POLYMERASE CHAIN REACTION. DNA Structure DNA consists of two molecules that are arranged into a...
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Transcript of POLYMERASE CHAIN REACTION. DNA Structure DNA consists of two molecules that are arranged into a...
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.