POLYMERASE CHAIN REACTION

DNA StructureDNA consists of two molecules that are

arranged into a ladder-like structure called a Double Helix.

A molecule of DNA is made up of millions of tiny subunits called Nucleotides.

Each nucleotide consists of:1. Phosphate group2. Pentose sugar3. Nitrogenous base

Nucleotides

Phosphate

Pentose

Sugar

Nitrogenous

Base

Nucleotides

The phosphate and sugar form the backbone of

the ladder, whereas the bases form the “ladder

steps”.

here are four types of nitrogenous bases.

Nucleotides

A

Adenine

T

Thymine

G

Guanine

C

Cytosine

NucleotidesEach base will only bind with one other specific

base.

Adenine (A)Thymine (T)

Cytosine (C)Guanine (G)

Form a base pair.

Form a base pair.

The bonds between the bases are

hydrogenous bonds.

Three bonds between G & C and two bonds

between T & A.

Because of this complementary base pairing,

the order of the bases in one strand

determines the order of the bases in the other

strand.

G

G

A

T

T

A

A

C

T

G

C

A

T

C

To recognize the genetic code found in DNA

we need to look at the sequence of bases.

The bases are arranged in triplets called

codons.

A G G - C T C - A A G - T C C - T A G

T C C - G A G - T T C - A G G - A T C

A gene is a section of DNA that codes for a protein.

Each unique gene has a unique sequence of bases.

This unique sequence of bases will code for the production of a unique protein.

It is these proteins and combination of proteins that give us a unique phenotype.

Protein

DNA

Gene

Trait

The polymerase chain reaction

(PCR) is a molecular biology technique

for isolating and amplifying a fragment

of DNA, via enzymatic replication,

without using a living organism (such as

E. coli or yeast).

Polymerase Chain Reaction

Polymerase: DNA polymerase

DNA polymerase duplicates DNA.

Before a cell divides, its DNA must be

duplicated.

Chain Reaction:

The product of a reaction is used to amplify the

same reaction.

Results in rapid increase in the product.

ApplicationsPCR is now a common technique used in medical

and biological research labs for a variety of tasks,

such as;

The sequencing of genes and the diagnosis of

hereditary diseases.

The identification of genetic fingerprints (used in

forensics and paternity testing)

The detection and diagnosis of infectious diseases

Basic components1. DNA polymerase ( Taq polymerase) Cannot assemble a new strand from components.

Needs a pre-existing DNA to duplicate called “template

DNA”.

Can only extend an existing piece of DNA called

“primers”

DNA strands are anti-parallel1. One strand goes in 5’ 3’2. The complementary strand is opposite

DNA polymerase always moves in one direction (from 5’ 3’)

2. DNA template that contains the

region of the DNA fragment to be

amplified.

3. One or more primers, which are

complementary to the DNA

regions at the 5' and 3' ends of the

DNA region that is to be amplified.

4.Deoxynucleotide

triphosphates, (dNTPs) from

which the DNA polymerase builds

the new DNA.

dCTP

dTTP

dCTPdGTPdATP

dGTP

dCTP

dTTP

dATP

dGTP

dCTP

dTTP

dATP

dATP

dGTPdCTP dTTPdATP dGTP

dATP

dGTP

dTTP

dATP

dCTP

dTTP5’ 3’

3’ 5’

5.Buffer solution, which provides a

suitable chemical environment for

optimum activity and stability of the

DNA polymerase.

6.Divalent cation, magnesium or

manganese ions.

7.Monovalent cation potassium ions.

8. Thermal cycler

The DNA, DNA polymerase, buffer,

nucleoside triphosphates, and primers are

placed in a thin-walled tube and then these

tubes are placed in the PCR thermal cycler

This is a machine that heats and cools the

reaction tubes within it to the precise

temperature required for

each step of the reaction.

PCR usually consists of a series of 30 to 40

cycles.

There are three main steps:

Denaturation at 94oC to 98oC for 1

minute.

Annealing at 54°C to 64oC for 45

seconds.

Extension/Elongation at 70°C to 74oC

for 2 minute.

Main idea

Heating separates the

double stranded DNA

"Denaturation”

Slow cooling anneals the

two strands “Annealing”

1. DenaturationIn this step double stranded DNA molecules are melted to yield single stranded DNA (template strand).

2. AnnealingIn this step the reaction temperature is

lowered so that the primers can anneal to the single-stranded DNA template.

Stable bonds are only formed when the primer sequence very closely matches the template sequence.

To this short section of double-stranded DNA the polymerase attaches and begins DNA synthesis.

3. Extension/Elongation In this step DNA polymerase synthesizes

new DNA strands complementary to the DNA template strands.

Primers that have annealed to DNA regions with mismatching bases dissociate from the template and are not extended.

4. PCR amplification

Reverse Transcription PCR (RT-PCR)

Is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA.

The PCR reaction is preceded by a reaction using reverse transcriptase to convert RNA to cDNA.

DETECTION OF AMPLIFICATION PRODUCTS BY GEL ELECTROPHORESIS

Agarose gel electrophoresis is commonly employed for size separation of the PCR products.

This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field.

Shorter molecules move faster and migrate further than longer ones.

The size's of PCR products is determined by comparison with a DNA ladder, which contains DNA fragments of known size, ran on the gel along with the PCR products.

Lane 1 : PCR fragment is

approximately 1850 bases

long.

Lane 2 and 4 : the fragments

are approximately 800

bases long.

Lane 3 : no product is

formed

Lane 5 : multiple bands are

formed because one of the

primers fits on different

places.

Gel electrophoresis can be used for the separation of DNA fragments of 50 base pairs up to several megabases (millions of bases). However, it is normally used in a range of 100 bp to 20 kbp.

Typical run times are about an hour.

Agarose is purified from agar. Different purities of agarose are commercially available with different melting properties.

High purity low melt agarose is often used if the DNA is to be extracted from the gel.

After electrophoresis the gel is illuminated with an ultraviolet lamp.

The ethidium bromide fluoresces pink in the presence of DNA.

The DNA band can also be cut out of the gel and can then be dissolved to recover the purified DNA.