Swab 5/10/2012 Life Technologies 37
38 AmpFlSTR NGM SElect Express Kit Instrument Compatibility Study 25 cycles Buccal samples on Omniswab 5/10/2012 Life Technologies 38
39 AmpFlSTR NGM SElect Express Kit Species Specificity Study Dog Cow Cat Pig Horse Rat Sheep Rabbit 10 ng input DNA, 28 cycles 5/10/2012 Life Technologies 39
40 AmpFlSTR NGM SElect Express Kit Negative Control Reaction No artifacts within the read region Low level peaks in the NED dye channel occasionally observed at ~ 70 bp 5/10/2012 Life Technologies 40
41 Test Site Results Test Site 1 Sample Type Cycle # CE Platform PCR Success Rate Number of Full Profiles First Pass Success Rate Interpretation Success Rate Number of Full Profiles First Pass Success Rate xl 60/60 100% % 2 FTA Paper xl 60/60 100% % xL 60/60 98% 57 98% xL 57/60 95% 57 95% 5 Copan 4N6 FLOQ Swabs xl 60/60 100% % Life Technologies xl 60/60 100% % Reproducibility Study Either 10 buccal swabs or 10 buccal samples on treated paper amplified in replicates of 6 5/10/2012 Life Technologies 41
42 A Complete Set of Direct Amplification Workflows Collect Samples Treated Paper Untreated Paper Buccal Swabs Punch 1.2 mm Disc Add PCR Reagents Add Prep-n-Go Buffer Punch 1.2 mm Disc Add PCR Reagents Lyse Swab in Prep-n-Go Buffer Add PCR Reagents Transfer Lysate Amplify Electrophorese 5/10/2012 Life Technologies 42
43 Maximising Direct Amplification Results & Workflow Efficiency 5/10/2012 Life Technologies 43
44 Factors Influencing Direct Amplification Results Choice of Kit Choice of Sample Type/Substrate Sample Transfer Efficiency Punch Size and Position Data Analysis Parameters Cycle Number 5/10/2012 Life Technologies 44
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 45
46 Optimised Parameters = More Efficient Analysis Unoptimised Optimised 5/10/2012 Life Technologies 46
47 Maximized Efficiency for Single Source Sample Processing Collection Extraction Quantitation/ Normalization Amplification Detection Analysis Time to Result (hrs/per 24 samples) ~ ~ ~ ~ ~1 Total Time to Result = ~7-12 hrs 5/10/2012 Life Technologies 47
48 Maximized Efficiency for Single Source Sample Processing Collection Amplification Detection Analysis Time to Result (hrs/per 24 samples) ~ ~ ~ ~ ~1 Total Time to to Result = = ~7-12 ~2 hrs hrs 5/10/2012 Life Technologies 48
49 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. 5/10/2012 Life Technologies 53
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