The AutoMate Express Forensic DNA Extraction System. Brad Dixon Forensic Science Applications Group
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1 The AutoMate Express Forensic DNA Extraction System Brad Dixon Forensic Science Applications Group
2 Presentation Overview Chemistry and Instrument Overview PrepFiler Express & PrepFiler Express BTA Forensic DNA Extraction Kits Protocol Overviews PrepFiler LySep Column and Cartridge Configuration AutoMate Express Instrument DNA Isolation Developmental Validation Data 2 8/9/ Life Technologies Corporation
3 AutoMate Express Forensic DNA Extraction System Developed specifically to enable maximum yield of high quality genomic DNA from forensic samples using the PrepFiler Express Forensic DNA Extraction Kits Processes 13 samples in ~30 minutes Two protocols on a single script card PrepFiler Lysis Buffer and Bone Tooth Adhesive (BTA) Tip and tube rack Cartridge rack 3 8/9/ Life Technologies Corporation
4 AutoMate Express DNA Extraction Workflow PrepFiler Lysis Buffer (PFLB) (majority of samples including trace evidence samples) high extraction efficiency Lysate separation from substrate PFLB or BTA Protocol DNA for downstream applications PrepFiler BTA Lysis Buffer (Bone, tooth, sticky tape, cig. butt) performing delicate lysis DNA DNA Binding Binding Wash Wash DNA DNA Elution Elution 4 8/9/ Life Technologies Corporation
5 What is the PrepFiler Express Kit? Contains all reagents needed to perform online DNA isolation in an AutoMate Express System specific cartridge A combination of uniquely structured magnetic particles and a multi-component surface chemistry optimized to provide extremely efficient DNA binding capacity and maximum DNA recovery No No organic extraction No No silica-based purification No No ion ion exchangers No No direct direct precipitation Advantages of the Magnetized DNA Complex Efficient DNA binding capacity and maximum DNA recovery Stable binding during wash steps while removing PCR inhibitors Consistent yields of purified DNA separated from the magnetic particles at the elution step 5 8/9/ Life Technologies Corporation
6 PrepFiler Express and Express BTA Forensic DNA Extraction Kits
7 Kit Configurations PrepFiler Express Forensic DNA Extraction Kit* PrepFiler Lysis Buffer PrepFiler Express Cartridges** Sample Tubes Elution Tubes PrepFiler LySep Columns Tips and tip sheaths PrepFiler Express BTA Forensic DNA Extraction Kit* PrepFiler BTA Lysis Buffer Proteinase K PrepFiler Express Cartridges** Sample Tubes Elution Tubes PrepFiler LySep Columns PrepFiler Lysis Tubes (for bone) Tips and tip sheaths *1M DTT also required but not included in the kit ** Isopropanol and prepared PrepFiler Kit wash buffers are included in cartridge format *** Available as a plastics only kit 7 8/9/ Life Technologies Corporation
8 PrepFiler Express /Express BTA Cartridge Configuration Cartridges are foil sealed and pierced after protocol start -1 Elution Tube 0 Sample Tube Elution Solution PrepFiler Lysis Magnetic Particles Wash Solution Buffer For BTA protocol Isopropanol 9 8/9/ Life Technologies Corporation
9 PrepFiler Express /Express BTA Kit Off-Line Lysis
10 PrepFiler Express Kit Protocol Overview Add 500 L Complete PrepFiler Express Lysis Solution to Sample in PrepFiler LySep Column Assembly Incubate 70ºC for 40 minutes with shaking (750 rpm) Centrifuge (10000 g for 2 minutes) PrepFiler LySep Column Assembly Transfer lysate and Sample Tube to AutoMate Express Instrument Perform PrepFiler Express Standard Instrument Purification Script 11 8/9/ Life Technologies Corporation
11 The PrepFiler LySep Column Simple off-line Lysis & Substrate Removal Current Spin Basket Method Spin basket Manual transfer Spin DNA purification Sample tube 12 8/9/ Life Technologies Corporation
12 The PrepFiler LySep Column Simple off-line Lysis & Substrate Removal Add Sample, Lysis Buffer & DTT* to PrepFiler LySep Column Remove PrepFiler LySep Column and discard Load on Instrument Incubate/ Shake High-speed centrifugation enables simple & efficient separation of lysate from substrate 13 8/9/ Life Technologies Corporation
13 PrepFiler LySep Filter Column Allows incubation of substrate with lysis buffer in assembled PrepFiler LySep Column+Sample Tube configuration Filter unit plus membrane seal prevents any lysis buffer drainage during incubation High-speed centrifugation enables separation of lysate from substrate Centrifugation force bursts membrane seal enabling lysate flow-through Discard PrepFiler LySep Column containing substrate and load hingeless sample tube directly onto the AutoMate Express Instrument Eliminates the need for a Tip Dance protocol 14 8/9/ Life Technologies Corporation
14 PrepFiler Express BTA Kit Protocol: Adhesives Add 230 L Complete PrepFiler BTA Lysis Solution to Sample in PrepFiler LySep Filter Column Assembly Incubate 56ºC for 40 minutes with shaking (750 rpm) Centrifuge (10000 g for 2 minutes) PrepFiler LySep Column Assembly Transfer lysate and Sample Tube to AutoMate Express Instrument Perform PrepFiler Express BTA Purification Instrument Script 15 8/9/ Life Technologies Corporation
15 PrepFiler Express BTA Kit Protocol: Bones and Teeth Add 230 L Complete PrepFiler BTA Lysis Solution to 50mg bone/tooth powder in 2mL screw cap tube Incubate 56ºC for at least 2 hours with shaking (1100 rpm) Centrifuge (10000 g for 2 minutes) screw cap tube Transfer supernatant into Sample Tube and load onto AutoMate Express Instrument Perform PrepFiler Express BTA Purification Instrument Script 16 8/9/ Life Technologies Corporation
16 PrepFiler Express BTA Kit Protocol: Bone Samples Screw Cap: Needed during lysis due to bone effervescence which can pop open snap cap tubes. Also, prevents loss of volume during incubation 50mg Bone Powder: Optimal input to recover the highest yield of DNA per mg of starting material. Higher starting weight of bone may increase yield but may decrease available lysate volume 2 hour Incubation: Minimum time for incubation but can do up to 18 hours without adverse results. Supernatant Processing: After incubation, centrifugation required to pellet bone powder Process supernatant only 17 8/9/ Life Technologies Corporation
17 AutoMate Express Instrument DNA Isolation
18 Setup AutoMate Express Instrument Ensure Protocol Card is properly inserted Do Not Turn on Instrument without card inserted! Do Not Remove IC Card if the instrument is on! Power on AutoMate Express Instrument 19 8/9/ Life Technologies Corporation
19 AutoMate Express Instrument Run 1. Load cartridge rack with appropriate number of reagent cartridges 2. Load tip and tube rack Sample lysate Tips and tip holders Elution tube 3. Select appropriate PrepFiler Express Protocol 4. Start run 20 8/9/ Life Technologies Corporation
20 AutoMate Express DNA Isolation Wash 3X -1 Elution Tube 0 Sample Tube Lysis solution for BTA protocol Elution Tube Sample Tube Magnetic Particles Isopropanol Wash Buffer x3 Elution Buffer *Not to Scale 21 8/9/ Life Technologies Corporation
21 Instrument Run and Completion Both the PrepFiler Express Standard and BTA Kits take approximately 30 minutes to complete When the instrument has completed the run, the instrument will beep briefly and display Finished Protocol on the display screen. Open the door, remove samples and store short term at 4 o C or long term at -20 o C Discard the sample tubes, cartridges, tips, and tip holders into biohazardous waste containers 22 8/9/ Life Technologies Corporation
22 Developmental Validation Data
23 Sensitivity Linearity DNA yield (ng) y = x R 2 = Blood Sample Volume (ul) Average DNA Yield, ng (n=4) Liquid blood sample volume (µl) XB 0.00 N=4 for each blood sample The blood series demonstrates a proportional increase in DNA yield as a function of blood input. 24 8/9/ Life Technologies Corporation
24 Sensitivity STR Data: Representative Profiles 5 ul 1 ul 0.25 ul 0.1 ul ul 25 8/9/ Life Technologies Corporation
25 Correlation Study-Low Input Samples DNA Yield (ng) AutoMate Express System Method A Method B 0 Blood on cotton cloth (0.3 µl) Semen on cotton cloth (1 µl) Bone (50 mg) Extraction blank 26 8/9/ Life Technologies Corporation
26 Correlation Study-High Input Samples DNA Yield (ng) AutoMate Express System Method A Method B 20 0 Blood on cotton cloth (2 µl) Blood on FTA paper (2 µl) E-cells on swab (50 µl) Blood on blue denim (1 µl) Extraction blank 27 8/9/ Life Technologies Corporation
27 Correlation Study-Total Allele Count 100 AutoMate Express Method A Method B % Alleles Recovered Blood on cotton cloth (0.3 µl) Blood on cotton cloth (2 µl) Blood on FTA paper (2 µl) E-cells on swab (50 µl) Semen on cotton cloth (1 µl) Blood on blue denim (1 µl) Bone (50 mg) 28 8/9/ Life Technologies Corporation
28 Reproducibility-Low Input Samples 9 8 Total DNA Yield (ng) Day 1 Day 2 Day 3 0 Epithelial-cell fraction (5 µl) Semen on cotton cloth (1 µl) Bone (50 mg) Extraction blank (PrepFiler Express kit) Extraction blank (PrepFiler Express BTA kit) 29 8/9/ Life Technologies Corporation
29 Reproducibility-High Input Samples Total DNA Yield (ng) Day 1 Day 2 Day Blood on cotton (2 µl) Tooth (5 mg) Extraction blank (PrepFiler Express kit) Extraction blank (PrepFiler Express BTA kit) 30 8/9/ Life Technologies Corporation
30 Case Type Samples DNA Yield (ng) Blood on acetate Blood on leather Blood on rayon Blood on silk Blood on wool Diluted blood on cotton Hair root Saliva on FTA DE-s fraction Gum Cigarette butt Extraction blank (PF kit) Extraction blank (PF BTA kit) 31 8/9/ Life Technologies Corporation
31 Summary Robust, easy-to-use system optimized for use with PrepFiler Chemistry Effectively isolates high quality genomic DNA from a broad range of forensic samples Successfully removes inhibitors to PCR commonly found in forensic samples maximizing the performance of downstream applications. Extracted DNA validated for use with the Quantifiler and AmpFlSTR kits 32 8/9/ Life Technologies Corporation
32 For Research, Forensic, or Paternity Use Only. Not intended for any animal or human therapeutic or diagnostic use. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. NOTICE TO PURCHASER: Limited Use Label License The products shown in this presentation may be covered by one or more Limited Use Label License(s). Please refer to the respective product documentation or the Applied Biosystems website under for the comprehensive license information. By use of these products, the purchaser accepts the terms and conditions of all applicable Limited Use Label Licenses. These products are sold for research use only, and are not intended for human or animal diagnostic or therapeutic uses unless otherwise specifically indicated in the applicable product documentation or the respective Limited Use Label License(s).
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