Www.scienceboard.net qPCR SNAPSHOTS LIVE MAY 13, 2003

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qPCR SNAPSHOTS LIVE

MAY 13, 2003


Agenda

• Introduction

• Objectives & Methodologies

• Detection Chemistries

• Instrumentation

• Conclusion


Real-Time PCR

• Ability to detect PCR product as it is synthesized

• Requires fluorescence-based detection chemistries and specialized detection instrumentation

•Advantages:

Increased analytical sensitivity

Faster results

Broad applicability

Introduction


Study Demographics

Introduction

<1%

<1%

2%

2%

5%

8%

18%

21%

44%

0% 5% 10% 15% 20% 25% 30% 35% 40% 45%

Academic

Pharmaceutical/Biotechnology

Hospital or UniversityMedical Center

Government

Private Research

Contract Research

Commercial Testing Lab

Medical Device/Diagnostics

Healthcare Network/Facility


Quantitative vs. Qualitative Applications

Quantitative only31%

Both (qualitative and quantitative)

58%

Qualitative only11%

Objectives & Methodologies


Template Use

DNA, cDNA and RNA as templates

34%

Only RNA or cDNA as a template

49%Only DNA as a template

17%



Research Objectives

4%

11%

13%

10%

14%

10%

13%

14%

5%

5%

9%

11%

10%

13%

17%

24%

0% 5% 10% 15% 20% 25% 30%

SNP genotyping

Other

Allelic discrimination

Gene duplication or other DNA quantification

Bacterial detection/identification

Viral load detection

Gene expression-confirmation of microarray data

Gene expression-primary validation

DNA, cDNA and RNA as a template

Only DNA as a template



Kits vs. Components: Preferences by Template


Individualcomponents

34%

A commercially available qRT-PCR kit

53%

Both13%

Both22%

A commercially available qPCR kit (or qPCR master mix)

57%

Individual components

21%

DNA

RNA


Kits vs. Components: Reasons for Selection


• Convenience

• Guaranteed optimized system

• Cost effective

• Greater flexibility

• Less expensive

• Have own system

• Too little reagent volume in kits

Kits Components


Detection Chemistry Preferences

Detection Chemistries

Both SYBR Green and fluorescent probes or

primers33%

Fluorescent probes or primers only

36%SYBR Green only31%


Commonly Used Fluorescent Probes

Detection Chemistries

2%

3%

9%

9%

15%

19%

78%

0% 10% 20% 30% 40% 50% 60% 70% 80%

TaqMan probes

Molecular Beacons

FRET probes

LUX fluorogenic primers

MGB Eclipse probes

Other

Scorpion probes


• Sensitivity

• Linear dynamic range

• Time-to-results

• Throughput

• Software

• Block/sample capacity

• Size

• User interface

Factors Influencing Instrumentation Purchase

Performance Parameters Product Specifications


Commonly Used Real-Time PCR Instrumentation

Instrumentation

<1%

1%

2%

2%

2%

3%

7%

9%

12%

16%

18%

26%

0% 5% 10% 15% 20% 25% 30%

ABI PRISM 7700 Sequence Detection System

ABI PRISM 7000 Sequence Detection System

LightCycler System

iCycler iQ Real Time PCR Detection System

ABI PRISM 7900HT Sequence Detection System

GeneAmp 5700 Sequence Detection System

Smart Cycler System

DNA Engine Opticon (or Opticon 2) System

Rotor-Gene

Mx4000 Multiplex Quantitative PCR System

Other

R.A.P.I.D. System


Conclusions

• Real-time PCR is dominated by gene expression studies and quantitative applications.

• The majority of researchers prefer using kits rather than individual components for real-time PCR amplification.

• Fluorescent probes/primers and SYBR Green dye are nearly equal in popularity as detection chemistries.

• TaqMan probes are the most popular choice for users of fluorescent probes/primers.

• Software, sensitivity and user interface are the most important features of real-time PCR instrumentation.

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Www.scienceboard.net qPCR SNAPSHOTS LIVE MAY 13, 2003