Introducing: The AmpF STR NGM SElect Express Kit From Sample to Result in 2 Hours* Presenter Information * Typical workflow for 24 samples
Direct Amplification for the Expanded ESSL AmpF STR NGM SElect Express Kit A fast, direct amplification version of the NGM SElect Kit 1.2 mm Paper Punch Swab* Lysate ± Prep-n-Go Buffer PCR Reagents Prep-n-Go Buffer PCR Reagents * Copan Flocked and Whatman Omni swabs included in developmental testing 5/10/2012 Life Technologies 2
Direct Amplification for the Expanded ESSL AmpF STR 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 3
AmpF STR NGM SElect Express Kit Configuration & Cycling Conditions Components Master Mix Primer Set Control DNA 007 (2 ng/µl) Allelic ladder Cycling Time: ~45* min! PCR Conditions 95 C/ 1 min 94 C/ 3 sec 25-28 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 4
AmpF STR NGM SElect Express Kit Allelic Ladder 5/10/2012 Life Technologies 5
AmpF STR NGM SElect Express Kit Control DNA 007 5/10/2012 Life Technologies 6
AmpF STR NGM SElect Express Kit Example Profiles: Treated Paper Buccal on FTA Indicating Card Blood on FTA Classic Card 5/10/2012 Life Technologies 7
AmpF STR NGM SElect Express Kit Example Profiles: Untreated Paper Buccal on Bode Buccal Collector Blood on 903 Paper 5/10/2012 Life Technologies 8
AmpF STR NGM SElect Express Kit Example Profiles: Swabs Buccal on Whatman Omniswab Buccal on Copan Flocked Swab 5/10/2012 Life Technologies 9
AmpF STR NGM SElect Express Kit Instrument Compatibility Study 25 cycles Buccal samples on Omniswab 5/10/2012 Life Technologies 10
AmpF STR 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 11
AmpF STR 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 12
Test Site Results: Reproducibility Study 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 26 3130xl 60/60 100% 60/60 100% 2 FTA Paper 28 3130xl 60/60 100% 60/60 100% 3 27 3500xL 57/60 95% 53/60* 88% 4 25 3500xL 57/60 95% 57/60 95% 5 Copan 4N6 FLOQ Swabs 26 3130xl 60/60 100% 60/60 100% Life Technologies 25 3130xl 60/60 100% 60.60 100% 1 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 13 * Lower success rate caused by instrument loss of resolution issues
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 14
Maximising NGM SElect Express Kit Results 5/10/2012 Life Technologies 15
Maximising NGM SElect Express Kit Results Swab Substrate Handling Ensure swabs are fully dried before storing or processing with Prep-n-Go Buffer Ensure swabs are stored correctly to prevent excessive degradation over time Thermal Cycling Platform Optimised for use on the 9700 with silver or gold-plated silver block and the Veriti 96-Well thermal cycler Not supported for use on the 9700 with Aluminium block Not supported for use on the Veriti 96-well Fast thermal cycler Thermal Cycling Parameters Use Max mode on the 9700 thermal cycler Use 100% mode on the Veriti 96-well thermal cycler Choice of Cycle Number Choose a cycle number that prevents allele drop-out and minimizes off-scale alleles 5/10/2012 Life Technologies 16
Optimising Data Analysis Parameters for Analysis of NGM SElect Express Kit Data 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 17
Optimised Parameters = More Efficient Analysis Unoptimised Optimised 5/10/2012 Life Technologies 18
Improvements in Database Sample Processing Collection Extraction Quantitation/ Normalization Amplification Detection Analysis Time to Result (hrs/per 24 samples) ~1-3.5 0 ~0-2 0 0.75 ~4 0.55 ~1.5 0.1 ~1 Total Time to to Result = = ~7-12 ~2 hrs hrs 5/10/2012 Life Technologies 19
The AmpF STR NGM SElect Express Kit Supports rapid, direct amplification on treated paper and untreated paper/swab substrates in conjunction with Prep-n-Go Buffer Facilitates high throughput & automated processing Eliminates extraction, purification and quantification costs and helps reduce labour and consumable costs Contains a new fast capable enzyme that allows completion of amplification in ~45 mins Maximizes first pass success rate, reducing the need for time consuming repeat amplifications Part of a complete integrated solution for efficient single source sample processing available from Life Technologies Enables laboratories to go from sample to result in only 2 hours*! 5/10/2012 Life Technologies 20 * Typical workflow for 24 samples
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. 5/10/2012 Life Technologies 21