1. Quantitative PCR Session 2: Overview of qPCR
Presented by:
Robert O'Brien
Training Specialist – Forensic Biology

2. Session 2- Overview of qPCR
Quantitative PCR
Session 2- Overview of qPCR
Differences between PCR and qPCR
Why Real- Time PCR
PCR Mechanism
Phases of qPCR amplification
qPCR chemistry
qPCR process
Relationship between the CT value and the quantity of DNA

3. Differences between PCR and qPCR
PCR (end point PCR)
Products are analyzed after the cycling is completed (static) using gels, CE, UV or fluorescence detection
End point assay means results are viewed at end, the end result is all that is important

4. Differences between PCR and qPCR
qPCR products are monitored as the PCR is occurring (dynamic)
Monitoring is occurring once per cycle
Fluorescence is measured
Kinetics of the system- what is happening during the entire process is what is important, not the end product

5. Why Real-Time PCR (Advantages and Disadvantages) Advantages
Availability of commercial qPCR kits and instrumentation
Higher throughput and less user intervention
Simple data analysis
Software rapidly analyzes data

6. Why Real-Time PCR Advantages
Process sensitive to same inhibitors as end point amplification due to presence of an internal control
No post PCR manipulation
High sensitivity
Large range of quantities that can be detected
Assays can be made to target different types of DNA and in some cases can be multiplexed

7. Why Real-Time PCR Disadvantages of qPCR
At very low or high levels of DNA precision may suffer.
Can give the incorrect quantity if DNA sample is degraded
qPCR assumes the sample is quantified at same efficiency as the calibrant sample
Results are calculated based on the calibrants. If the calibrants are made up wrong then the quantities of the samples will be wrong.

8. PCR Mechanism PCR- Polymerase Chain Reaction
Result of the PCR reaction is an exponential increase in PCR products
The amount of DNA present theoretically doubles with every cycle

9. PCR Mechanism Starting with one copy of DNA
Doubling after one cycle to give 2 copies
After two cycles another doubling to produce 4 copies and so on

10. PCR Mechanism

11. PCR Mechanism

12. Phases of qPCR amplification
There are 4 Phases that occur during the PCR process.
1st Phase- Lag phase
2nd Phase- Exponential phase
3rd Phase- Linear phase
4th Phase- Plateau phase

13. Phases of qPCR amplification
Lag Phase, Exponential Phase, Linear Phase and Plateau Phase

14. Phases of qPCR amplification
Lag Phase
This is the first phase in PCR process
During this phase there is an increase in PCR product but fluorescence produced by this increase is too low to be detected
During lag phase the baseline is being set

15. Phases of qPCR amplification
Exponential phase
Also known as the geometric phase
During the exponential phase PCR efficiency is at 100%
There is an abundance of all reaction components - polymerase, free dNTPS, template DNA, primers etc.
As PCR product continues to increase the ratio of Ampli Taq Gold DNA polymerase to PCR product decreases

16. Phases of qPCR amplification
Exponential phase
The signal is in direct proportion to the increase of PCR product
A plot of DNA concentration vs cycle number on a log scale should approximate a straight line

17. Phases of qPCR amplification
Linear phase
One or more components of PCR have decreased below a critical concentration therefore amplification begins to decrease
Phase is called linear because amplification approximates an arithmetic progression rather than a geometric increase
Amplification efficiency is continually decreasing during linear phase

18. Phases of qPCR amplification
Linear Phase
Slope of amplification plot decreases significantly

19. Phases of qPCR amplification
Plateau Phase
PCR reaction has stopped
There is no increase in PCR product therefore no more fluorescence
Amplification plot is a straight flat line

20. qPCR Chemistry Overview Two 5’ nuclease assays
One for target of Total Human DNA or Human male DNA
One for target of Internal PCR Control (IPC)

21. qPCR Chemistry Components of Target Specific Assay
Two primers for amplifying total human DNA or male human DNA
One Taqman ® MGB probe labeled with FAM dye for detecting the amplified sequence

22. qPCR Chemistry Targets of PCR Total Human
Human Telomerase reverse transcriptase gene (hTERT gene)
Location 5p15.33
Amplicon length- 62 bases
Non translated region is amplified
Diploid

23. qPCR Chemistry Targets of PCR Human male
Sex determining region Y gene (SRY)
Location Yp11.3
Amplicon length 64 bases
Non-translated region is amplified
Haploid

24. qPCR Chemistry IPC assay components
IPC template- DNA is synthetic not found in nature
Two primers for amplifying the IPC template DNA
One Taqman ® MGB probe labeled with VIC dye for detecting amplified DNA

25. qPCR Chemistry Taqman ® MGB Probe Made up of a reporter dye
FAM dye or VIC dye linked to the 5’end of the probe
Has a Minor groove binder (MGB) at the 3’ end of the probe
Non-fluorescent quencher (NFQ) at the 3’ end of the probe

26. qPCR Chemistry Taqman ® MGB Probe Importance of MGB at 3’end of probe
Increases melting temperature of probe without increasing length
Melting temperature needs to be high so probe is not destroyed during heating cycles of PCR
If probe is too long quencher dye and reporter dye will be too far so signal from reporter dye will not be suppressed

27. qPCR Chemistry Taqman ® MGB probe
The proximity of the reporter dye to the quencher dye allows for suppression of the fluorescence from the reporter dye by a Förster type energy transfer.

28. qPCR process
5’ nuclease assay process (Nuclease activity is on the 5’ end of the probe)
1- Occurs during PCR amplification
2- Occurs in every cycle
3- Does not interfere with exponential accumulation of product

29. qPCR process
Components of qPCR

30. qPCR process
Step 1- Taqman ® MGB Probe anneals specifically to a complementary sequence between the forward and reverse primer sites

31. qPCR process Step 2 - qPCR process begins
Ampli Taq Gold DNA ploymerase begins its polymerase activity incorporating free dNTPS
Ampli Taq Gold DNA polymerase has a 5’ nuclease activity

32. qPCR process
As Ampli Taq Gold DNA polymerase progresses on the strand of DNA it cleaves off the reporter dye on the 5’ end of probe.
As the reporter dye is cleaved it is no longer close enough to the quencher dye for suppression of fluorescence to occur.
As a result fluorescence from reporter dye can be detected

33. qPCR process

34. qPCR process
As the Ampli Taq Gold DNA polymerase progresses it will displace the probe

35. qPCR process IPC IPC undergoes same process as template DNA
Because it is a synthetic sequence and labeled with a VIC dye its fluorescence is detected separately

36. Relationship between the CT value and the Quantity of DNA
CT value is the cycle at which the fluorescence reaches a high enough level to cross the threshold.
A low CT value means it took less cycles for the fluorescence to cross the threshold
If every cycle is a doubling of product then the product did not have to be doubled many times to reach a certain level

37. Relationship between the CT value and the Quantity of DNA
Therefore the input of DNA would be more if the CT was low.
Therefore the more DNA present the lower the CT value.
Thus the CT value and quantity of DNA are inversely proportional to each other
The software is able to calculate the input amount of DNA based on the CT value of the sample.

38. Relationship between the CT value and the Quantity of DNA
To obtain the quantity of DNA from the CT value a curve is created using known concentrations of DNA vs their CT values
From this curve the quantity of DNA from unknown samples can be calculated based on their CT value
The calculation is based on a doubling of PCR product.

39. Relationship between the CT value and the Quantity of DNA
Therefore the portion of the amplification plot looked at is when the PCR reaction is occurring at 100% efficiency
This occurs during the exponential phase
The standard curve is a graph of the CT of the standards vs the Log of the Concentration of the standards

40. Relationship between the CT value and the Quantity of DNA
The quantifiler software calculates the regression line by calculating the best fit curve with the quantification standard data points
Regression line formula
CT = m (log Qty) + b
Fits formula for a straight line
Y= mx +b
Y= CT, m=slope, x = log qty, b= y intercept

41. Relationship between the CT value and the Quantity of DNA
Calculating Quantity
CT= m (log Qty) + b
CT -b = m (log Qty)
(CT -b)/m =log qty
10 [ (CT –b)/m] = Qty

42. Relationship between the CT value and the Quantity of DNA
Qty= starting quantity of DNA
R² value- Measure of the closeness of the fit between the standard curve regression line and the individual Ct data points of the standards. A value of 1.00 means a perfect fit. R² value > 0.99 represents a close fit.
Slope indicates the PCR amplification efficiency for the assay.
A slope of -3.3 indicates 100% amplification efficiency.
The Standard curve shows all this information and it is used to assess if the standards for the plate were set up correctly.

43. Relationship between the CT value and the Quantity of DNA

44. Quantitative PCR Reaction efficiency = [10 (-1/m) ]-1
Reaction efficiency of 100% = 1
1= [10 (-1/m) ]-1
Solving for m (slope)
2 = 10 (-1/m)
Log 2 = -1/m
-1/log 2 = m
-3.3= m
Y intercept indicates expected CT value for a sample with qty = 1ng/µl