Automated Extraction of High Quality Genomic DNA from ... · 0.5 uL blood swab 0.3uL blood blue denim 0.3uL blood + inhibitor mix ... • AutoMate ExpressTM benchtop system developed

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.

Automated Extraction of High Quality Genomic DNA from Forensic Evidence Samples Using a Cartridge-Based System

© 2010 Life Technologies CorporationFebruary 26, 2009


2

Disclosure

• This presentation includes commercial products from• Applied Biosystems (part of Life Technologies)• Other Laboratory Suppliers



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



4

• Suitable for multiple sample types• Effective removal of inhibitors• High recovery for minimal input

samples• Simple to use with minimal hands on

operations

DNA ExtractionDNA Extraction

SampleSample

DNA quantificationDNA quantification

STR profilingSTR profiling

Main Steps in Forensic DNA Analysis



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



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Cartridge Configuration

1 2 3 4 5 6 7 8 9 10 11 12

Sample

Tube

Elution

Tube

0-11 2 3 4 5 6 7 8 9 10 11 12

Sample

Tube

Elution

Tube

0-1

Magnetic Particles Wash solution

Elution solution

Isopropanol

Universal cartridge suitable for DNA extraction from lysatesgenerated from both PrepFiler™ and PrepFiler BTA™ Lysis Buffers

Lysis bufferFor BTA protocol(500ul)



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AutoMate ExpressTM

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



8

Overview of the Workflows

PrepFiler™ Lysis Buffer(majority of samples including trace evidence samples) – high extraction efficiency

PrepFiler BTA™Lysis Buffer(Bone, tooth, sticky tape, cig. butt) performing delicate lysis

Lysate separation from substrate

DNA for downstream applications

PFLB protocol

BTA protocol



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Sample tube

Spin basket

Manualtransfer

SpinDNA purificationSpin basket

PrepFiler LySep™ column: Lysis and substrate separation in a single device

PrepFilerLySep™ column

Add Sample, Lysis Buffer & DTT* to PrepFiler LySep™Column

Remove PrepFiler LySep™ Column and

discard

Load on Instrument

Incubate/ Shake

Protocol Options



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Protocol 1: PrepFiler Express™ Extraction

Quantifiler Duo Results

0

2

4

6

8

10

0.5 uL bloodswab

0.3uL bloodblue denim

0.3uL blood +inhibitor mix

1uL semencotton

E-cell swab

Tota

l DN

A y

ield

(ng)

HumanMale

PrepFilerLySep™Column

PF ExpressInstrument

ProtocolPrepFiler

Lysis buffer

Inhibitor mix: 12.5mM Indigo, 0.5mM Hematin, 2.5mg/mL Humic acid and 300mg/mL urban dust

*

*



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0.5 uL blood swab

1uL semen cotton

Identifiler® STR Kit Profiles

E-cell swab

0.3uL blood blue denim

0.3uL blood w. inhibitors

1ng

1ng

1ng

1ng

1ng



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Protocol 2: PrepFiler Express BTA™Bones/Teeth Sample Extraction


0.0

0.5

1.0

1.5

2.0

50mg bone

Tota

l DN

A y

ield

(ng)

HumanMale


0

20

40

60

80

100

120

10mg tooth

Tota

l DNA

yie

ld (n

g)

Human

Bone & Tooth Lysate Tube

PF Express BTAInstrument

ProtocolPrepFiler BTA

Lysis buffer



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1ng

1ng

1ng

270pg

50mg bone

10mg tooth

160pg

190pg




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Protocol 3: PrepFiler Express BTA™ Adhesive Sample Extraction

PrepFilerLySep™Column

PF Express BTAInstrument

ProtocolPrepFiler BTA

Lysis buffer


0

10

20

30

40

50

Cigarette_butt Gum Envelope

Tota

l DNA

yie

ld (n

g)

HumanMale



16

Cigarette butt

Chewed Gum

Envelope

1ng

0.5ng

1ng




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Differentialextraction

E-cellfraction

Spermfraction

PrepFiler ExpressInstrument

Protocol

PrepFiler™Lysis buffer

PrepFiler™Lysis buffer

PrepFiler Express™ Extraction Protocol for Processing of Sexual Assault Samples



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0.12

3.2

0.14

4.4

0.15

5

0

1

2

3

4

5

sperm1 E-cell-1 sperm2 E-cell-2 sperm3 E-cell-3

DN

A C

onc.

(ng/

uL)

HumanMale

Mock sexual assault samples: E-cell/sperm ratio, 20:1

Quantification results

Sample 1 Sample 2 Sample 3



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Sperm reference

Sperm fraction

E-cell fraction


Cross-Contamination, Sensitivity and Instrument Variation Studies



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Cartridge rack 1 Cartridge rack 2 Cartridge rack 31 1 1

2 2 2

3 3 3

4 4 4

5 5 5

6 6 6

7 7 7

8 8 8

9 9 9

10 10 10

11 11 11

12 12 12

13 13 13

Cross-Contamination Study

-AutoMate Express™ positions alternate with 10uL blood and 10uL 1xPBS-MiniFiler profiles for all extraction blanks did not show cross-contamination

10uL 1xPBS10uL Blood



22

Sensitivity Study

0.7

1.5

3.6

67

18

0

1

10

100

5uL 1uL 5uL (1:20dil) 5uL(1:50dil) 5uL(1:200dil)

Blood

DN

A (n

g)

N=4



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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

28.8 28.7 28.7 28.7 28.6

0

5

10

15

20

25

30

35

40

45

Instr-1 Instr-2 Instr-3 Instr-4 Instr-5

DN

A y

ield

(ng)

0

5

10

15

20

25

30

IPC

Ct

HumanMaleIPC Ct



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Representative STR Profiles

Instr-1

Instr-2

Instr-3

Instr-4

Instr-5



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Conclusions:• AutoMate ExpressTM 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



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Acknowledgements

Lori Hennessy

Lisa Calandro

Chang Zhong

Robert Lagacé

Clarissa Kao

Thomas McElroy

© 2010 Life Technologies Corporation. 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

Automated Extraction of High Quality Genomic DNA from ... · 0.5 uL blood swab 0.3uL blood blue denim 0.3uL blood + inhibitor mix ... • AutoMate ExpressTM benchtop system developed

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