Automated Extraction of High Quality Genomic DNA from Forensic Evidence Samples Using a Cartridge-Based System
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1 Automated Extraction of High Quality Genomic DNA from Forensic Evidence Jason Yingjie Liu1, Ph.D.*, Maxim Brevnov1, Ph.D., Allison Holt1, Ph.D., James E. Stray1, Ph.D., Declan Donovan2, Ph.D., Alan B. Dixon1, MSFS, Jim C. Nurse1,BS, Manohar R. Furtado1, Ph.D., and Jaiprakash G. Shewale1, Ph.D.
2 Disclosure This presentation includes commercial products from Applied Biosystems (part of Life Technologies) Other Laboratory Suppliers 2
3 Presentation Outline Introduction of automated benchtop DNA extraction system PrepFiler LySep column for workflow simplification Protocols and examples of DNA extractions using these protocols System studies such as cross contamination study, sensitivity study and instrument variation study Conclusions 3
4 Main Steps in Forensic DNA Analysis Sample Sample DNA Extraction Suitable for multiple sample types Effective removal of inhibitors High recovery for minimal input samples Simple to use with minimal hands on operations DNA quantification STR STR profiling 4
5 Automated Benchtop Forensic DNA Extraction System: Highlights Core reagents are from the PrepFiler Forensic DNA Extraction Kit utilizing magnetic particle based nucleic acid purification technology PrepFiler LySep column developed to perform lysis and separation of substrate in a single device PrepFiler lysis buffer for lysis of majority of forensic sample types PrepFiler BTA Lysis buffer for lysis of bone, tooth or samples containing adhesive All required DNA extraction reagents are in a sealed disposable plastic cartridge Benchtop instrument for automated purification of DNA 5
6 Cartridge Configuration Universal cartridge suitable for DNA extraction from lysates generated from both PrepFiler and PrepFiler BTA Lysis Buffers -1 Elution Tube 0 Sample Tube Lysis buffer For BTA protocol (500ul) Magnetic Particles Isopropanol Wash solution Elution solution 6
7 AutoMate Express TM Benchtop Instrument Processes 13 samples in about 30 minutes Elution Vol: 50 µl Two protocols (PFLB protocol and BTA protocol) on a single script card Cartridge rack Tip and tube rack 7
8 Overview of the Workflows PrepFiler Lysis Buffer (majority of samples including trace evidence samples) high extraction efficiency Lysate separation from substrate PFLB protocol DNA for downstream applications PrepFiler BTA Lysis Buffer (Bone, tooth, sticky tape, cig. butt) performing delicate lysis BTA protocol 8
9 PrepFiler LySep column: Lysis and substrate separation in a single device Spin basket Spin basket Manual transfer Spin DNA purification Sample tube PrepFiler LySep column Add Sample, Lysis Buffer & DTT* to PrepFiler LySep Column Load on Instrument Incubate/ Shake Remove PrepFiler LySep Column and discard 9
10 Protocol Options
11 Protocol 1: PrepFiler Express Extraction PrepFiler Lysis buffer PrepFiler LySep Column PF Express Instrument Protocol Quantifiler Duo Results Total DNA yield (ng) Human Male ul blood swab 0.3uL blood blue denim 0.3uL blood + inhibitor mix* 1uL semen cotton E-cell swab 11 * Inhibitor mix: 12.5mM Indigo, 0.5mM Hematin, 2.5mg/mL Humic acid and 300mg/mL urban dust
12 Identifiler STR Kit Profiles 1ng 0.5 ul blood swab 1ng 0.3uL blood blue denim 1ng 0.3uL blood w. inhibitors 1ng 1uL semen cotton 1ng E-cell swab 12
13 Protocol 2: PrepFiler Express BTA Bones/Teeth Sample Extraction PrepFiler BTA Lysis buffer Bone & Tooth Lysate Tube PF Express BTA Instrument Protocol Total DNA yield (ng) Quantifiler Duo Results 50mg bone Human Male Total DNA yield (ng) Quantifiler Duo Results Human 10mg tooth 13
14 Identifiler STR Kit Profiles 270pg 160pg 50mg bone 190pg 1ng 1ng 10mg tooth 1ng 14
15 Protocol 3: PrepFiler Express BTA Adhesive Sample Extraction PrepFiler BTA Lysis buffer PrepFiler LySep Column PF Express BTA Instrument Protocol Total DNA yield (ng) Quantifiler Duo Results Cigarette_butt Gum Envelope Human Male 15
16 Identifiler STR Kit Profiles 1ng Cigarette butt 0.5ng Chewed Gum 1ng Envelope 16
17 PrepFiler Express Extraction Protocol for Processing of Sexual Assault Samples E-cell fraction PrepFiler Lysis buffer Differential extraction PrepFiler Express Instrument Protocol Sperm fraction PrepFiler Lysis buffer 17
18 Mock sexual assault samples: E-cell/sperm ratio, 20:1 Quantification results Human Male 5 DNA Conc. (ng/ul) sperm1 E-cell-1 sperm2 E-cell-2 sperm3 E-cell-3 Sample 1 Sample 2 Sample 3 18
19 Identifiler STR Kit Profiles Sperm reference Sperm fraction E-cell fraction 19
20 Cross-Contamination, Sensitivity and Instrument Variation Studies
21 Cross-Contamination Study 10uL Blood 10uL 1xPBS Cartridge rack 1 Cartridge rack 2 Cartridge rack AutoMate Express positions alternate with 10uL blood and 10uL 1xPBS -MiniFiler profiles for all extraction blanks did not show cross-contamination 21
22 Sensitivity Study DNA (ng) N=4 0 5uL 1uL 5uL (1:20dil) 5uL(1:50dil) 5uL(1:200dil) Blood 22
23 Instrument to Instrument Variation Study 5 AutoMate Express instruments tested 2uL liquid blood processed in 12 replicates on each instrument using PrepFiler Express protocol Quantifiler Duo and Identifiler kits used for downstream analysis Consistent DNA yield obtained from each instrument DNA yield (ng) IPC Ct 0 Instr-1 Instr-2 Instr-3 Instr-4 Instr-5 0 Human Male IPC Ct 23
24 Representative STR Profiles Instr-1 Instr-2 Instr-3 Instr-4 24 Instr-5
25 Conclusions: AutoMate Express TM benchtop system developed for extraction of DNA from forensic samples 13 samples can be processed simultaneously in about 30 minutes PrepFiler LySep column greatly simplifies lysis workflow and reduces risk for contamination Minimal instrument to instrument variability PrepFiler Express chemistry capable of extracting DNA free of PCR inhibitors from samples containing low quantities of biological material Dedicated PrepFiler Express BTA lysis buffer for extracting DNA from challenging samples like bones, teeth, and adhesives. High quality STR profiles are generated from a broad range of forensic samples 25
26 Acknowledgements Lori Hennessy Lisa Calandro Chang Zhong Robert Lagacé Clarissa Kao Thomas McElroy 26
27 . All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. For Research, Forensics or Paternity Use Only. Not intended for any animal or human therapeutic or diagnostic use
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