1. POLYMERASE CHAIN REACTION (PCR): PRINCIPLES AND APPLICATIONS
DR. IRAM SIDDIQUE
ASSISTANT PROFESSOR

2. What is PCR?
The polymerase chain reaction (PCR) is a technique to amplify a piece of DNA very rapidly outside of a cell.
Starting with a single molecule of DNA, 25 rounds or cycles of PCR will produce about 10 million identical DNA molecules!!

3. It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993.

4. P: POLYMERASE
C: CHAIN
R: REACTION

5. What is PCR? : Why “Polymerase”?
It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.

6. What is PCR? : Why “Chain”?
It is called “chain” because the products of the first reaction become substrates of the following one, and so on.

7. What is PCR? : The “Reaction” Components
1) Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that define the sequence
to be amplified.
3) dNTPs - deoxynucleotidetriphosphates: DNA building
blocks.
4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction
5) Mg++ ions – cofactor of the enzyme
6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme

8. DNA DNA Sugar DNA has four nitrogen bases.
Deoxyribonucleic acid
DNA has four nitrogen bases.
Two are purines ( 2 ringed base )
Adenine ( A ), Guanine ( G )
Two are pyrimidines ( 1 ringed base )
Cytosine ( C ), Thymine ( T )

9. DNA
A purine always links with a pyrimidine base to maintain the structure of DNA.
Adenine ( A ) binds to Thymine ( T ), with two hydrogen bonds between them.
Guanine ( G ) binds to Cytosine ( C ), with three hydrogen bonds between them.

10. DNA
These four bases are linked in a repeated pattern by hydrogen bonding between the nitrogen bases.
The linking of the two complementary strands is called hybridization.

11. DNA Example of bonding pattern. Primary strand CCGAATGGGATGC
GGCTTACCCTACG
Complementary strand

12. DNA Molecule
Adenine
Thymine
Cytosine
Guanine

13. PCR PCR is a technique that takes a specific
sequence of DNA of small amounts and
amplifies it to be used for further testing.
PCR Targets:
The targets in PCR are the sequences of DNA
on each end of the region of interest, which
can be a complete gene or small sequence.

14. TTAAGGCTCGA . . . . AATTGGTTAA
PCR Targets
The number of bases in the targets can vary.
TTAAGGCTCGA AATTGGTTAA
The Represents the middle DNA sequence

15. PCR Requirements Magnesium chloride: 0.5-2.5mM Buffer: pH 8.3-8.8
dNTPs: µM
Primers: µM
DNA Polymerase: units
Target DNA:  1 µg

16. PCR Cycles Review Denaturalization: 94°- 95°C
Primer Annealing: 50°- 65°C
Extension of DNA: 72° C
Number of Cycles: 25-40

17. PCR Primers A primer for each target sequence on the end
of your DNA is needed. This allows both
strands to be copied simultaneously in both
directions.

18. PCR Primers TTAACGGCCTTAA . . . TTTAAACCGGTT
AATTGCCGGAATT >
and
< AAATTTGGCCAA

19. PCR Primers The primers are added in excess so they will
bind to the target DNA instead of the two
strands binding back to each other.

20. PCR Taq DNA Polymerase Taq stands for Thermus aquaticus, which is a
microbe found in 176°F hot springs in Yellow
Stone National Forest.
Taq produces an enzyme called DNA
polymerase, that amplifies the DNA from the
primers by the polymerase chain reaction, in
the presence of Mg.

21. PCR Denaturing Denaturing is the first step in PCR, in which
the DNA strands are separated by heating to
95°C.

22. PCR Annealing Annealing is the process of allowing two
sequences of DNA to form hydrogen bonds.
The annealing of the target sequences and
primers is done by cooling the DNA to 55°C.

23. Extension
DNA polymerization by a thermostable DNA polymerase at 72 degrees C.

24. The Reaction
PCR tube
THERMOCYCLER

26. Lower temperature to 56oC Anneal with primers
Denature (heat to 95oC)
Lower temperature to 56oC Anneal with primers
Increase temperature to 72oC DNA polymerase + dNTPs

28. PCR Cycles

29. PCR Cycles

30. PCR Cycles

31. PCR Cycles

32. PCR Cycles

36. Applications of PCR Detection of pathogens Classification of organisms
DNA fingerprinting
Drug discovery
Genetic matching
Genetic engineering
Pre-natal diagnosis
Classification of organisms
Genotyping
Molecular archaeology
Mutagenesis
Mutation detection
Sequencing
Cancer research

37. Some applications of PCR.
Forensic medicine.
Preimplantation Genetic Diagnosis (PGD).
Archeology.
Paternity testing.

38. Forensic uses of PCR
PCR can be used to amplify DNA from a small amount of cells (about 1000 cells).
The amplified DNA from cells can be used in DNA fingerprinting analysis to determine who was at the crime scene.

39. DNA fingerprinting using PCR in forensic investigations.
DNA is isolated from blood at a crime scene and amplified by PCR.
The amplified DNA is digested with restriction enzymes and resolved on an agarose gel.
Southern blot analysis is performed to give a DNA fingerprint.

40. MOLECULAR IDENTIFICATION:

41. Detection Of Pathogens
Molecular Identification:
Detection Of Pathogens

42. Detection Of Pathogens
Molecular Identification:
Detection Of Pathogens
Sensitivity of detection of PCR-amplified M. tuberculosis DNA.
(Kaul et al.1994)

43. Sensitivity of detection of PCR-amplified M. tuberculosis DNA.
(Kaul et al.1994)