Introduction to PCR Polymerase Chain Reaction Dr.Firas Al- Tae

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

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.

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

(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

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

Exponential multiplication

Exponential multiplication

Exponential multiplication

Exponential multiplication

Exponential multiplication

Exponential multiplication

Exponential multiplication

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

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

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

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

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

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

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

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

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

Extension DNA polymerase duplicats DNA Optimal temperature 72C

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

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

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

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”.

Conventional Vs Real -Time PCR Conventional PCR Real –Time PCR