Next generation PCR, leading by digital PCR, Cast PCR and ...cgs.hku.hk/portal/files/GRC/Events/Seminars/2011/20110516/digital … · A pair of assays: mutation assay and a gene reference

Next generation PCR, leading by digital PCR, Cast PCR and OpenArray ® PCR, Cast PCR and OpenArray ®

Kevin MunnellySr. Director, qPCR PlatformLife Technologies

Agenda• Introduction

• castPCR Overview

• OpenArray Overview− Red Blood Cell Genotyping Study− Breast Cancer Biomarker Study− miRNA in Plasma Study

2

− miRNA in Plasma Study− Single Cell Gene Expression Study in Stem cells

• Digital PCR Overview− Detection of West Nile Virus

| Life Technologies Proprietary & Confidential | 5/12/2011

Individual Assays in TubesFlexible, any instrument

TaqMan® Assay Plates5 formats – FAST & Std96 well optical platesCustom Plated; 135 inventoried

Choice of 4 formats

GeneAssist ToolsSearch for assays in agiven pathway or workflow

Software tools simplifyAssay selection & data analysis

UmapItSearch for assays that map to microArray probes

Genomic Assays Portfolio

3

TaqMan® Arrays384 well microfludic cards>80 pre-defined pathways Custom AssaysMedium and large projects

Custom Plated; 135 inventoriedPredefined gene sets

map to microArray probes

DataAssist, StatMinerData analysis softwareStatistical analysis

TaqMan OpenArray® Plates3072 well array plate; highestThroughput/lowest cost per rxn

SNP Browser

AutoCaller SNP Analysis

CopyCaller CNV Analysis

Next-Gen qPCR technology now..

TaqMan assays for any sample, any size research project!

4

• mRNA, microRNA, epigenetic target discovery & validation• DNA variant discovery & validation• Toxicity• Dosing profiles

• Clinical screening with consolidated human pathogen panels• Host-pathogen response• Antibiotic screening

• Genetic epidemiology• Copy number variation• Gene discovery• siRNA knockdown validation• Stem cell biomarkers

Drug Development Translational Clinical Research

Pathogen ID and Quantification

Consolidation and Standardization of Molecular Information Across Multiple Markets

• Biomarker selection and validation• Companion diagnostic development• Predisposition screening• Treatment selection• Recurrence monitoring

Life Science Research

5

• Dosing profiles• Mechanism of action

• Recurrence monitoring

Multiple applications enabled by spatially multiplexed, multi-specimen quantitative PCR analysis

A New Era in High-Productivity qPCR

● Integrated TaqMan® Array block

● Responsive touch-screen interface

6

● Rapid block change

● Intuitive software with ReadiApp™ Application and QuickStart functions

● OptiFlex™ System

Most Flexible Optical TechnologyOptiFlex™ System

Emission

Excitation

● Maximal multiplexing capability− 6x6 excitation and emission filters

for up to 21 combinations− Run TaqMan® and SYBR®

chemistry-based assays on the same run

7

• Sensitivity and Uniformity

− Read 384-wells at volumes to 1uL

(TaqMan® Array Cards)

− Detect 1.5 fold change

− Enables consistent results: run-to-

run and instrument-to-instrument

How to Detect Cancer Early?

Early Late

Cancer invasionAngiogenesisMetastasis

Diagnosis

Premalignant progression

8

• Circulating tumor cells (CTCs) in blood carry mutations.

• CTCs could be used as diagnostic markers. • No. of CTCs in blood is low. Highly specific

and sensitive assays are needed for early cancer detection.

AAAA

G

A

Normal DNA

Mutant DNA

A pair of assays: mutation assay and a gene reference assay

Both assays with FAM MGB probe

Mutation Assay− Allele specific primer (ASP)

GC

Mutation specific PCR

ASP

LSP

LST

MGBASB

Competitive Allele-Specific TaqMan ® PCR

castPCR™: C ompetitive A llele-S pecific T aqMan® PCR

9

− Allele Specific MGB blocker (ASB)− locus-specific TaqMan® probe (LST)− locus-specific PCR primer (LSP)

Gene Reference Assay− Standard TaqMan® assay− Two locus specific primers− Locus specific TaqMan® Probe

AMGBASB

CastPCR: TaqMan-like Sensitivity and Dynamic Range

Amplification plot Standard curve

Cq

25

30

35

40

SNP: NRAS-181C>A

Cq

1.00

∆Rn

Y = -3.5x + 34.2R2 = 0.9999

107 106 105 104 103 102 10 1 copies

10

DNA copy number (log10)

Cq

0

5

10

15

20

0 1 2 3 4 5 6 70 5 10 15 20 25 30 35 40 45

Cycles

Cq0.10

0.01

∆Rn

Assay Specificity – Conventional AS-PCR vs. castPCR

50.048.228.5G

ATC

50.048.228.5G

ATC

37.328.127.2G

ATC

37.328.127.2G

ATC

Conventional allele-specific-PCR CastPCR

Primer base at 3′ Primer base at 3′

11

28.15050T

5028.548.5A

50.048.228.5G

28.15050T

5028.548.5A

50.048.228.5G

27.330.528.1T

37.128.128.5A

37.328.127.2G

27.330.528.1T

37.128.128.5A

37.328.127.2G

gDN

A te

mpl

ate

Ave. ∆∆∆∆Cq (Cq _mm – Cq_pm) = 4.1 Ave. ∆∆∆∆Cq (Cq _mm – Cq_pm) = 21.1gD

NA

tem

plat

e

SNP: hCV11711720

Specificity of CastPCR Assays – Tumor gDNA Spiking

20

30

40

Cq

y = -3.572x + 38.9

R2 = 0.999

A1/A1

A2/A2A2/A2

12

Mutant 1 10 102 103 104 105 106 copies

Conclusion: KRAS castPCR assays can detect up to 1 copy of mutant DNA in the background of 2.5 µg wild-type genomic DNA

Wild-type 2.5 2.5 2.5 2.5 2.5 2.5 2.5 µg

20

A1/A1

Detection of Mutations in Heterogeneous Tumor Samples

• Study 1: KRAS Mutation in Colorectal FFPE Samples ( n=21)

G12A G12C G12D G12R G12S G12V G13D WT

castPCR™ 2 1 4 0 0 4 8 2

Sanger Sequencing

2 1 4 0 0 4 8 2

13

• Study 2: BRAF, KRAS and EGFR Mutation in Cell Line, FFPE and Fresh Frozen tumor samples (n=33)

BRAFEGFR T790M

EGFR L858R

EGFR G719D

EGFR Del E746-A750

(6223)

EGFR Del E746-A749

(6225)

7 KRAS mutations

WT

castPCR™ 1 1 3 1 2 3 18 6

Allele-specificPCR

1 1 2 1 2 3 17 7

Sanger sequencing

1 1 2 1 2 3 17 7

Detection of BRAF 1799T>A Mutation in Plasma of Recurrent Melanoma Cancer Patients

14

Note: 20 ng plasma DNA per PCR reaction

TaqMan® Mutation Detection Assays Product Overview

• 70 castPCR™ assays for:– EGFR (30 mutation + 25 wild type assay)– KRAS (14 mutation + 14 wild type assay)– BRAF (1 mutation + 1 wild type)

• Single assay may cover multiple deletions• Three gene specific reference assays• IPC reagents

Single tube assays(Full launch- July 2011)

Fixed set of TaqMan® Mutation Detection assays in 96-well FAST Plates(Early Access- April 2011)

KRAS-BRAF plate & EGFR plate

15

Data Analysis

• IPC reagents

Real-time PCR instruments

KRAS-BRAF plate & EGFR plate

The OpenArray® System for Real-Time PCR and TaqMan® Genotyping

16

OpenArray® Plates

Hydrophilic

Hydrophobic

17

Data Points per/day/FTE

Genotyping

Gene Expression

�70,000

�32,000

OpenArray Plate Layout

Subarrays 48 (12 x 4)

Through-holes 64 per subarray (8 x 8)

Reaction Volumes

Total 33nL

What is the OpenArray® System?

• The OpenArray Real Time PCR System now delivers the proven, market leading chemistry of TaqMan® products in a faster, more flexible format.

• TaqMan OpenArray Real-Time PCR products enabling dense gene coverage and sample throughput

− 32,000 data points in just one work day

• SYBR® OpenArray Real-Time PCR products offers pathway panels

18

• SYBR® OpenArray Real-Time PCR products offers pathway panels and custom plating enabling unprecedented gene coverage and sample throughput

− 24,000 data points in just one work day

• TaqMan OpenArray Genotyping products utilizes TaqMan chemistry for allelic discrimination

− > 70,000 data points in just one work day

Single-Plex PCR

PCR tests are isolated and independent

19

‘Swappable’ independent tests facilitates rapid optimization and

implementation of pathogen detection panels.

No need to re-optimize test panel if changed

“Plug and play”

TaqMan® SNP Genotyping Assays

- 160,000 validated assays with 20 million associated genotypes (Mapping SNPs)

- 3.5 million HapMap SNPs providing 70% HapMap coverage

4.5 million pre-designed assays for high-density, genome wide coverage pre-mixed at a 40x and 80x concentration

20

- 70,000 coding SNP assays for the detection of SNPs within coding regions, including many putative functional SNPs

- >2,600 Drug Metabolism assays for 220 drug metabolism and transporter genes

SNP Panels

SNP Panel Name Suggested Format SNP Panel Details

Sample Tracking Panel 32-64 Markers identified at U Miami, represented on Illumina and AffySNP panels

Ancestry Informative Marker 128 Markers identified by the Seldin

21

Ancestry Informative MarkerSNP Panel

128 Markers identified by the Seldin Lab at UC Davis

Universal Individual IdentificationSNP Panel

128 Markers identified by the Kidd Lab at Yale

Cytochrome P450SNP Panel

128 Pharmacologically relevant polymorphisms in the cytochrome p450 family of genes

TaqMan® OpenArray® Genotyping Formats

22

Genotyping WorkflowAt Life Technologies

23

At the Researcher’s Lab

Results!

TaqMan® Genotyper Software

24

Red Blood Cell Antigen Genotyping• Serological methods are labor intensive and expensive

• About 5% of transfusion patients lack a common blood group antigen and risk developing an antibody to the missing blood antigen.

− Hemolysis of subsequently transfused blood

• Is there a high throughput method to screen blood donors for antigen genotypes?

25

genotypes?− Molecular genotyping has a better analytic specificity and sensitivity then

serological methods.− High throughput SNP genotyping methods can first screen a donor

population to identify donors of specific blood types and then followed up by a confirmatory serological test.

Hopp K, Weber K, Bellissimo D, Johnson ST, Pietz B. (2010). High-throughput red blood cell antigen genotyping using a nanofluidic real-time polymerase chain reaction platform. Transfusion. 50:40-46.

Study Design• Antigens

− Four antigens chosen for genotype/phenotype concordance study for providing blood to Sickle Cell patients

> Rh E/e; Fya /Fyb; Jka /Jkb ; K/k

− Three antigens and one SNP responsible for silencing expression of the Fy antigen were chosen to detect rare antigen negative phenotypes

> Jsa /Jsb; Joa; Hy; Fy (GATA)

26

− Two antigens selected due to the scarcity of reliable antisera (antiserum)

> Lua /Lub; Doa /Dob

• Patients− 427 African American donors

Results− >99% Reproducibility− 99.7% concordance with predicted phenotype generated by

interpretation of assay genotype results

> 4 discordant resolved by repeat serology

> 1 discordant resolved after repeat genotyping

27

Typical Allelic Discrimination plot generated by the TaqMan® OpenArray® Genotyping Analysis Software

OpenArray Genotyping System • More samples for more genetic power

− Process 24 arrays per day with minimal hands-on time

• Simple workflow − Preloaded with assays of your choice—just add sample, cycle, and

image

• Best experimental flexibility− Choose any combination of TaqMan SNP Genotyping or DME Assays

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− Choose any combination of TaqMan SNP Genotyping or DME Assays

• Reliable− Uses TaqMan Genotyping Assay chemistry

• Easy-to-understand pricing− No oligo setup fee or hidden costs

• Easy ordering− Online Custom Assay Design Tool and configurator tool

• Powerful Data Analysis− TaqMan® Genotyper Software

TaqMan® Gene Expression AssaysInventoried Assays

Species Assay CountHuman 29,222

Mouse 19,100

29

Mouse 19,100

Rat 10,862

Other (15 species) 2,118

Total 61,302

Minimum 10-pack order for all formats with 10-pack increments

Look forward to a TaqMan® OpenArray Hs Housekeeping Panel

TaqMan OpenArray Pathways Panels• Validated Content

• Gene Selection from literature

• Off the Shelf Product

30 | Life Technologies Proprietary & Confidential | 5/12/2011

OpenArray® Real-Time Formats

OpenArray® Real-Time qPCR Plate, 18



A1 1 2 3 4 5 6 7 8

a 1 2 3 4 5 6 7 8

b 9 10 11 12 13 14 15 16

c 17 18 19 20 21 22 23 24

d 25 26 27 28 29 30 31 32

e 33 34 35 36 39 39 39 40

f 41 42 43 44 45 46 47 48

g 49 50 51 52 53 54 55 56

h 57 58 59 60 61 62 63 64

Subarray:

31




Gene Expression WorkflowAt Life Technologies

32

At the Researcher’s Lab

Results!

Confidential4.0

6.0

8.0

Open

Arr

ay

OpenArray vs ABI7900(Colon Tumor vs Normal Colon)

Ope

nArr

ay (∆∆

Ct)

R2=0.99

Ct (OpenArray)

Ct(7900)

20

25

30

35

15 20 25 30

OpenArray (Ct)

AB

7900

(C

t)

Correlation=0.996

Platform Comparisons

33nL Reactions vs. 20 uL Reactions

33 33

-6.0

-4.0

-2.0

0.0

2.0

-6.0 -4.0 -2.0 0.0 2.0 4.0 6.0 8.0

∆∆

CtO

pen

Arr

ay

∆∆Ct 7900

Correlation =0.98

ABI7900 (∆∆Ct)

Ope

nArr

ay (

Correlation=0.974R

otor

-Gen

e (C

t)

OpenArray (Ct)

Gene Expression: Methods• 44 patients enrolled at 2 medical facilities

− Undergoing mastectomy: cancer, ipsi- and contra lateral normal tissues were collected (histologically examined to ensure correct histology)

• 20 patients enrolled at private practices− Undergoing breast reduction (tissues histologically examined, less than

60% fat content)

• Samples were frozen within 1 hr post surgery

34

• Samples were frozen within 1 hr post surgery− RNA isolated using standard homogenization and column methods− Analyzed to confirm

> 28S/18S ratio = 1 ±0.2 (using Agilent Bioanalyzer)

> A260/A280 ratio > 1.6

Comparison of Breast Cancer to Healthy Control Tissue Discovers Novel Markers

withPotential for Prognosis and Early

Detection

Samples: 9 normal controls and 13 invasive tumorsNormalization: 3 Housekeeping genesConclusions:

- High correlation with previous qPCR data

35

- High correlation with previous qPCR data- Identified 101 new genes normal/breast cancer- Better patient stratification

“Applying the same criterion as for the

original set … both our original analysis and

BioTrove analysis of the reduced set would

have selected the same differentially

expressed genes.”

Schummer M, et al. (2010) PLoS ONE 5(2): e9122. doi:10.1371/journal.pone.0009122

Comparison of Breast Cancer to Healthy Control Tissue Discovers Novel Markers withPotential for Prognosis and Early Detection

“

“


“

Schummer M, et al. (2010) PLoS ONE 5(2): e9122. doi:10.1371/journal.pone.0009122

Next-Gen miRNA Profiling PlatformTaqMan® OpenArray® miRNA Panels

• Broad coverage− Run three samples per OpenArray® plate− 818 assays per sample (758 unique assays)

> 3 positive and 1 negative/process control per subarray

• High-throughput − 9 samples per ~2.5 hr instrument run Sample

Sample 3

37

− 9 samples per ~2.5 hr instrument run− 36 samples per 8hr working day

• Cost effective− Rapid sample screening with minimal hands-on time

• Minimal sample requirement− 100ng of total RNA recommended for a full profile

Sample 1

Sample 2

OpenArray ® miRNA Profiling Workflow

Sample 1

Sample 2

Megaplex™ RT Primer Pool A

Megaplex™ Preamp Primer

Pool A

OpenArray® MicroRNA Panel

Reverse Transcription•TaqMan® MicroRNA RT Kit

Preamplification•TaqMan® PreAmp Master Mix

qPCR•GeneAmp Fast MM

38

Sample 3

3 Samples≈ 750 Targets

Megaplex™ RT Primer Pool B

Megaplex™ Preamp Primer

Pool B

Sample 1 Total RNA Sample

OpenArray® 384-well Sample Plate

150 min. 90 min.

150 min.*(*up to 3 arrays/9 samples

run simultaneously)

Total time = < 8 hours

30 min.

Concordance With Individual AssaysOpenArray ddCq(Brain-Lung) vs. Single Assay ddCq(Br ain-Lung)

y = 0.9198x - 0.1703

R2 = 0.9476

0

2

4

6

Sin

gle

Ass

ay d

dCq

(Bra

in-L

ung)

39

-8

-6

-4

-2

0

-8 -6 -4 -2 0 2 4 6 8

OpenArray ddCq (Brain-Lung)

Sin

gle

Ass

ay d

dCq

(Bra

in-L

ung)

Human Plasma: Full Workflow Reproducibility

y = 0.9448x + 1.3014

R² = 0.9508

0

5

10

15

20

25

30

0 10 20 30

S1 vs S2

y = 0.9507x + 1.1552

R² = 0.9341

0

5

10

15

20

25

30

0 10 20 30

S1 vs S3cy = 0.9994x - 0.0128

R² = 0.973

0

5

10

15

20

25

30

0 10 20 30

S2 vs S3c

R2 S1 S2 S3C S4 S5 S8

S1 1.0000

• 6x 200ul aliquots from the same human plasma sample• RNA isolated using mirVana™ PARIS™ Kit• Ran through protocol independently• Single replicates of each sample run on OpenArray• R2 plotted for Ct <25


y = 0.9295x + 1.7689

R² = 0.9335

0

5

10

15

20

25

30

0 10 20 30

S1 vs S4

y = 0.9189x + 2.0114

R² = 0.9302

0

5

10

15

20

25

30

0 10 20 30

S1 vs S5

y = 0.9332x + 1.6659

R² = 0.9139

0

5

10

15

20

25

30

0 10 20 30

S1 vs S8

y = 0.9785x + 0.6125

R² = 0.9683

0

5

10

15

20

25

30

0 10 20 30

S2 vs S4

y = 0.9602x + 0.9855

R² = 0.9662

0

5

10

15

20

25

30

0 10 20 30

S2 vs S5

y = 0.9682x + 0.8481

R² = 0.9604

0

5

10

15

20

25

30

0 10 20 30

S3c vs S4

y = 0.9945x - 0.1071

R² = 0.9612

0

5

10

15

20

25

30

0 10 20 30

S3c vs S5

y = 0.9724x + 0.4203

R² = 0.9496

0

5

10

15

20

25

30

0 10 20 30

S3c vs S8

y = 0.9828x + 0.3159

R² = 0.9692

0

5

10

15

20

25

30

0 10 20 30

S4 vs S5

y = 0.9691x + 0.6817

R² = 0.966

0

5

10

15

20

25

30

0 10 20 30

S4 vs S5y = 0.9639x + 0.8227

R² = 0.9598

0

5

10

15

20

25

30

0 10 20 30

S5 vs S8y = 0.9826x + 0.4829

R² = 0.9566

0

5

10

15

20

25

30

0 10 20 30

S2 vs S8

S1 1.0000

S2 0.9508 1.0000

S3 0.9341 0.9730 1.0000

S4 0.9335 0.9683 0.9604 1.0000

S5 0.9302 0.9662 0.9679 0.9692 1.0000

S8 0.9139 0.9566 0.9660 0.9660 0.9777 1.0000

• All R2 above 0.9• Average 0.966

miRNA Detection By qRT-PCR In Single mESCs


Violin plot showing the distribution of miRNA expression levels. miRNAshave been sorted according to expression frequency and all zero valueshave been removed showing a correlation between expression level andexpression frequency.

Hierarchical clustering analysis of theexpression of 61 miRNAs in 24 single cellsreveals a variety of expression patterns.

Courtesy of Fred Kolling IV et al, Dpt Molecular and Cell Biology, University of Connecticut

Cell Heterogeneity: Materials and Methods

• Samples: FACS-sorted CellSensor MA-180 cells(single cells, n=420; 10 cells, n=20, 100 cells, n=20, no cells, n=20)

Treatment: ±EGF, activation of ras and MAPK pathwaysOA: 56 Taqman assays

42

Sample protocol: optimized Cells-to-Ct and gene-specific pre-amplificationqPCR platform: AB7900 and OpenArrayTest sites: Austin and WoburnTotal # of samples tested: 480/treatment

Single Cell-to-C T kit is compatible with OpenArray Platform

Product Name Part Number

Package

Single Cell Lysis Kit 4458235 100 rxn

Single Cell-to-CT™ Kit 4458236 400 rxn

Single Cell-to-CT™ Kit 4458237 50 rxn

Additions to a single tube

43 | Life Technologies Proprietary & Confidential |

Single Cell

Reverse Transcription

***Taqman Multiplex

PreAmplification

Taqman OpenArray Real-Time

qPCR

Additions to a single tube

Single Cell-to-CT®

* **Pre-Amp primers provided through Speedboat Group

Single Cell Gene Expression Profiles (OA)

44

The analysis of single cells clearly show cellular heterogeneity for these genes


Digital PCR™ at Life Technologies

• Dilute-Distribute-Amplifiy-Count

Digital PCR™ OpenArray ®

• 3072 thru-holes• Runs TaqMan ®Chemistry

• MxN Flexibility

•Acquired Cytonix Corp. •Acquired BioTrove for the

46

•Acquired Cytonix Corp. for IP related to digital polymerase chain reaction technology

•Acquired BioTrove for the OpenArray technology

Digital OpenArray

• Flexible Digital solutions using TaqMan

Analog PCR (qPCR) and Digital PCR: Absolute Quantitation

Analog Digital

47 8/16/10© 2010 Life Technologies. All Rights Reserved. | Proprietary and Confidential.

47

... relative quantitation

# positive, negative wells

# copies present in sample

Cycle

Rn

Ct value

Standards, endogenous

controls

Results from other samples

∆Ct, ∆∆Ct, …

... absolute quantitation

OpenArray Digital PCR Product Concept

Digital PCR answer:# copies / uL

Customer sample

TaqMan® OpenArray® Digital PCR

Master Mix (2X)OpenArray® Digital PCR

Software

New Digital PCR Products

48 8/16/10© 2010 Life Technologies. All Rights Reserved. | Proprietary and Confidential.

48

OA Real-Time PCR System

C t b y P o s i t i o n

1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

5

1 0

1 5

2 0

2 5

3 0

AccuFill(or AutoLoader I)

Primary analysis

TaqMan assay

say

TaqMan OpenArray ®

Digital PCR Master MixOpenArray ® Digital

PCR Plates

Set-up file

Software

Problem: WNV• Blood/tissue samples in early stages of infection present at low levels

− Sometimes, availability of sample volume is low further confounding the issue

• 50ul Standard qPCR yields consistent results with Ct greater than 36.

• Is the detection real and how many copies of WNV are in the actual

49

• Is the detection real and how many copies of WNV are in the actual sample?

| Life Technologies Proprietary & Confidential | 5/12/2011

dPCR of West Nile Virus (Standard curve) and HIV Samples

A

B

C

D

EF

Z21

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

HIV+ MM+ABI03

HIV+ MM+HIV4Rev

HIV+ MM+ABI12

WNV dil 1-5 +WNV 1-28098 WNV dil 1-5 +

WNV 2-28099

50

DNTC

dPCR AnalysisGroup

Average Copies per Reaction

Lower Confidence

Interval

Upper Confidence

Interval

Total Replicates

Total Negatives

Confidence Interval Range

WNV1_WNV1-28098 1.5562 1.2547 1.9301 128 27 1.2547 - 1.9301WNV2_WNV1-28098 0.9808 0.7808 1.232 128 48 0.7808 - 1.2320WNV3_WNV1-28098 0.5894 0.4529 0.7669 128 71 0.4529 - 0.7669WNV4_WNV1-28098 0.3087 0.2203 0.4327 128 94 0.2203 - 0.4327WNV5_WNV1-28098 0.1515 0.0954 0.2406 128 110 0.0954 - 0.2406NTC_WNV1-28098 NaN NaN NaN 128 128 NaN - NaN

51

28098 NaN NaN NaN 128 128 NaN - NaNWNV1_WNV2-28099 NaN NaN NaN 128 0 NaN - NaNWNV2_WNV2-28099 2.213 1.7699 2.7669 128 14 1.7699 - 2.7669WNV3_WNV2-28099 1.418 1.1425 1.7601 128 31 1.1425 - 1.7601WNV4_WNV2-28099 0.8267 0.6518 1.0484 128 56 0.6518 - 1.0484WNV5_WNV2-28099 0.3634 0.265 0.4982 128 89 0.2650 - 0.4982NTC_WNV2-28099 NaN NaN NaN 128 128 NaN - NaNHIVABI03 NaN NaN NaN 512 512 NaN - NaNHIVABI12 NaN NaN NaN 512 512 NaN - NaNHIVHIV4Rev NaN NaN NaN 512 512 NaN - NaN

Summary

OpenArray Systems accelerates molecular analysis

52

� Scalable molecular analysis platform

� Utilizes familiar PCR chemistries

� Eliminates need for complex multiplex PCR assay development

� Enabled reduced time, labor and reagent consumption

Legal StatementNOTICE TO PURCHASER: Limited Use Label License

The products shown in this presentation may be cove red by one or more Limited Use Label License(s). Please refer to the r espective product documentation or the Applied Biosystems website und er www.appliedbiosystems.com for the comprehensive license information.

By use of these products, the purchaser accepts the terms and conditions of all applicable Limited Use Label Licenses. These products are sold for research use only, and are not intended for human o r animal diagnostic or

53

research use only, and are not intended for human o r animal diagnostic or therapeutic uses unless otherwise specifically indi cated in the applicable product documentation or the respective Limited Use Label License(s).

© 2010 Life Technologies Corporation. All rights re served.

The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. TaqMan is a registered trademark of Roche Molecular Systems, Inc.

Fore research use only. Not intended for human or an imal therapeutic or diagnostic use

Documents

Next generation PCR, leading by digital PCR, Cast PCR and ...cgs.hku.hk/portal/files/GRC/Events/Seminars/2011/20110516/digital … · A pair of assays: mutation assay and a gene reference