1 Introduction to PCR Polymerase Chain ReactionDr.Firas Al- Tae

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 cellsDNA 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. 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

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

7 Exponential multiplication

8 Exponential multiplication

9 Exponential multiplication

10 Exponential multiplication

11 Exponential multiplication

12 Exponential multiplication

13 Exponential multiplication

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

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

16 1. Preparation of PCR samplesDNA 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

17 1. Preparation of PCR samplesPCR 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

18 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

19 2. PCR machine PCR ThermocyclerNow, 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

20 What is going on inside PCR machine? Thermal CyclingA PCR machine controls temperature Typical PCR go through three steps Denaturation Annealing Extension

21 Denaturation Heating up to 95 °C separates the double stranded DNASlow cooling anneals the two strands Renaturation Heating t (95 °C) Cool

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

23 Extension DNA polymerase duplicats DNA Optimal temperature 72C

24

25 Example of thermocycler parameters used to amplify a particular gene of interestCycle 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

26 3. Visualization of PCR productNeed 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

27 3. Visualization of PCR productPositive PCR product should look like this

28 Conventional Vs Real-time PCRConventional 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”.

29 Conventional Vs Real -Time PCRConventional PCR Real –Time PCR