This document discusses polymerase chain reaction (PCR) and real-time PCR techniques. It begins with an overview of using PCR to study gene expression through RNA extraction, cDNA synthesis, and either end point PCR or real-time PCR. Real-time PCR allows for simultaneous amplification and quantification of specific nucleic acid sequences. It describes the basic components and steps of real-time PCR, including different chemistries used and quantification methods. The document emphasizes the importance of controls and melt curve analysis to validate real-time PCR results.

1. PCR AND
REAL TIME PCR
Ashikh Seethy
Senior Resident and PhD Scholar
Dept of Biochemistry
AIIMS – New Delhi.

2. OVERVIEW:
Studying Gene Expression using PCR:
RNA extraction
and
quantification
cDNA
synthesis
End Point PCR
or
Real Time PCR
▪ PCR
▪ Reverse Transcription PCR
▪ Real Time PCR
2

3. NUCLEIC ACID AMPLIFICATION
TECHNIQUES:
• Polymerase chain reaction (PCR)
• Transcription-mediated amplification (TMA)
 Self-sustained sequence replication (3SR)
 Nucleic acid sequence–based amplification (NASBA)
• Strand displacement amplification (SDA)
• Loop-mediated amplification (LAMP)
• Helicase dependent amplification (HDA)
• Recombinase polymerase amplification (RPA)
• Whole-genome amplification (WGA)
 Multiple displacement amplification (MDA)
• Antisense RNA amplification (aRNA)
• Branched DNA (bDNA)
• Rolling circle amplification (RCA)3

4. NASBA:
4

5. Polymerase Chain
Reaction

6. SEQEUNCE SPECIFIC AMPLIFICATION
6

7. 7
LSD-using, AIDS-denying, UFO-believing, yet somehow Nobel Prize-
winning biochemist Kary Mullis.

8. 8

9. COMPONENTS OF PCR:
▪ Template DNA
▪ Polymerase
▪ Primers
▪ dNTPs
▪ Buffer
▪ Cations
9
Questions:
1. What are the polymerases used in PCR?
2. What makes the thermostable polymerases “thermostable”?
3. What is the ideal length of a primer?
4. Why is a high concentration of dNTPs inhibitory?
5. What is the role of divalent cations?
6. What is a hot-start polymerase?

11. “How will you proceed
with a PCR reaction?
11

12. 12
95°C 95°C
Annealing
72°C 72°C

13. 13
Questions:
1. What is the use of a heated lid?
2. Why is it better to add the polymerase as the last
component?

14. “How will you confirm a
successful PCR reaction?
14

16. “What are the precautions for
preventing contamination of
PCR reaction?
16

17. ▪ Prepare the DNA sample, set up the PCR mixture, perform
thermal cycling and analyze PCR products in separate
areas
▪ Laminar flow cabinets equipped with an UV lamp
▪ Wear fresh gloves for DNA purification and reaction set up
▪ Reagent containers dedicated for PCR.
▪ Pipette tips with aerosol filters
▪ “No Template Control” (NTC)
17

18. STUDYING GENE EXPRESSION:
RNA extraction
and
quantification
cDNA
synthesis
End Point PCR
or
Real Time PCR
18
No of copies of mRNA ≡ No of copies of cDNA
REVERSE TRANSCRIPTION PCR
(RT-PCR)

20. COMPONENTS OF RT-PCR:
20

21. PRIMING STRATEGIES:
21
Which strategy
will you prefer?
What is the
primer for RT in
HIV?

22. REVERSE TRANSCRIPTASES:
22
▪ Avian myeloblastosis virus reverse transcriptase (AMV RT)
▪ Moloney murine leukemia virus RT (M-MLV RT)
▪ Which one to choose?
AMV RT MMLV RT
Temperature 42°C 37°C, 55°C
RNase H activity ++ Minimal
Processivity 25 nucleotides 1500 nucleotides
Fidelity Relatively low 1/15000

23. AFTER REVERSE TRANSCRIPTION:
23
1000 mRNAs
of gene X
Other mRNAs
RNA
cDNA
100 mRNAs
of gene X
Other mRNAs
RNA
cDNA
If you have started the cDNA
synthesis with equal amounts
of total RNA,
what is the relative abundance
of cDNA of gene X in blue
compared to yellow?

24. “How will you estimate the
abundance of cDNA of your
GOI?
Perform a PCR
Two choices:
1. End Point PCR
2. Real Time PCR
24

25. END POINT PCR:
25
Amplicons Amplicons
Primers for Gene X
Perform a conventional PCR
Gel electrophoresis
cDNA cDNA

26. “What is the problem with End
Point PCR?
26

27. PHASES OF A PCR:
27

29. PHASES OF A PCR:
29

30. END POINT PCR:
30
Amplicons Amplicons
Primers for Gene X
Perform a conventional PCR
Gel electrophoresis
cDNA cDNA

31. DISADVANTAGES OF
END POINT PCR:
31
Poor precision Low sensitivity
Low resolution
Non - automated Size-based discrimination
Use of ethidium bromide
What is the use of end point PCR?

32. Alternative?
Real Time PCR
Uses fluorescence detecting thermal-cyclers to
amplify specific nucleic acid sequences and measure
their concentrations simultaneously

33. REAL TIME PCR:
33

34. REAL TIME PCR: OVERVIEW
• Chemistries used in Real Time PCR
• The process of Real Time PCR
• Absolute and relative quantification
• Melting curve
• Precautions
• Applications
34

35. 35
DNA Binding
dyes
Taqman
Probes
Molecular
Beacons
Scorpion
Primers
CHEMISTRIES IN REAL TIME PCR:

36. DNA BINDING DYES
What are the limitations of dye based methods?
What are the limitations of SYBR Green?
• Increased background
• Inhibition of the PCR reaction
Other dyes:
SYTO 9
Evagreen
36

37. TaqMan PROBES:
Advantage: Multiplexing
37

38. MOLECULAR BEACONS:
38

39. SCORPION PRIMERS:
39

40. INSTRUMENTATION:
40

41. PERFORMING REAL TIME PCR:
• What is the function of ROX?
• Primer considerations:
41

42. PERFORMING REAL TIME PCR:
42

43. “No Real Time PCR is valid
without Melt Curve and
adequate Controls
43

44. ONE STEP vs TWO STEP qPCR:
44

45. QUANTIFICATION:
45
1  2  4  8  16  32  64  128  256  512
Yield = Original conc. X 2 cycle no.
Yield = Original conc. X (1+Efficiency)cycle no.
 
01020408 16  32  64  128  256  512 1024
08163264128256512102420484096
06
09

46. QUANTIFICATION:
46

47. QUANTIFICATION:
47
1 copy of template
2 copies of template
4 copies of template
For every 1 cycle difference in Ct, there is a
2 fold difference in the initial copy number.
For every n cycles difference in Ct, there is a
2n fold-difference in the initial copy number.

48. ABSOLUTE QUANTIFICATION:
48

49. RELATIVE QUANTIFICATION:
49
• ΔCt method
• Livak’s method
• Pfaffl’s method
ΔCt method:
Relative gene expression(test/control) = 2-ΔCt
= 2-(Ct test - Ct control)

50. NORMALISATION WITH A REFERENCE GENE:
50
1000 mRNAs
of gene X
Other mRNAs
RNA
cDNA
100 mRNAs
of gene X
Other mRNAs
RNA
cDNA
X≡ 1000 X≡ 100
X≡ 100X≡ 1000

51. 51
1000 mRNAs
of gene X
Other mRNAs
RNA
cDNA
100 mRNAs
of gene X
Other mRNAs
RNA
cDNA
X≡ 1000 X≡ 100
X≡ 100X≡ 900
NORMALISATION WITH A REFERENCE GENE:

52. 52
1000 mRNAs
of gene X
Other mRNAs
RNA
cDNA
100 mRNAs
of gene X
Other mRNAs
RNA
cDNA
X≡ 1000 X≡ 100
X≡ 100X≡ 900
NORMALISATION WITH A REFERENCE GENE:
Y≡ 100
Y≡ 90
Y≡ 100
Y≡ 100

53. 53
1000 mRNAs
of gene X
Other mRNAs
RNA
cDNA
100 mRNAs
of gene X
Other mRNAs
RNA
cDNA
X≡ 1000 X≡ 100
X/Y≡ 1X/Y= 10
NORMALISATION WITH A REFERENCE GENE:
Y≡ 100 Y≡ 100

54. “How will you select a
reference gene for
normalisation?
54

55. RELATIVE QUANTIFICATION:
55
Livak’s method
• Relative gene expression = 2-ΔΔCt
• ΔΔCt = (CtTarget-CtRef)Test - (CtTarget-CtRef)Control
= (CtTest-CtControl)Target - (CtTest-CtControl)Ref
= ΔCtTarget - ΔCtRef
Pfaffl’s method
•

56. CONTROLS IN REAL TIME PCR:
56
• Non-Template Control/ Negative Control
• Minus RT control
• Positive control- exogenous/ endogenous
• Internal/ Normalisation control
• Passive reference dye

57. “How will you make sure that
the signal generated is due to
the intended product?
57

58. MELT CURVE ANALYSIS:
Increase the
temperature
incrementally
DNA
denatures
Monitor the
reduction in
fluorescence

59. MELT CURVE ANALYSIS:

60. MELT CURVE ANALYSIS:

61. APPLICATIONS OF REAL TIME PCR:
61
• Quantification of gene expression
• Quantification of viral load
• Validation of microarray data
• Detection of pathogens
• Genotyping
• Response to treatment

62. PRECAUTIONS:
62
• Primers incorporating exon-exon junctions
• Avoid primer dimerization and misfolding:
 GC clamps
 Inverted repeats
• Product length
• Melting Curve Analysis
• Controls
• SYBR Green I is light sensitive
• Avoid marking on PCR tubes
• Prepare a cocktail
• Pipetting

63. AFTER A qPCR RUN:
63

64. THANK YOU