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Fundamentals of Fundamentals of Real-Time RT-PCR

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Fundamentals of Real-Time RT-PCR David Chappell, PhD Field Applications Scientist

Fundamentals of

Real-Time RT-PCR

David Chappell, PhD

Field Applications Scientist

Real-time PCR

Workflow

Sample

prep

Real-time

PCR

Analysis

4 1 2

Assay

3

design

PCR

PCR – Geometric amplification of target

95 C

15 s

58 C

30 s

72 C

60 s

Real-time PCR

World’s First Real-Time PCR Instrument

t

Introduced both

- mechanical innovation

and

- conceptual innovation

1995: ABI PRISM ® 7700

Sequence Detection System

World’s First Real-Time PCR Instrument

t

Introduced both

- mechanical innovation

and

- conceptual innovation

1995: ABI PRISM ® 7700

Sequence Detection System

Detection fluorescence in real-time

Optics

Detection

Device

excitation

emission

96 well plate

Detection of

fluorescence

Optics to excite and

Quantitation

through caps read the plate of fluorescence

PCR doubles template each cycle

Fluorescence

(Copy #)

Cycle #

Finite reagents cause deviation from doubling curve

Fluorescence

(Copy #)

Cycle #

Finite reagents cause deviation from doubling curve

Exponential phase

(Good)

Non-Exponential phase

(Bad)

Fluorescence

(Copy #)

Cycle #

Exponential phase easier to see in log view

Copy #

Log

Copy #

Cycle #

Lower limit of detection also easier to see in log phase

Copy #

Bad

Good

Bad

Log

Copy #

Cycle #

World’s First Real-Time PCR Instrument

t

Introduced both

- mechanical innovation

and

- conceptual innovation

1995: ABI PRISM ® 7700

Sequence Detection System

Concept of Ct

Instead of measuring change in fluorescence

at a set cycle number,

escence

og Fluore Lo

0 5 10 15 18 20 22 25 30 35 40

Cycle #

Concept of Ct

Instead of measuring change in fluorescence

at a set cycle number,

escence

og Fluore Lo

0 5 10 15 18 20 22 25 30 35 40

Cycle #

Concept of Ct

We measure the number of cycles

it takes to reach a set fluorescence threshold (Ct)

escence

og Fluore Lo

0 5 10 15 18 20 22 25 30 35 40

Cycle #

Thus, real-time PCR is superior to regular PCR because:

1) The threshold is always set in the exponential phase,

Giving greater accuracy

Thus, real-time PCR is superior to regular PCR because:

2)

We are measuring cycle number,

therefore very large dynamic range

Results are usually given in table form

indicating Ct value

the higher the Ct, the lower the target copy number

Raw Ct indicates APPROXIMATE copy number

10 12

10 11

10 10

10 9

10 8

10 7

10 6

10 5

10,000

1000

100

10

1

0 10 20 30 40

Because the exact Ct values may change between

users, reagents and labs and therefore only

indicates an approximate copy number,

Ct values are not normally published

However, if we compare Cts from the SAME RUN or

EXPERIMENT, then we can be extremely accurate.

Quantitative real-time PCR analysis measures the

DIFFERENCE in the Cts

Either the difference between Sample Cts and Std Cve Cts (Absolute)

Or, the difference between sample Cts directly (Relative)

Log Fluorescence

0 5 10 15 18 20 22 25 30 35 40

Cycle #

SYBR ® Green

Visualization – Fluorescent dyes

Intercalating

agents

Minor

Groove

Binder

Ethidium

Bromide

SYBR

® Green

(10-20 x

more signal)

Problem with DNA-binding Dyes

Bind non-specifically to any double-stranded DNA

Therefore specificity of the amplifications must be checked

Can detect non-specific amplification i by

performing dissociation curves

scence

Fluores

Cycle number Increasing temperature

PCR reactions with non-specific peaks

should not be used for quantification

Specific amplification Non-specific amplification

(1 peak) (>1 peak)

Exact position of peak affected by:

size of fragment

nucleotide composition

ionic environment (ie: [Mg 2+ ])

Non-specific amplification promoted by high primer

concentration

Optimal primer concentration

Primer concentration too high

Primer-dimer formation reduced d by minimizing

i i i

primer concentration

Forward Primer

Primer Matrix 25nM 50nM 100nM

rse Prim mer

Reve

25nM

50nM

100nM

25/25 25/50

25/100

50/25

50/50

50/100

100/25 100/50 100/100

TaqMan ®

TaqMan ® assays were developed

to address the problem of specificity

These assays are also fluorescence-based

Fluorescence molecules absorb light and emit

light at a longer wavelength (ie lower energy)

Excitation

Emission

FAM

TM

TET TM

JOE TM

VIC ®

HEX TM

CY3 TM

NED TM

TAMRA TM

ROX TM

TEX.R ®

CY5 TM

Note: these are approximations only

Behavior of 1 fluorescent molecule

Molecule 1

1

Molecule 1 absorbs blue and emits green

Behavior of 2 fluorescent molecules

Molecule 1 Molecule 2

1

2

Molecule 1 absorbs blue and emits green

Molecule 2 absorbs yellow and emits red

Behavior of 2 fluorescent molecules l may produce:

FRET (Fluorescent Resonant Energy Transfer)

Molecule 1 Molecule 2

1

2

Quenching

IF:

1) Em of molecule 1 = Abs of molecule 2

2) The molecules are adjacent

FRET effect is determined by proximity of molecules

1 2

Quenching

TaqMan® Assay Uses FRET by attaching fluorescent

molecules to a probe

Reporter dye

Quencher dye

5’ -Nuclease Activity Digests Probe

Reporter

Fluoresces

Fluorescent signal is directly proportional to

template amplification

Advantages of TaqMan ® Assays:

1) Fluorescence is specific to target gene

Advantages of TaqMan® Assays:

2) Allows use of more than one probe in the same tube

(multiplex)

Why Multiplex

Increase throughput

Reduce reagent and sample usage

Endo

Endo/Target

Target

Why Multiplex

Increase throughput

Reduce reagent and sample usage

Endo

Endo/Target

Target

These advantages decrease with increasing number of genes in the experiment.

Why Multiplex

Increase throughput

Reduce reagent and sample usage

Endo

Target

Increase in number

of samples = 100%

Endo/Target

These advantages decrease with increasing number of genes in the experiment.

Endo

Endo/

/T Target 1

Target 1

Endo/ Target 2

Target 2

Increase in number

Endo/ Target 3

Target 3

Endo/ Target 4

Target 4

of samples = 12.5%

Endo/ Target 5

Target 5

Endo/ Target 6

Target 6 Endo/ Target 7

Target 7

Endo/ Target 8

Why Multiplex

Increase throughput

Reduce reagent and sample usage

Endo

Target

Increase in number

of samples = 100%

Endo/Target

Increased precision

Multiplexing with the normalizer should

eliminate sample pipet error in theory.

Problems with multiplex:

Multiplexing is usually limited by the chemistry

not by the instrument

The extent of useful multiplexing is limited by:

• Primer/primer or primer/probe interaction

• Competition for reagents

• Reduced dynamic Range

FAM TM

Common AB TaqMan ® dyes

Emission wavelength

approximations

only

VIC ®

TAMRA TM

NFQ

Non Fluorescent Quencher

ROX TM 1 2 3 4

Filter

MGB

Probing for specific sequence in a pool of

completely random sequences

Probing for specific sequence in a pool of

completely random sequences

Longer probes increase specificity

it

Probing for specific sequence in a pool of

similar sequence families

Probing for specific sequence in a pool of

similar sequence families

Shorter probes increase selectivity

“State of the Art” MGB Probe

FAM

TM

NFQ

Minor

Groove

Binder

Increases binding affinity for target

Allows for a shorter probe

Primers ~ 20-30bp

regular probes ~30-40bp

MGB probes ~13-22bp

Short MGB probes allow

robust single nucleotide specificity

ie: SNP assays

A

T

ROX TM Dye

Resolution of the assay is determined by precision

– how reproducible is the data

This precision is obviously better….

Resolution of the assay is determined by precision

– how reproducible is the data

….than this precision

Common sources of variation of light signal

excitation

optics

condensation

cover

bubble

volume

Variation negated by normalizing to a

Passive Reference dye

ROX TM is a Passive Reference dye

Greatly improves precision i of replicates.

Rn = Normalization = Reporter / Reference

Targ get

X TM

ROX

Targ get

X TM

ROX

Well 1 Well 2

Rn

R n

Well 1 Well 2

ROXTM

TM Passive Reference increases precision

36 Replicates analyzed without ROXTM

dye

36 Replicates analyzed with ROXTM

dye

Multicomponenting

Filters are assigned to particular dyes

1 Filter

2 Filter

Dye

VIC

Dye

FAM

3

ROX

TM

Filter

Dye

However, if more than one dye is present, there

is spectral overlap

1 2 3

Filter

Dye

FAM VIC ROX TM Total FL

If not addressed, this would introduce large

inaccuracies

Quantitative multicomponenting

During installation, a plate of pure dyes is read and the spectra recorded

1) During the run, the fluorescence is read through ALL filters

This produces a data file with ALL spectral data

2) When the data file is analyzed, the appropriate dyes are chosen

Multicomponenting algorithm determines

individual dye fluorescence

Multicomponent View

This is not the fluorescence through each filter, but the

calculated specific fluorescence of each dye

Advantages of Multicomponenting

1) Accurate determination of individual dye fluorescence

2) If an error is made in dye designation, the data is still

stored and error may be corrected

3) Not reliant on dye-specific filters sets - If using a new

dye, measure pure dye spectrum

Applied Biosystems

Real-time PCR

Instruments

Applied Biosystems Real-Time PCR Instruments

Filters

Step

HP

Em 2 100 !l tubes

10-30!l

One TM LED 1 3

48 fast 10-30!l

Hal. Filters

200 !l tubes

Lamp Ex 3

7300 1 2Em

4

1

96 well

25-100!l

Step

One

Filters 100 !l tubes

10-30!l

3

HP

2Em

4

Plus TM LED 1 96 fast 10-30!l

Hal.

Lamp

2 3 4

Filters

2 3 4

7500 1 Ex Em

5 1 5

200 !l tubes

96 well

25-100!l

96 fast 10-30!l

Argon laser

96 well 25-100!l

7900 96 fast

Scan Head

10-30!l

384 well 5-20!l

Interchangeable TaqMan® TM Low

blocks

Density Arrays

Applied Biosystems

Trademarks

For Research Use Only. Not for use in diagnostic procedures.

The patented t 5’ Nuclease Process and the dsDNA Binding-dye d Process are

covered by patents owned by or licensed to Applera Corporation. For further

information, contact the Director of Licensing, Applied Biosystems, 850 Lincoln

Centre Drive, Foster City, California 94404, USA.

Applera,Applied Biosystems, AB (Design), ABI PRISM, GeneAmp, and VIC are

registered trademarks and FAM, HEX, JOE, NED, ROX, StepOne, TAMRA, TET,

VeriFlex, and Veriti are trademarks of Applera Corporation or its subsidiaries in the

US and/or certain other countries.

TaqMan® is a registered trademark of Roche Molecular Systems, Inc.

SYBR® (R) and Texas Red are registered trademarks of Molecular Probes, Inc.

CY is a trademark of GE Healthcare

Ct – measured where curve crosses threshold

Rn

Noise

Baseline

Ct

Threshold

Rn

Threshold

Noise

Baseline

Ct

Applied Biosystems StepOne TM Optics

HP LED

mirror

2

excitation

lens

emission

1

3

Emission

filters

CCD

48 well plate

Applied Biosystems StepOnePlus TM Optics

HP LED

mirror

2 3

excitation

lens

emission

1

4

Emission

filters

CCD

96 well plate

Applied Biosystems 7300 Optics

tungsten lamp

Excitation

filter

mirror

excitation

lens

emission

2 3

1

4

Emission

filter wheel

CCD

96 well plate

Applied Biosystems 7500 Optics

tungsten lamp

2 3 4

1 5

Excitation

filter wheel

mirror

2 3 4

lens 1 5

excitation

emission

Emission

filter wheel

CCD

96 well plate

excitation

emission

Applied Biosystems 7900 Optics

Argon

laser

Scan

Head

Spectro

graph

CCD

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