Q-PCR allows for quantitative analysis of DNA amplification in real-time using fluorescence detection. It monitors accumulation of fluorescent signals during each PCR cycle, allowing quantification of starting DNA template. Common probe-based methods include TaqMan probes with a fluorophore-quencher pair and molecular beacons which become fluorescent upon target binding. SYBR Green also detects amplification nonspecifically by binding double-stranded DNA. Q-PCR provides advantages over conventional PCR such as greater precision, sensitivity, and ability to quantify initial template amounts.

1. Khuda Bakhsh
FA14-R02-002
Q-PCR

2. OUTLINE
 What is PCR and purpose of it?
 What is Q-PCR and purpose of it?
 How does Q-PCR work?
 Types of Q-PCR probes and comparison
 Advantages and Disadvantages of Q-PCR vs.
PCR
 Questions

3. What is PCR?
 Stands for Polymerase
Chain Reaction
 For the amplification of
DNA fragments

4. Purpose of PCR
 Easy to sequence more copies
 For comparing DNA fragments
 It is used to clone specific genes

5. Material:
 PCR reagents:
 A DNA template:
 Primers
 10x PCR buffer
 dNTPs
 Taq polymerase
 MgCl2

6. Steps of PCR
Denaturation:
 This step is the first regular cycling event and
consists of heating the reaction to 94–98 °C
Annealing
 The reaction temperature is lowered to4 5–60 °C for
primer attachment
Extension/elongation:
 At 72 °C for taq plymerase working
Final elongation:

7. What is Q-PCR?
Stands for Quantitative Polymerase Chain Reaction
Assay that monitors accumulation of DNA from a PCR
reaction
Important technique to quantify RNA(mRNA)
Similar to PCR except the progress is monitored by a
camera or detector

8. What Type of Instruments are
used with Real-Time PCR?
Real-time PCR instruments consist of TWO main
components:
1. Thermal Cycler (PCR machine)
2. Optical Module (to detect fluorescence in the tubes
during the run)

10. Amplification Plot

11. Types of Q-PCR
Hydrolyzation based Assays
Taqman,
Beacons,
DNA-binding agents
SYBR Green

12. SYBR green
 It is used as a dye for the
quantification of double stranded DNA
in some methods of quantitative PCR
 It is also used to visualise DNA in gel
electrophoresis

13. SYBR GREEN DYE

14.  SYBR Green
 Advantages:
 Relative low cost of primers.
 No fluorescent-labeled probes
required.
 Disadvantages:
 Less specific
 Not possible to multiplex multiple gene
targets.

15. SYBR GREEN DYE

16. TaqMan Probes
 Double- Dye Oligonucleotides or dual
labeled probes.
 Consists of a ssDNA probe that is
complemenatry to one of the amplicon
strands
 A fluorophore is attached to one end of the
probe and a quencher to the other end.

18. Uses for TaqMan Probes
 DNA Quantitation
 Mutation Detection-Probe designed to
hybridize over mutation
 Gene Expression
 Can be multiplexed
 Dark Quenchers- absorb emitted energy,

19. Molecular beacons
 Oligonucleotide hybridization probes
 Report the presence of specific
nucleic acids in homogenous
solutions.
 Molecular beacons are hairpin shaped
 whose fluorescence is restored when
they bind to a target nucleic acid
sequence

20. Molecular beacon

21.  Applications of molecular beacons
 SNP detection
 Real-time PCR quantification
 Allelic discrimination and identification
 Multiplex PCR assays

22. Taqman vs. SYBR Green I
TaqMan Probe
Advantages:
 Increased specificity
 Use when the most accurate
quantitation of PCR product
accumulation is desired.
 Option of detecting multiple
genes in the same well
(multiplexing).
Disadvantages:
 Relative high cost of labeled
probe.
 SYBR Green
 Advantages:
 Relative low cost of
primers.
 No fluorescent-labeled
probes required.
 Disadvantages:
 Less specific
 Not possible to
multiplex multiple gene
targets.

23. Application of Q-PCR
 Gene expression
 DNA quantification
 Pathogen detection
 Mutation detection
 SNP detection

24. Method of quantification
 Absolute method
 Relative quantification

25. Imagining
Real-Time
PCR
Measuring
Quantities
We describe the position of the lines with a value that
represents the cycle number where the trace crosses
an arbitrary threshold.
This is called the “Ct Value”.
Ct values are directly related to the starting quantity of
DNA, by way of the formula:
Quantity = 2^Ct
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
23 25
28
Ct Values:
0 5 10 15 20 25 30 35 40

26. Standard curve

27. SNPs detection

28. Q-PCR vs. PCR
Some of the problems with End-Point
Detection:
 Poor Precision
 Low sensitivity
 Non - Automated
 Size-based discrimination only
 Results are not expressed as numbers