the evolution of direct amplification: from sample to ... · the evolution of direct amplification:...

The Evolution of Direct Amplification: From Sample to Result in 2 Hours*

Nicola Oldroyd Forensic Applications Consultant Human Identification

* Typical workflow for 24 samples

5/10/2012 | © Life Technologies™ 2

Traditional Single Source Sample Workflow

Time to Result (hrs/per 24 samples)

~1-3.5 ~0-2 ~4 ~1.5 ~1

Total Time to Result = ~7-12 hrs

Collection Sample

Preparation Quantitation/ Normalization

Amplification Detection Analysis


Development of Direct Amplification on Treated Paper Substrates


Evaluation of Direct Amp Capability of the Identifiler® Kit

Control DNA 007

25 μL Reaction Volume

1.2 mm Blood on FTA® Disc

No full profiles under direct amp conditions


Identifiler® Kit Direct Amp Performance Evaluation

Possible explanations for poor performance

− Insufficient DNA?

− Excess PCR inhibitors?

0.75 mm disc ~ 22.08 ng DNA


1.2 mm disc ~ 56.52 ng DNA Height = 1mm

1 μL blood ~ 50 ng DNA

Sufficient DNA still available

Inhibitor concentration reduced by >5-fold



Effect of Reducing Disc Size on Identifiler® Kit Direct Amp Performance

0.5 mm blood on FTA® disc


Control DNA 007 (1 ng)


But: − No automated

0.5 mm punch head options

− 0.5 mm disc size too small for buccal samples

− 0.5 mm discs very hard to handle


Optimisation of Reaction Buffer for 1.2 mm Discs

Component 1

Response Component 2

Use of Design of Experiments (DOE) Approach


Optimisation of Identifiler® Direct Kit Mastermix Buccal sample on 1.2 mm FTA® disc before DOE


Optimisation of Identifiler® Direct Kit Mastermix Buccal sample on 1.2 mm FTA® disc after DOE


− 3 x liquid blood samples

− 80 μL of blood onto FTA® Classic (passive diffusion)

− Sample 1.2 mm discs from the center to the edge of the blood stain

− Perform replicates for each position

Effect of Punch Position on Sample Peak Heights

1 3

2 4 5


FTA® Technology by Whatman

FTA® Classic Card

EasiCollect ® System

Identifiler® Direct Kit

Identifiler® Kit

Exam

ple

1

Exam

ple

2

Identifiler®Direct Kit

Identifiler® Kit

Blood on FTA® samples


Identifiler® Direct Kit Validation: External Test Site Results

Sample Type

PCR Success Rate Interpretation Success Rate Number of

Samples Tested Range of Success rates

Mean Success Rate

Range of Success rates

Mean Success Rate

VTS Stu

dy

Blood 99.4 % 99.4 % 95.7 – 98.8% 97.3 % 414

Buccal 91.8 – 99.4% 97.1 % 84.2 – 95.5% 90.9 % 653

CTS Stu

dy

Blood 100% 100 % 98.8 – 100% 99.8 % 437

Buccal 98.7 – 100% 99.0 % 91.7 – 100% 94.7 % 703

1st Pass Success Rate Definition

− All profile peaks higher than specified threshold

− Off scale peaks produce no artifacts which interfere with profile interpretation

> OL-labelled pull-up peaks <20% of highest peak of the marker

> No split, double called peaks

> Stutter peaks < 20% of marker or < marker stutter cut off, whichever is higher


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NUCLEICard™ System by Copan

NUCLEICard™ System

Chemically-treated paper-based

substrate to:

Lyse cell membranes and denature

proteins upon contact

Prevent the growth of bacteria and

other microorganisms

Maintein the integrity of nucleic acids

Allow direct PCR amplification from a

card punch

Certified human DNA, DNAse, RNAse

free and EtO treated


NUCLEICard™ Performance Study

PCR Success Rate Interpretation Success Rate

Substrate N Number of

Full Profiles

First Pass

Success Rate

Number of

Full Profiles

First Pass

Success Rate

FTA® Card 39 39/39 100% 39/39 100%

NUCLEICard™ Lot 1 40 40/40 100% 40/40 100%

NUCLEICard™ Lot 2 40 40/40 100% 39/40 97.5%

NUCLEICard™ Lot 3 40 40/40 100% 40/40 100%

Blood samples on FTA® Paper and NUCLEICard™ substrates

All data run on 3500 series platforms


Blood on FTA® Card Blood on NUCLEICard™

Profile Comparison: NUCLEICard vs. FTA® Paper

Identifiler® Direct Kit Amplifications


Expansion of the Direct Amplification Workflow to Non-FTA Substrates

Untreated Paper


Identifiler® Direct Kit development and optimisation performed on FTA® substrates only

Direct amplification workflow proven to deliver time and cost savings for database sample processing

Many laboratories in the US and international jurisdictions want to take advantage of direct amplification but use alternative substrates and/or alternative marker sets

Expansion of the Direct Amplification Workflow to Non-FTA® Substrates

Goal: Enable direct amplification of non-FTA® buccal samples

Prep-n-Go™ Buffer


Minimal additional workflow steps

Efficient cell lysis without the requirement for heat incubation

Good sample peak heights

High quality, well-balanced STR profiles

No introduction of artifacts

Optimized for Bode Buccal DNA Collector™ and Identifiler® Direct

Tested on buccal swabs and other STR chemistries

Prep-n-Go™ Buffer: Development Requirements & Considerations


Direct Amp Workflow: Untreated Paper Substrates

Collect Samples on Untreated Paper

One New Step

Electrophorese

Amplify

Punch 1.2 mm disc

Add PCR reagents

Add Prep-n-Go™ Buffer


Bode Buccal DNA Collector™ Samples Amplified with the Prep-n-Go™ Buffer and the Identifiler® Direct kit

2 different individuals amplified for 26 PCR cycles Well-balanced profiles within each dye colour


Performance Study Results Summary

Test Site Number of

Samples Tested

PCR Cycle

Number

CE Platform


Number of Full Profiles

First Pass Success Rate

Number of Full Profiles

First Pass Success Rate

1 82* 27 3130xl 81/82 98.8% 78/82 95.1%

2 80 26 3130xl 78/80 97.5% 78/80 97.5%

3 84* 26 3130xl 83/84 98.8% 79/84 94.0%

4 84* 26 3130xl 80/81 98.8% 76/81 93.8%

5 84* 26 3730 83/84 98.8% 83/84 98.8%

6 84 26 3500 82/84 97.6% 74/84 88.1%

Life Technologies

84 26 3130xl 82/84 97.6% 79/84 94.0%

Total 582 569/582 97.8% 547/582 94.0%

* Used real offender samples


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Buccal DNA Collector™ Performance Study

Peak Heights Intracolor Balance


Identifiler® Direct Kit w/ Prep-n-Go™ Buffer Identifiler® Direct Kit w/Other Buffer

1200 1200

Lysis Buffer Performance Comparison

No allelic drop-out using Prep-n-Go™ Buffer


Expansion of the Direct Amplification Workflow to Non-FTA Substrates

Buccal Swabs


Pros

− Easy to use

− Inexpensive

Cons

− High performance variability among swab types

− Limited opportunities for automation increasing labour requirements and the risk of contamination

− Result quality dependent on collection process and storage conditions

Buccal Swabs


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Total recovery of DNA from FLOQSwabs™

Maximum efficiency in collection capacity and sample release

Available with different anatomical and ergonomic design

Human DNA, DNASE and RNASE free certified

ETO treated (Ethylene Oxide)

• Patented worldwide: Copan WO 2004/086979

Pre-scored breaking point

Fibre v Floq swab


Direct Amp Workflow: Buccal Swabs

Collect Samples on Buccal Swab

Lyse swab in Prep-n-Go™ Buffer

Add PCR Reagents

Transfer Lysate

New steps

Electrophorese

Amplify

Stand @ RT for 20 minutes


Copan 4N6 Swab Puritan® Cotton Swab Whatman® Omni Swab

Buccal Swab Performance Comparison

Identifiler® Direct Kit Amplifications


Expansion of the Direct Amplification Workflow to Other STR Marker Sets


Direct Amplification: The NGM™ & NGM SElect™ Kits

NGM SElect™ Kit NGM™ Kit

Amplification of buccal samples on Bode Buccal Collector™ treated with Prep-n-Go™ Buffer


Utilises the same primer sequences as the NGM™ and NGM SElect™ Kits

Includes a new mastermix optimised specifically to support direct amplification of swab and treated/untreated paper substrates

Delivers rapid cycling times through the introduction of a new fast-capable enzyme (< 1 hr)

May be amplified using the Veriti® 96-Well or 9700 (silver or gold-plated silver block) thermal cyclers

− Veriti® 96-Well Thermal Cycler (standard) now supported for use with all existing AmpFℓSTR® kits

Direct Amplification for the Expanded ESSL

AmpFℓSTR® NGM SElect™ Express Kit


AmpFℓSTR® NGM SElect™ Express Kit Configuration & Cycling Conditions

Components Master Mix Primer Set

Control DNA 007 Allelic ladder

PCR Conditions 95°C/ 1 min 94°C/ 3 sec 59°C/ 16 sec 65°C/ 29 sec 60°C/ 5 min

25-28 x

*Cycle number requires optimisation by each laboratory for each sample type/substrate, additional cycles will increase the cycling time slightly

Cycling Time: ~45* min!


AmpFℓSTR® NGM SElect™ Express Kit Allelic Ladder


AmpFℓSTR® NGM SElect™ Express Kit Control DNA 007


AmpFℓSTR® NGM SElect™ Express Kit Example Profiles: Treated Paper

Blood on FTA® Classic Card

Buccal on FTA® Indicating Card


AmpFℓSTR® NGM SElect™ Express Kit Example Profiles: Untreated Paper

Blood on 903 Paper

Buccal on Bode Buccal Collector™


AmpFℓSTR® NGM SElect™ Express Kit Example Profiles: Swabs

Buccal on Copan Flocked Swab

Buccal on Whatman Omniswab


AmpFℓSTR® NGM SElect™ Express Kit Instrument Compatibility Study

25 cycles

Buccal samples on Omniswab


AmpFℓSTR® NGM SElect™ Express Kit Species Specificity Study

Dog

Cat

Horse

Sheep

Cow

Pig

Rat

Rabbit

10 ng input DNA, 28 cycles


AmpFℓSTR® NGM SElect™ Express Kit Negative Control Reaction

Low level peaks in the NED dye channel occasionally observed at ~ 70 bp

No artifacts within the read region


Test Site Results

Reproducibility Study

− Either 10 buccal swabs or 10 buccal samples on treated paper amplified in replicates of 6

Test Site Sample

Type Cycle #

CE Platform


Number of

Full Profiles

First Pass

Success Rate

Number of

Full Profiles

First Pass

Success Rate

1

FTA® Paper

26 3130xl 60/60 100% 60 100%

2 28 3130xl 60/60 100% 60 100%

3 27 3500xL 60/60 98% 57 98%

4

Copan 4N6 FLOQ Swabs

25 3500xL 57/60 95% 57 95%

5 26 3130xl 60/60 100% 60 100%

Life Technologies

25 3130xl 60/60 100% 60 100%


A Complete Set of Direct Amplification Workflows

Collect Samples

Electrophorese

Amplify

Punch 1.2 mm Disc

Add PCR Reagents

Treated Paper

Add Prep-n-Go™ Buffer

Punch 1.2 mm Disc

Add PCR Reagents

Untreated Paper

Lyse Swab in Prep-n-Go™ Buffer

Add PCR Reagents

Transfer Lysate

Buccal Swabs


Maximising Direct Amplification Results & Workflow Efficiency


Factors Influencing Direct Amplification Results

Choice of Kit

Choice of Sample Type/Substrate Sample Transfer Efficiency

Punch Size and Position

Cycle Number

Data Analysis Parameters


Optimising Data Analysis Parameters for Single-Source Samples

Use of a global cut-off filter can reduce significantly the amount of editing required for single-source sample data

Peak Quality settings may also be adjusted to better reflect the characteristics of single-source samples


Optimised Parameters = More Efficient Analysis

Un

op

tim

ised

Op

timised



~1-3.5 ~0-2 ~4 ~1.5 ~1

Maximized Efficiency for Single Source Sample Processing


0 0 0.75 0.55 0.1


Collection Extraction Quantitation/ Normalization

Amplification Detection Analysis



~1-3.5 ~0-2 ~4 ~1.5 ~1

Maximized Efficiency for Single Source Sample Processing


0 0 0.75 0.55 0.1


Collection Amplification Detection Analysis

Total Time to Result = ~2 hrs


Thank You

© 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. EasiCollect and FTA are registered trademarks of Whatman Limited. Buccal DNA collector is a trademark of Bode Technology. FLOQswabs is a trademark of Copan Italia S.P.A.

Refer to product page on the Life Technologies website for Limited Use License.

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