Tools for Improving DNA Extraction Efficiency
Human Identification Life Technologies
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Efficient Solutions for Forensic DNA Analysis
PrepFiler® Forensic DNA Extraction Kit − Designed specifically to maximize the yield and quality of DNA obtained
from forensic samples − Provides superior DNA yield compared to organic methods and
common commercial DNA extraction kits − Extracted DNA is optimized and validated for use with the Quantifiler®
and AmpFℓSTR® kits to streamline integration − Protocols available for both manual and automated extraction to suit all
laboratory throughput requirements
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Hypothesis of DNA Binding to the PrepFiler® Magnetic Particles: Schematic Presentation
PrepFiler® Magnetic Particles utilize a multi-component surface chemistry
DNA binds to the magnetic particles by formation of a complex between the polymer present on the surface of the particles, soluble polymer and DNA
The complex formation is accomplished in presence of high salt, detergent and isopropanol
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PrepFiler® & PrepFiler® BTA DNA Extraction Chemistry: Designed for Wide Variety of Forensic Sample Types
Routine Sample Types Liquid samples
− Blood, saliva and semen
Body fluid stains − Blood on cotton, rayon and nylon
fabric, denim − Saliva on cotton fabric − Semen on cotton fabric
Vaginal swabs w/ semen
Pulled hair roots
Casework reference samples − Buccal swabs − Blood/Buccal on FTA® Card
Challenged Sample Types
Bone and teeth
Cigarette butt and chewing gum
Fingernail clippings, scrapings
Formalin fixed tissue
Touch Evidence-Type Samples
Swabs from firearms/knives
“Wear area” swabs/cuttings
Steering wheel swab
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PrepFiler® Chemistry Platform Options
A single extraction chemistry available in several formats … − Provides flexibility in selecting a platform capable of
required sample throughput − Supports more straightforward conversion to different
platform, if throughput needs or applications should change
PrepFiler® Chemistry Platform Options − Manual − AutoMate Express™ DNA Extraction System − HID EVOlution™ System − Other Robotic Platforms (with scripts provided and
validated/verified by HID Professional Services Team)
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PrepFiler® Forensic DNA Extraction Kit
Options and Developments for Manual, Semi-Automated & Automated Workflows
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Alternative Chemistries Protocol Comparison
PrepFiler®/ PreFiler BTA ® DNA IQ QIAamp DNA
Investigator
Phenol Chloroform
(PCI)
Prelim
/Prep
Lysis C
ollection/Isolation
Purchase DTT Purchase Proteinase K
Purchase Ethanol Custom Bone Buffer (BB)
Custom Incubation Buffer (IB) T & H Kit
Pre-soak in 0.5% SDS (E)
Purchase DTT Purchase Proteinase K Make Bone Buffer (BB) Make Lysis Buffer (LB)
Purchase DTT Purchase Isopropanol/Ethanol
Purchase DTT Purchase Ethanol
Custom Bone Buffer (BB) Custom Incubation Buffer (IB)
T & H Kit
40 mins-2 hr @ 56-70°C
40 mins @ 70°C
1 hr @ 56°C in BB (B, To) 30 min @ 56°C in LB (G, Ta)
30 min @ 56°C in IB (C) 30 min @ 70°C in LB (E)
18 hr @ 56°C in LB (B, To)
3 hr @ 56°C in LB (G)
1 hr @ 56°C in LB (C, Ta, E)
1 hr @ 56°C in SB (B, To, G) 10 mins @ 56°C in SB
(C, Ta, E)
18 hr @ 56°C in BB (B, To)
1.5 hr @ 56°C in LB (C, G, Ta, E)
Add Binding Solution
Bind / Mag-Wash 3X
Bind / Spin-Wash 3X Phenol:CHCl3:IAA
YM30 Filter / Spin
Elute DNA Elute DNA Elute DNA
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PrepFiler® Kit: Comparison with Other Methods
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New! PrepFiler® BTA Manual Forensic DNA Extraction Kit
Improves performance of DNA extraction from calcified tissues, as well as certain adhesive and paper-containing samples
− Bones and teeth − Cigarette butts − Envelope flaps, gum, tape
PrepFiler® Manual Kit Updates
Available Now
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PrepFiler® BTA Kit: Protocol Highlights
Optimized performance with bones, teeth, as well as other adhesive and paper-containing samples; comparable or better yields & purity than conventional phenol-chloroform-based methods
50 to 200 mg of bone sample is optimal input material
Involves fewer tube transfers and greater overall ease of use than conventional methods to minimize the opportunity for sample mix-ups or contamination
2 hour lysis time for bone samples compared to 18 to 48 hours by traditional and alternative commercial chemistry methods
Simple, robust protocol facilitates training of users, and is more “tolerant” to range of experience levels
Utilizes PrepFiler® chemistry for isolation of DNA and removal of PCR inhibitors
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PrepFiler® BTA Manual Kit: Bone Sample: DNA Yield
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PrepFiler® BTA Manual Kit: Bone Sample: Information Recovery
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PrepFiler® BTA Manual Kit: Cigarette
Vendor 1 Vendor 2
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PrepFiler® BTA Manual Kit: Cigarette
Site A BTA
Site A PCI
Site B BTA
Site B PCI
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Case Study 1: Missing Persons Case
Figure 1: Bone sample extracted with QIAamp® Micro DNA kit and amplified with the Identifiler® kit. Neither the Identifiler® nor the Identifiler® Plus kits produced any STR results
Figure 2: Bone extracted with the PrepFiler® BTA extraction kit and amplified with the Identifiler® kit. Y scales between 200-400 RFU
Figure 3: Bone extracted with the PrepFiler® BTA extraction kit and amplified with the Identifiler® Plus kit. Y scales between 600-1000 RFU
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Case Study 2: Identification of Missing Persons from Spanish Civil War
• Recovery of Historical Memory − Identification of victims from the Spanish Civil war − 24 bodies deposited in a well on March 19th, 1937
• PrepFiler® BTA Forensic DNA extraction chemistry combined with Next Generation DNA profiling kits to significantly improve the quantity and quality of results
PrepFiler® BTA DNA Extraction and AmpFℓSTR® NGM™ kit
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Semi-Automated Extraction using the AutoMate™ Express DNA Extraction System
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The 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 up to 13 samples in ~30 minutes − PFLB and BTA protocols on a single script card
AutoMate Express™ Instrument +
PrepFiler® Express & PrepFiler® Express BTA Forensic DNA
Extraction Kit
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AutoMate Express™ DNA Extraction System
Cartridge rack
Tip and tube rack
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DNA for downstream applications
PFLB or BTA Protocol
AutoMate Express™ DNA Extraction Workflow
DNA Elution
Wash
DNA Binding
PrepFiler™ Lysis Buffer (PFLB) Majority of samples including trace evidence samples, sexual assault samples – high extraction efficiency
PrepFiler BTA™ Lysis Buffer Bone, tooth, sticky tape, cig. butt – for samples requiring delicate lysis
Lysate separation from substrate
(including differential lysate
fractions if necessary)
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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
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Identifiler® Kit Profiles Routine Sample Types
0.5 μL blood swab
All amplifications performed using 1 ng input DNA concentration
0.3 μL blood on blue denim
0.3 μL blood w/inhibitors
1.0 μL semen on cotton
E-cell swab
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Identifiler® Kit Profiles Bone and Tooth Samples
1ng
1ng
1ng
270pg
160pg
190pg
50 mg bone
10 mg tooth
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Standard Differential extraction
E-cell fraction
Sperm fraction
PrepFiler® Express PFLB Protocol
PrepFiler®
Lysis buffer
PrepFiler® Lysis buffer
PrepFiler® Express Extraction Protocol Processing of Sexual Assault Samples
The offline lysis protocol associated with the AutoMate Express™ DNA Extraction System allows for the same basic protocol, to be used across multiple sample types including differential
extraction of sperm and e-cell fractions from sexual assault cases
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Mock Sexual Assault Samples Quantifiler® Duo Kit Results
E-Cell/Sperm Ratio, 20:1
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Identifiler® Kit Profiles
Sperm Reference
Sperm Fraction
E-Cell Fraction
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Correlation Study: PrepFiler® Express Kit
0
20
40
60
80
100
120
Blood on cotton cloth(2 µL)
Blood on FTA paper(2 µL)
E-cells on swab(50 µL)
Blood onblue denim
(1 µL)
Extraction blank
DN
A Y
ield
(ng)
AutoMate Express™ SystemMethod AMethod B
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Correlation Study: Total Allele Count
40
50
60
70
80
90
100
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)
% A
llele
s R
ecov
ered
AutoMate Express™ Method A Method B
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Plastic Consumable Cleanliness Initiatives
All plastics, including PrepFiler® LySep columns, are assembled and packaged in a clean room environment, with minimal human interaction
Plastic components undergo “decontamination” by ethylene oxide for removal of any potential extraneous DNA
These consumables are screened for the presence of any extraneous human DNA, using internal kit QC methods on every batch of kits produced, and by external testing conducted by an independent third party on a quarterly basis
PrepFiler® Express Kit Updates
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PrepFiler® Automated Forensic DNA Extraction Kit
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The PrepFiler® Kit High Throughput Automation System
Multiple platform options now available to support the use of the PrepFiler® Automated Forensic DNA Extraction Kit
− HID EVOlution™ System (Tecan) − Forensics STARlet ABI Workstation (Hamilton) − Other Robotic Platforms (with scripts provided and
validated/verified by HID Professional Services Team)
Suitable for high throughput operations where releasing analyst time for more operator-dependent processes is critical
Key parameters: reproducibility, consistency and minimal risk of contamination
Packaging improvements for the manual PrepFiler® Kit have also been applied to the automated kit
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Reproducibility and Concordance- Tecan
0
10
20
30
40
0
2
4
6
5 μL Blood Stained Cotton
50 μL Saliva on Swab
1 μL Semen Stained Cotton
5 μL Blood Stained Cotton + Inhibitors
5 μL Blood Stained Denim
Extraction Blank
IPC
CT
DN
A Y
ield
(ng/
mL)
Sample Type
Experiment 1 Experiment 2 Experiment 3 Manual Extraction
Experiment 1 Experiment 2 Experiment 3 Manual Extraction
DNA Yield
IPC CT
Samples were run in replicates of 8 and 3 for automated and manual operations, respectively
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Automated extraction of DNA from 8 challenging samples - 2 µL of blood stain on cotton in presence of inhibitors
PrepFiler® Kit Automated: Consistency of DNA Quality- Tecan
3000 RFU
3000 RFU
3000 RFU
3000 RFU
3000 RFU
3000 RFU
3000 RFU
3000 RFU
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Cross-Contamination Study- Tecan
1 2 3 4 5 6 7 8 9 10 11 12
A 10.82 0.00 9.77 0.00 11.06 0.00 16.63 0.00 11.63 0.00 14.76 0.00
B 0.0004 19.93 0.00 11.64 0.00 9.25 0.00 10.37 0.00 12.52 0.00 11.12
C 14.99 0.00 13.34 0.00 20.96 0.00 11.07 0.00 8.86 0.00 7.26 0.00
D 0.00 10.80 0.00 16.03 0.00 13.67 0.00 20.76 0.00 10.49 0.00 8.98
E 15.38 0.0005 8.74 0.00 9.94 0.0004 14.23 0.00 12.80 0.00 14.84 0.0006
F 0.00 20.61 0.00 11.83 0.00 8.57 0.00 9.49 0.00 15.45 0.00 9.74
G 14.46 0.0005 11.72 0.00 19.65 0.0009 10.60 0.00 8.41 0.00 5.64 0.00
H 0.00 7.22 0.00 15.71 0.00 11.92 0.00 19.72 0.00 11.28 0.00 10.67
Checkerboard extraction of DNA from 6 blood donors (10 μL in replicates of 8) and 48 blank samples (concentration shown in ng/µL)
None of the blank wells exhibited an STR profile when amplified with the MiniFiler™ kit
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PrepFiler® DNA Extraction Verification – Hamilton
Blood on Cotton
Saliva on Cotton
Indirect Dried Saliva
Diluted Blood on Swab
Diluted Saliva on Swab
Mock sample study – NGM SElect™ Kit profiles
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PrepFiler® & PrepFiler® BTA Forensic DNA Extraction Chemistry: Summary New developments and improvements to support the use of the PrepFiler®
chemistry for all types of forensic DNA extraction − PrepFiler® and PrepFiler® BTA kits available for manual and AutoMate
Express™ workflows to provide maximum flexibility for routine and challenging sample types
− Introduction of new wash buffer and kit packaging changes to improve performance, usability, and ordering experience
− PrepFiler® kit available for fully automated workflows on different
platforms such as Tecan and Hamilton to support high throughput of routine sample types
− HPS available to facilitate validation & implementation of various HT platforms
− New initiatives introduced to improve cleanliness of AutoMate Express™ plastics
Thank You For Forensic or Paternity Use Only. Not for use in any animal or human therapeutic or diagnostic procedure. © 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.