Real Time PCR

1. What is Real-Time PCR?
Real-time PCR is the continuous collection of fluorescent signal
from one or more polymerase chain reactions over a range of
cycles.

2. Real-Time PCR
 Real-time PCR is a variant of PCR technology that
allows the detection of PCR products as they
accumulate in "real-time" during the PCR
amplification process.
 All real-time PCR systems rely upon the detection and
quantitation of a fluorescent reporter
 The signal of which increases in direct proportion to
the amount of PCR product in a reaction

4. RT-PCR:
Reverse Transcription Polymerase Chain Reaction
- Or –
Real-Time Polymerase Chain Reaction?
RT-PCR = Reverse Transcription Polymerase Chain Reaction (may
be applied to both conventional and Real-time PCR systems).
qPCR = Real-Time PCR when used as a quantitative tool.
A commonly misused acronym …

5. An Overview of General PCR
DNA
Extraction PCR
Visualization of
PCR Products by
Electrophoresis

6. What is in the reaction ?
 Template DNA/RNA
 Reaction buffer
(Tris, ammonium ions/and or potassium ions,
magnesium ions, bovine serum albumin)
 Nucleotides (dNTPs)
 Primers
 Taq DNA polymerase

8. Limitations of General PCR
 Poor precision
 Low sensitivity
 Low resolution
 Non Automated
 Size based discrimination only
 Results are not expressed as numbers
 Post PCR processing required
 Not quantitative

9. Advantage of Real-Time PCR
 Post-PCR processing not required
 Quantitative
 Precise
 Accurate

10. Real-Time PCR Chemistries
 SYBR Green
 TaqMan
 Molecular beacon
 Scorpion primer & probe
All real-time PCR systems rely upon the detection
and quantitation of a fluorescent reporter

11. CYBR Green Chemistry
 CYBR Green is the most widely used double-strand DNA
specific dye
 It binds to the minor groove of the DNA double helix
 In solution, the unbound dye exhibits very little
fluorescence
 When CYBR Green dye binds to double stranded DNA,
the fluorescent is substantially enhanced
 As more double stranded amplicons are produced SYBR
green dye signal will increse

13. CYBR Green Chemistry
 Template DNA
 Reaction buffer
 Nucleotides (dNTPs)
 Primers (Forward & Reverse)
 Taq DNA polymerase
What’s in general PCR ? What’s new in CYBR Green
 Template DNA
 Reaction buffer
 Nucleotides (dNTPs)
 Primers (Forward & Reverse)
 Taq DNA Polymerase
 CYBR Green Dye

15. Advantage and disadvantage of CYBR Green
Method
Advantage
 Inexpensive
 No probe is required
 Simple to design
Disadvantage
 SYBR Green will bind to any double stranded DNA (e.g.
primer dimers, non-specific reaction products)
 Overestimation of target concentration
 Non-specific background in very late cycles

16. TaqMan Chemistry
TaqMan chemistry requires two more
additional components with traditional PCR
 Template DNA
 Reaction buffer
 Nucleotides (dNTPs)
 Primers (Forward & Reverse)
 Taq DNA polymerase
What’s in general PCR ? What’s new in TaqMan ?
 Template DNA
 Reaction buffer
 Nucleotides (dNTPs)
 Primers (Forward & Reverse)
 TaqMan Probe

17. TaqMan Probe
 TaqMan probe is a short DNA sequence with a high energy
dye called reporter dye at the 5´ end and a low energy dye
called quencher at the 3´ end
 When this probe is intact and excited by a light source, the
reported dye emission is suppressed by the quencher dye as
a result of close proximity of the dyes. This is known as
FRET

19. Fluorescence Resonance Energy Transfer
When a high energy dye is in close
proximity of a low energy dye, there will be
a transfer of energy from high to low

34. The increase in reporter signal is captured by the
sequence detection system and displayed by the
software
The signal of which increases in direct proportion to
the amount of PCR product in a reactionreaction

35. Molecular Beacon
Molecular beacons are short segments of single stranded
DNA that forms a hairpin in its free form
The loop portion of the molecular beacon is composed of
bases that are complimentary to one strand of the PCR
product the investigator wants to detect and quantify
Attached to opposite end of the beacon are a fluorescent
reported dye and a quencher dye

36. Molecular Beacon
When the molecular beacon is in the hairpin conformation, any
fluorescence emitted by the reporter is absorbed by the quencher
dye and no fluorescence is detected.

37. Molecular Beacon
As the PCR continues, the
newly synthesized PCR
products are denatured by
high temperatures
At the same time the
molecular beacon also is
denatured so the hairpin
structure is disrupted.
As the temperatures cool for
the next round of primer
annealing, the molecular
beacon is capable of
forming base pairs with the
appropriate strand of the
PCR product

38. Molecular Beacon
 Molecular beacons that bind to the PCR product remove the
ability for the quencher to block fluorescence from the reporter
dye
 Molecular beacons that do not bind to the PCR product
reform the hairpin structure and thus unable to fluoresce
 Therefore, as PCR product accumulates, there is a linear
increase in fluorescence.

39. Scorpion probe
 Scorpion probe is a bifunctional molecule in which a primer is
covalently linked to the probe
 That is why they are sometimes known as “Scorpion primer &
probe”
 The probe has a self complimentary stem sequence with a
fluorophore at one end and a quencher at the other end
 In the initial PCR cycle the primer hybridizes to the target and
extension occurs due to the action of polymerase
 After denaturation and cooling, the specific probe sequence is
able to bind to its complement within the extended amplicon
thus opening up the hairpin loop
 The fluorescent dye and quencher are separated, FRET does
not occur, and the fluorescent dye emits light upon irradiation

40. Scorpion primer & probe

41. Fluorescence dyes used in
Real-Time PCR
 Reporter dye
 Quencher dye
 Reference dye
The passive reference dye does not participate in the 5’
nuclease PCR, but instead provides an internal reference to
which the reporter-dye signal can be normalized during data
analysis. This is necessary to correct for fluorescent
fluctuations due to changes in concentration or volume in the
wells.

42. Fluorescence dyes used in
Real-Time PCR
Reporter dye
FAM™ (6-carbxyfluorescin)
TET™
JOE™
VIC™
Texas Red
SYBR® Green
Quencher dye
TAMRA™
Black Hole Quencher™
Reference dye
ROX™

43. Applications of Real-Time PCR
 Applications that would have been less
effective with traditional PCR
 Viral quantitation
 Quantitation of gene expression
 Drug-therapy efficacy
 DNA damage measurement
 Pathogen detection
 Genotyping

44. How Real-Time PCR Works
To understand how real-time PCR works, let’s start by examining a sample
amplification plot
-- In this plot, the PCR cycle number is shown on the x-axis,
and the fluorescence from the amplification reaction,is shown on the y-
axis.
… which is proportional to the amount of amplified product in the tube,
The amplification plot shows two phases, an exponential phase followed by
a nonexponential plateau phase.
- During the exponential phase, the amount of PCR product approximately
doubles in each cycle. As the reaction proceeds, however, reaction
components are consumed, and ultimately one or more of the components
becomes limiting. At this point, the reaction slows and enters the plateau
phase (cycles 28–40 in Figure 1.1).

45. 1. The signal measured during these
PCR cycles is used to plot the
threshold.
2. The threshold is calculated as 10
times the standard deviation of
the average signal of the baseline
fluorescent signal.
3. A fluorescent signal that is
detected above the threshold is
considered a real signal that can
be used to define the threshold
cycle (Ct) for a sample.
4. The Ct is defined as the
fractional PCR cycle number at
which the fluorescent signal is
greater than the minimal detection
level.
How the signal of Real time PCR is quantified

46.  The real-time PCR is based on the detection and
quantitation of a fluorescent reporter .
 This signal increases in direct proportion to the amount
of PCR product in a reaction.
 By recording the amount of fluorescence emission at
each cycle, it is possible to monitor the PCR reaction
during exponential phase where the first significant
increase in the amount of PCR product correlates to the
initial amount of target template.

47.  The higher the starting copy number of the
nucleic acid target, the sooner a significant
increase in fluorescence is observed.
 A significant increase in fluorescence above
the baseline value measured during the 3-15
cycles indicates the detection of accumulated
PCR product.

49. What does Ct mean?
 The Ct (cycle threshold) is defined as the
number of cycles required for the fluorescent
signal to cross the threshold (ie exceeds
background level).

50.  Cts < 29 are strong positive reactions
indicative of abundant target nucleic acid in
the sample
 Cts of 30-37 are positive reactions indicative
of moderate amounts of target nucleic acid
 Cts of 38-40 are weak reactions indicative of
minimal amounts of target nucleic acid which
could represent an infection state or
environmental contamination.

54. Bio-Rad
iCYCLER
ABI 7500 Fast Cepheid
SmartCycler 1600

55. Bio-Rad
iCYCLER iQ5
Pros:
• High throughput support (96 well format)
• Low Cost
• Software is user friendly
• 5 targets at one time
• Single Tube or Plate
Cons:
• Only 1 assay running at a time.

56. ABI 7500 Fast
Pros:
• High throughput support (96 well format)
• Feature rich software
• +/- interpretation of results
• ‘Fast’ when using certain mastermix
• Desktop or laptop PC Included
Cons:
• Plate only (no tubes)
• Only 1 assay running at a time.

57. Cepheid
SmartCycler
1600
Pros:
• Multiple assays can be performed
simultaneously
• Approved in many regulatory SOPs.
Cons:
• Low sample load support (only 16
wells per unit).
• Bench space hog when ‘daisy chained’ together
• Proprietary reaction tubes ($.50 each) & centrifuge.