Real-Time PCR

AMOUNT OF DNA
1
2
4
8
16
32
64
128
256
512
1,024
2,048
4,096
8,192
16,384
32,768
65,536
131,072
262,144
524,288
1,048,576
2,097,152
4,194,304
8,388,608
16,777,216
33,554,432
67,108,864
134,217,728
268,435,456
536,870,912
1,073,741,824
1,400,000,000
1,500,000,000
1,550,000,000
1,580,000,000

1600000000
1400000000
AMOUNT OF DNA

CYCLE NUMBER
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34

1200000000
1000000000
800000000
600000000
400000000
200000000
0
0

10

15

20

25

PCR CYCLE NUMBER

30

35

Whats Wrong With

Agarose Gels?
*
*
*
*
*

Low sensitivity
Low resolution
Non-automated
Size-based discrimination only
Results are not expressed as numbers
based on personal evaluation
Ethidium bromide staining is not very
quantitative
End point analysis

Real-time Principles
based on the detection and quantitation of
a fluorescent reporter
In stead of measuring the endpoint we focus on the first
significant increase in the amount of PCR product.
The time of the increase correlates inversely to the initial
amount of DNA template

500nm

Wavelength (nm)

660nm

SYBRGreenIChemistry
Polymerization
5'

Forward
Primer

3'

5'

5'

3'
Reverse
Primer

5'

Polymerization completed
5'
3'

5'

5'

3'
5'

3.
intensifie
r

1. halogen
tungsten
lamp

2b. emission
filters

2a.
excitation
filters

5. ccd
detect
or
350,00
0
pixels

4. sample
plate

www.biorad.com

Data analysis
Cycle Threshold
* cycle threshold or the CT value is the cycle at which
a significant increase in Rn is first detected
* it is the parameter used for quantitation
* CT value of 40 or more means no amplification and
cannot be included in the calculations

FRET (Fluorescence Resonance Energy Transfer)

TaqMan Chemistry
-Hydrolysis Probes-

Probe

Polymerization

5
3

Forward
Primer

Probe

R = Reporter
Q Q = Quencher
3

5
Reverse
Primer

3
5

Strand Displacement
R
5
3

5
3
5

Cleavage
R
5
3
5

5
3
5

Polymerization Completed
R

5
3

(www)

real-time
real-time
PCR
real-time
Summary

ADVANTAGES OF REAL-TIME PCR

Rapid cycling times (1 hour)

High sample throughput (~200 samples/day)
Low contamination risk (sealed reactions)
Very sensitive (3pg or 1 genome eq of DNA)
Broad dynamic range (10 - 1010 copies)
Reproducible (CV < 2.0 %)
Allows for quantitation of results
Software driven operation
No more expensive than in house PCR ($15/test)

real-time
real-time
PCR
real-time
Summary

DISADVANTAGES OF REAL-TIME PCR

Current technology has limited capacity for multiplexing.

Simultaneous detection of 2 targets is the limit.

Development of protocols needs high level of technical

skill and/or support. (Requires R&D capacity and capital)

High capital equipment costs ($ 50,000 -160,000).

Real-Time PCR Applications

* quantitation of gene expression
* drug therapy efficacy / drug monitoring
* viral quantitation
* pathogen detection

real-time
real-time
real-time PCR
real-time

hardware

5700
Applied Biosystems

iCycler
BioRad

7700
Applied Biosystems

LightCycler
Roche

FluorTracker
Stratagene

FluorImager
Molecular Dynamics