1. Introduction to PCR Polymerase Chain Reaction

2. Why Polymerase Chain Reaction (PCR)?
Polymerase: DNA polymerase
DNA polymerase is the only enzyme used in PCR and actions through duplication of DNA
Chain Reaction: The product of a reaction is used to amplify the same reaction
Results in rapid increase in the product

3. What is PCR PCR is a laboratory version of DNA replication in cells
DNA replication inside living cell
PCR in test tube
PCR is a laboratory technique used to: amplify specific region of DNA (gene) , in order to make a huge number of copies of that gene to be adequately tested.

4. Gene of interest
Genomic DNA
So PCR: 1. first detect the gene of interest in the genomic DNA 2. then amplifies it to make billions copies of that gene in just few hours

5. (How do we identify and detect a specific sequence in a genome?)
The Problem...
(How do we identify and detect a specific sequence in a genome?)
TWO BIG ISSUES:
There are a LOT of other sequences in a genome that we’re not interested in detecting. (SPECIFICITY)
The amount of DNA in samples we’re interested in is VERY small. (AMPLIFICATION)
To “see” a single “gene”, the DNA in a sample of
100 cells would have to be multiplied 180 million
times!!!!!

6. (How do we identify and detect a specific sequence in a genome?)
The Problem...
(How do we identify and detect a specific sequence in a genome?)
TWO BIG ISSUES:
There are a LOT of other sequences in a genome that we’re not interested in detecting.
The amount of DNA in samples we’re interested in is VERY small. (AMPLIFICATION)
PCR can solve BOTH of these issues!!!
PCR can make billions of copies of a specific
gene of interest in just few hours!!!!!!(Amazing)
Specificity
Amplification

7. Pattern of Amplification of gene product by PCR
PCR amplifies gene of interest through what is called exponential amplification 2 4 8 16 32
so on

8. Exponential multiplication

9. Exponential multiplication

10. Exponential multiplication

11. Exponential multiplication

12. Exponential multiplication

13. Exponential multiplication

14. Exponential multiplication

15. What you need to perform a PCR reaction?
Preparation of samples
PCR machine
Visualization of PCR product

16. 1. Preparation of PCR samples
DNA template
Needs a pre-existing DNA to duplicate
Cannot assemble a new strand from components

17. 1. Preparation of PCR samples
DNA Primers (for the detection of gene of interest)
Short nucleotide sequence (18-30 nucleotides)
Forward primer
Anneals to DNA anti-sense strand
Reverse primer
Anneals to DNA sense strand

18. 1. Preparation of PCR samples
PCR master mix
Taq polymerase
Enzyme that extends growing DNA strand complementary to DNA template
MgCl2
Provides ions needed for enzyme reaction
dNTP’s
Nucleotides (Adenine, Cytosine, Guanine, Thymine) building blocks for new DNA strands
Buffer
Maintains optimal pH for enzyme

19. Typical PCR sample
In a thin wall Eppendorf tube assemble the following
PCR components
Amount
Template DNA (5-200 ng)
1 mM dNTPs (200 uM final)
10 X PCR buffer
25 mM MgCl2 (1.5 mM final)
20 uM forward primer (20 pmoles final)
20 uM reverse primer (20 pmoles final)
5 units/uL Taq DNA polymerase (1.5 units)
Water
Final Volume
variable
10 uL
5 uL
3 uL
1 uL
0.3 uL
Variable
50 uL

20. 2. PCR machine PCR Thermocycler
Now, the DNA template, DNA polymerase, buffer, dNTPs and primers are placed in a thin-walled tube and then these tubes are placed in the PCR thermal cycler

21. What is going on inside PCR machine? Thermal Cycling
A PCR machine controls temperature
Typical PCR go through three steps
Denaturation
Annealing
Extension

22. Denaturation Heating up to 95 °C separates the double stranded DNA
Slow cooling anneals the two strands
Renaturation
Heating t (95 °C)
Cool

23. Annealing Two primers are supplied in molar excess
They bind to the complementary region
Optimal temperature varies based on primer length etc.
Typical temperature from 40 to 60 C

24. Extension
DNA polymerase duplicats DNA
Optimal temperature 72C

25. PCR Amplification
Exponential Amplification of template DNA

27. Example of thermocycler parameters used to amplify a particular gene of interest
Cycle step
Temperature
Time
Cycles
Initial denaturation
95°C
30 seconds 1
Denaturation
Annealing
Extension *
72°C
5-10 seconds
10-30 seconds
1 minute
30 cycles
Final extension
4°C
5 minutes
hold

28. 3. Visualization of PCR product
Need visualization system to confirm the presence of the PCR product
(Agarose Gel Electrophoresis) Small fraction of PCR product is loaded on agarose gel together with DNA loading dye
Agarose gel is then put on UV transiluminator to visualise PCR product

29. 3. Visualization of PCR product
Positive PCR product should look like this

30. Conventional Vs Real-time PCR
Conventional PCR
Real-time PCR
The amplified product is detected by an end-point analysis i.e. by running DNA on an agarose gel after the reaction has finished.
Real-time PCR allows the accumulation of amplified product to be detected and measured as the reaction progresses, that is, in “real time”.

31. Conventional Vs Real -Time PCR
Conventional PCR
Real –Time PCR