lab PCR

Introduction to PCR Polymerase Chain Reaction

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.


So PCR:1. first detect the gene of interest in the genomic DNA2. then amplifies it to make billions copies of that gene in just few hours

Genomic DNA

Gene of interest

The Problem...

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!!!!!

(How do we identify and detect a specific sequence in a genome?)

The Problem...
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)

(How do we identify and detect a specific sequence in a genome?)

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
• 1 uL
• 0.3 uL
• Variable
• 50 uL


2. PCR machine


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
PCR Thermocycler

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
Denaturation
Slow cooling anneals the two strands
Renaturation

Cool

Heating t (95 °C)


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

PCR Amplification

Exponential Amplification of template DNA

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
• 95°C
• *
• 72°C
• 5-10 seconds
• 10-30 seconds
• 1 minute
• 30 cycles
• Final extension
• 72°C
• 4°C
• 5 minutes
• hold
• 1


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
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

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