PCR Reverse transcription -PCR Real Time PCR Electrophoresis DNA sequencing RFLP Hybridization

1. 1
Clinical Laboratory Principle

2. Nucleic Acid Techniques
Polymerase Chain Reaction (PCR)
Reverse Transcription Polymerase Chain Reaction(RT- PCR)
Real-Time PCR
Electrophoresis
DNA Sequencing
Restriction Fragment Length Polymorphism
Hybridization
2

3. Nucleic acids structure and organization in Eukaryotic cells
3

4. Nucleic Acids structure
4

5. Polymerase Chain Reaction
(PCR)
5

6. Polymerase Chain Reaction
(PCR)
Target Region for PCR
chromosome
cell nucleus
Double stranded
DNA molecule
Individual
nucleotides
6

7. Polymerase Chain Reaction
(PCR)
7
A method widely used to rapidly make millions to
billions of copies of a specific DNA sample, allowing
biologists/scientists to take a very small sample of
DNA/RNA and amplify it to a large amount

8. Polymerase Chain Reaction
(PCR)
8
A technique to make many copies of a specific DNA/RNA region
in vitro (in a test tube rather than an organism).
PCR relies on a
Thermostable DNA polymerase (Taq polymerase)
DNA primers (18-22 bases)
PCR Buffer
dNTPs (dATPs, dGTPs, dCTPs, dTTPs)
Cations (Mg++)
Samples (DNA/RNA)

9. 9

10. 10
PCR Machine and Program

11. 11

12. 12

13. 13

14. 14

15. 15
Reference:
Tietz Fundamentals of Clinical Chemistry,
Sixth Edition. Chapter 17

16. 16
REVERSE TRANSCRIPTION–POLYMERASE CHAIN
REACTION (RT-PCR)

17. 17
REVERSE TRANSCRIPTION–POLYMERASE CHAIN
REACTION (RT-PCR)
Topics:
Definition
Introduction and General feature
Method
Applications

18. 18
Reverse Transcription –Polymerase Chain
Reaction (RT-PCR)
The discovery of
Retroviral reverse
transcriptase in the early
1970s ultimately made RT-PCR
possible.

19. 19
Reverse Transcription –Polymerase Chain
Reaction (RT-PCR)
Reverse transcriptase is an RNA-dependent DNA
polymerase, catalyzing DNA synthesis using RNA as the
template. The end product is known as
complementary DNA (cDNA).

20. 20
Reverse Transcription –Polymerase Chain Reaction
Introduction and General feature
 RNA as starting material for in vitro nucleic acid
amplification.
RNA extraction, when combined with RT-PCR,
make RNA analysis in the clinical laboratory
Rapid and equally sensitive as PCR-based DNA
amplification.

21. 21
Reverse Transcription PCR Method
 Used to amplify RNA targets.
 The RNA template is converted into complementary (c)DNA
by the enzyme reverse transcriptase.

22. 22
Reverse Transcription PCR Method
Primers
 Oligo (dT)
Random hexamer
Gene-specific primers
•Oligo (dT) primers are generally preferred as they
hybridize to the 3ʹ poly (A) tails in mRNAs (transcribed gene
sequences),

23. 23
Reverse Transcription –Polymerase Chain Reaction
(RT-PCR)

24. 24
Reverse Transcription –Polymerase Chain Reaction
(RT-PCR)

25. 25
Application of Reverse Transcription –Polymerase Chain
Reaction (RT-PCR)
Diagnosis and quantification of RNA virus infections
(E.g., human immunodeficiency virus and hepatitis C virus)
 Analysis of mRNA transcripts: commonly associated with
non-Hodgkin's lymphomas
Leukemias
Sarcomas.
Gene expression profiling :
Impact on molecular diagnostics and will depend on RNA
analysis using RT-PCR and possibly high-density arrays.

26. 26
Real-Time PCR

27. 27
Real-Time Polymerase Chain Reaction
 It monitors the amplification of a targeted DNA
molecule during the PCR, not at its end, as in
conventional PCR
Known as Quantitative- PCR

28. 28
PCR relies on a
Thermostable DNA polymerase (Taq
polymerase)
DNA primers (18-22 bases)
PCR Buffer
dNTPs (dATPs, dGTPs, dCTPs, dTTPs)
Cations (Mg++)
Samples (DNA/RNA)
Probes
Real-Time Polymerase Chain Reaction

29. 29
How does Real Time PCR work?
Probes :
A probe is an oligonucleotide that
is labeled with a fluorescent
Reporter and a Quencher
 The Quencher decreases the
fluorescent intensity

30. 30
Probes :
 During The PCR Cycle the Probe
denatures and anneals to the
target sequence
 For every amplification of the
target sequence, a fluorescence
Reporter is released from the
probe

31. 31
Real – Time PCR
 Real –Time Instruments
composed of
1- Thermal Cycler
2- Optical Module
Optical Module
Thermal Cycler

32. 32
Thermal Cycler (PCR - Machine)
Denaturation
Annealing
Elongation

33. 33
Elongation

34. 34
The Reporter molecule is only released when a DNA
strand is completely polymerized by Taq Polymerase

35. 35
2- Optical Module :
To detect fluorescence in the tubes
during the PCR run
This component measures the
fluorescence intensity in the tubes

36. 36
2- Optical Module :
Cycle after cycle; the fluorescence intensity
increases indicating the amplification of
specific region in a real-time
PCR cycle Number = 10 PCR cycle Number = 35

37. 37
The fluorescence of the cycles are measured and
calculated into an amplification curve/plot

38. Electrophoresis

39. Electrophoresis
• Analytical Technique
• The most commonly used in clinical applications.
• Separate and analyzed Ionize analytes
• Charged molecules migrate in a porous supporting
medium (agarose), after the sample is mixed with a
buffer solution, generates an electropherogram

40. Electrophoresis
Definition:
“ The migration of charged solutes or particles in a
liquid medium under the influence of an electrical
field.”

41. Theory of Electrophoresis
In an electrophoresis system, chemical take on electrical
charge and becomes ionized.
• Move toward electrode
Negative Electrode :Cathode
Positive Electrode : Anode
Positive ions(cations) migrate toward the Cathode
Negative ions (anions) migrate toward the Anode

42. Theory of Electrophoresis
CathodeAnode

43. Electrophoresis Apparatus
1. Buffer Tanks
2. Electrodes (Anode/Cathode)
3. Electrophoretic support for separation
4. Wick
5. Tank Cover
6. Power Supply

44. Electrophoresis Apparatus

45. Electrophoresis
 Agarose Gel Electrophoresis (AGE)
 Polyacrylamide Gels Electrophoresis (PAGE)

46. Agarose Gel Electrophoresis (AGE)
• Smaller DNA fragments have
migration rates in agarose that are
inversely proportional to the logs of
their molecular weights
• This relationship decreases as their
fragment size increases.

47. Loading of PCR Amplified Product in Agarose Gel

48. Direction
VerticalHorizontal

49. Agarose Gel Electrophoresis (AGE)

50. Agarose Gel Electrophoresis (AGE)

51. Visualization of PCR Amplified DNA Products

52. Polyacrylamide Gels (PAGE)
• Polyacrylamide is a polymer
• Prepared by heating acrylamide with a variety of catalysts,
with or without cross-linking agents.
• Polyacrylamide gel is
1. Thermostable
2. Transparent
3. Durable
4. Chemically Inert (uncharged)

53. Polyacrylamide Gels
• Capable of resolving DNA molecules that differ by as
little as 2% in length (1 bp in 50 bp).
• Accommodates a larger amount of sample (up to 10 fig) in
a single sample slot (well)
• DNA recovered from a polyacrylamide gel is extremely
pure, containing no inhibitors.
• Polyacrylamide is most useful for mixtures of smaller
DNA fragments and resolves fragments < 1 kbp

54. Apparatus for Polyacrylamide Gels (PAGE)