Tools for Improving DNA Extraction Efficiency · Forensic DNA Extraction System . AutoMate ....

Tools for Improving DNA Extraction Efficiency

2/14/2012 | © Life Technologies™ 2

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


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


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

Reference samples − Buccal swabs − Blood 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

Cell phone swab


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)

6 2/14/2012 | © Life Technologies™

PrepFiler® Forensic DNA Extraction Kit (Manual Chemistry)


Comparison of Alternative Chemistries and Protocols: Manual Extraction

PrepFiler® Forensic DNA Extraction Kit

Vendor 1 Vendor 2 Phenol

Chloroform (PCI)

Prelim

/Prep

Lysis C

ollection/Isolation

Purchase Proteinase K Custom Bone Buffer (BB)

Custom Incubation Buffer (IB) T & H Kit

Pre-soak in 0.5% SDS (E)

Purchase Proteinase K Make Bone Buffer (BB) Make Lysis Buffer (LB)

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 SB

Bind / Mag-Wash 3X

Bind / Spin-Wash 3X Phenol:CHCl3:IAA

YM30 Filter / Spin

Elute DNA Elute DNA Elute DNA


PrepFiler® Kit: Comparison with Other Methods


PrepFiler® BTA Kit …Now Available!

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® 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


Manual PrepFiler® BTA Chemistry: Bone Sample


Manual PrepFiler® BTA Chemistry: Bone Sample


Manual PrepFiler® BTA Chemistry: Cigarette

Vendor 1 Vendor 2


Manual PrepFiler® BTA Chemistry: Cigarette

Site A BTA

Site A PCI

Site B BTA

Site B PCI


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: Same bone sample extracted with the PrepFiler® BTA extraction kit and amplified with the Identifiler® kit. Y scales between 200-400 RFU.

Figure 3: Same bone sample extracted with the PrepFiler® BTA kit and amplified with the Identifiler® Plus kit.

Y scales between 600-1000 RFU.


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


PrepFiler® Express Forensic DNA Extraction Kit Using the AutoMate Express™ DNA Extraction System


The AutoMate Express™ Forensic DNA Extraction System AutoMate Express™ Instrument

+

PrepFiler® Express & PrepFiler® Express BTA Forensic DNA

Extraction Kit

The Automate Express™ Forensic DNA Extraction System is an easy-to-use, robust bench-top DNA purification system which utilizes PrepFiler® kit chemistry and is designed to maximize DNA yield

and PCR inhibitor removal across the widest range of casework samples


AutoMate Express™ 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 – Two protocols on a single script card

> PrepFiler® Lysis Buffer and Bone Tooth Adhesive (BTA)

Cartridge rack

Tip and tube rack


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 – high extraction efficiency

PrepFiler BTA™ Lysis Buffer Bone, tooth, sticky tape, cig. butt – for samples requiring delicate lysis

Lysate separation

from substrate


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


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


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


“Raising the Bar” on Plastics Quality

All plastics, including PrepFiler® LySep columns, are assembled and packaged in clean room environment, with minimal human interaction

Plastic components undergo “decontamination” by ethylene oxide for removal of any potential extraneous DNA

Kits are regularly certified by third party vendor for absence of detectable levels of human DNA (i.e. < 1 cell)


PrepFiler® Automated Forensic DNA Extraction Kit


The PrepFiler® Kit High Throughput Automation System: Main Components HID EVOlution™ Extraction System

− Tecan Freedom EVO® 150 robotic platform

8 channel liquid handling system & Combo (4 x 4)

Robotic manipulator (RoMa) for plate transfer

TeShake module for plate heating and shaking

O-ring magnetic platform for magnetic separation

Holders for disposable tips, plates and tubes

Reservoirs for reagents

Liquid waste system

For labs that “batch” large numbers of samples (up to 96); “centralized” point of DNA extraction


Reproducibility and Concordance

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


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

3000 RFU

3000 RFU

3000 RFU

3000 RFU

3000 RFU

3000 RFU

3000 RFU

3000 RFU


Cross-Contamination Study

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


PrepFiler® & PrepFiler® BTA Forensic DNA Extraction Chemistry: Summary

Effectively isolates genomic DNA from a broad range of forensic-type samples

Successfully removes inhibitors to PCR commonly found in forensic samples

A single extraction chemistry available in several formats providing flexibility when selecting a platform capable of required sample throughput and application

DNA purified is ready for downstream applications such as quantitative real-time PCR (Quantifiler® kits) and STR profiling (AmpFSTR® kits)


PrepFiler® Kit Updates Updated PrepFiler® Manual and Automated Forensic DNA Extraction Kits

− Repackaging of current on-market PrepFiler® kits to include new wash buffer and configuration improvements:

> Wash Buffer B (i.e. WB3) to maximize performance with samples containing a small amount of DNA

> Packaging to include all reagents, including magnetic particles, with a single expiration date

> Plastics sold separately (Spin Tubes, Filter Columns, 2 ml screw cap tubes, all plates)

− Updated kits will have new P/N’s

> Current P/N’s will be discontinued no sooner than October/November, 2012

Expected release: Manual kit (now available); Automated Kit (later in 2011)

Thank You For Research, Forensic or Paternity Use Only. Not for use in any animal or human therapeutic or diagnostic procedure. © 2011 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.

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