tools for improving dna extraction efficiency

Tools for Improving DNA Extraction Efficiency

Human Identification Life Technologies

8/16/2012 | © 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

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


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)


PrepFiler® Forensic DNA Extraction Kit

Options and Developments for Manual, Semi-Automated & Automated Workflows


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


PrepFiler® Kit: Comparison with Other Methods


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


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


PrepFiler® BTA Manual Kit: Bone Sample: DNA Yield


PrepFiler® BTA Manual Kit: Bone Sample: Information Recovery


PrepFiler® BTA Manual Kit: Cigarette

Vendor 1 Vendor 2


PrepFiler® BTA Manual Kit: 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: 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


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


Semi-Automated Extraction using the AutoMate™ Express DNA Extraction System


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


AutoMate Express™ DNA Extraction System

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, 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)


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


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


Identifiler® Kit Profiles Bone and Tooth Samples

1ng

1ng

1ng

270pg

160pg

190pg

50 mg bone

10 mg tooth


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


Mock Sexual Assault Samples Quantifiler® Duo Kit Results

E-Cell/Sperm Ratio, 20:1


Identifiler® Kit Profiles

Sperm Reference

Sperm Fraction

E-Cell Fraction


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


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


PrepFiler® Automated Forensic DNA Extraction Kit


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


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


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


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


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


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.

tools for improving dna extraction efficiency

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