Polymerase chain
Reaction (PCR)
Polymerase chain Reaction
(PCR )
• Polymerase chain reaction is a technique to amplify a
specific DNA segment by invitro procedure.
• Kary Mullis, the inventor of PCR, was awarded the 1993 Nobel
Prize in Chemistry
Requirements
• Taq DNA Polymerase
• dNTP (dATP, dGTP, dCTP,
Requirements
dTTP)
• Primers
• Taq polymerase is a
thermostable DNA
polymerase derived from
the thermophilic bacterium
“Thermus aquaticus” from
Yellow stone National Park
hot springs
Steps involved
• Strand separation /
Denaturation
• Primer Annealing
• New strand synthesis
1. Strand separation /
Denaturation
• DNA from the desired source is taken
and heated to 90oC.
• This treatment separates the double
stranded DNA into single strands
2. Primer Annealing (50o C)
• The primers are annealed by cooling to 50°C
for 0.5 to 2 minutes
• Two DNA primers each of about 15-20
nucleotides in length are added to the
single stranded DNA.
• These primers bear a sequence
complementary to the 3’ ends of the DNA
segment to be copied.
• Primers stick to the 3’ ends of the DNA this
process is called annealing.
3. New strand synthesis (72oC)
• To this mixture dATP, dGTP, dCTP, dTTP and Taq DNA
polymerase are added and incubated at 72oC for 30 seconds
• Taq DNA polymerase synthesizes 2 new strands (5’ 3’
direction) using the 2 single-stranded DNA as templates,
adding new nucleotides to the 3’ends of the primers.
• At the end of this step we get 2 copies of DNA.
• The above steps (Strand separation, Primer Annealing,
New strand synthesis) are repeated around 20 to 30
times.
• In each cycle DNA copies double and in 20 cycles we
get 2 20 (over a million copies) of the desired segment.
• After the amplification procedure, DNA hybridization technique
or Southern blot analysis with a suitable probe, shows the
presence of the DNA in the sample tissue.
Advantage
• Sensitive : Amplification of even trace of
DNA
• Speed: Faster than the conventional
cloning
Applications:
• Diagnosis of bacterial and viral diseases
• Medicolegal cases: PCR allows the DNA from a hair follicle or a
blood cell to be analyzed
• Diagnosis of genetic disorders
Detection of Variations and Mutations in Genes
• Detection of infectious diseases
• PCR is especially useful for prenatal diagnosis of inherited
diseases
• Cancer detection
• Fossil studies
Reverse Transcriptase PCR (RTPCR):
• It is the method used to amplify, isolate or identify a known sequence from a cell or tissue RNA
library. Essentially normal PCR is preceded by reverse transcription (to convert the RNA to cDNA).
Nested PCR:
• Nested PCR is intended to reduce the contamination in products due to the amplification of
unexpected primer binding sites. Two sets of primers are used in two successive PCR runs, the
second set intended to amplify a secondary target within the first run product. This is very
successful, but requires more detailed knowledge of the sequences involved.
Real Time PCR:
• By this method, quantitation of the number of virus present in a sample can be calculated., e.g.,viral
load in HIV or HBV. So, the treatment modalities can be planned and the response to treatment
could be assessed.
Multiplex-PCR:
• The use of multiple, unique primer sets within a single PCR reaction to produce amplicons of
varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional
information may be elicited from a single test run.
Blotting
Techniques
Definition: The transfer of electrophoretically
separated molecule onto a solid support medium
(nitrocellulose paper)
1. Southern Blotting
2. Northern Blotting
3. Western Blotting
Southern
Blotting
• Definition: Southern blotting is a technique to identify and
characterize a segment of DNA
• Detect mutations in DNA.
• It combines the use of restriction enzymes, Electrophoresis and
DNA probes
Procedure
Isolate DNA from suitable source
Cut with a restriction endonuclease (eg: ECORI)
Sort by Agarose electrophoresis
Agarose is very fragile so it is treated with dilute NaOH solution
to convert DNA into single stranded DNA
DNA fragments have to be transferred to a sheet of nitrocellulose membrane
Nitrocellulose is immersed in a buffer containing the 32P labelled probe
Probe hybridizes with Patient DNA
Autoradiograph: Location of the DNA fragment bound
to the probe is revealed as a dark line on the X-ray film
Summary - Southern Blot
Restriction Digest
Gel Electrophoresis
DNA Preparation:
Denaturation/Depurination
Transfer to filter: Blotting
Detecting DNA: Probing
Southern Blotting
1. ToApplications
detect DNA mutations such as deletion or insertion, or rearrangement
of nucleotide bases.
2. To detect point mutations (replacement of one nucleotide by another),
that cause the loss or gain of restriction sites. Such mutations cause the
pattern of bands to differ from the normal gene
3. To detect identity of an individual using DNA by RFLP (Restriction
Fragment length Polymorphism) and DNA finger printing
4. Gene mapping: Locating the gene on the chromosome.
5. To study related genes in other species
Northern Blotting
• Definition: Northern blotting is a technique to identify
and characterize a segment of RNA
• Nucleic acid analyzed is RNA
Northern Blotting Procedure
RNA purified and treated with formaldehyde to prevent secondary
structure formation
Agarose Gel electrophoresis
Transfer of RNA from gel to Nitrocellulose
Nitrocellulose membrane is immersed in a buffer containing the 32P
labelled deoxy ribonucleotide probe
The probe hybridize to those RNA having complementary sequence
Excess probe is washed and the sheet is exposed to X-ray film.
Northern Blotting Applications
• To study tissue wise distribution of RNA and its level in the
cells. To detect and estimate RNA in cells
• Different tissues contain different mRNA some of them are
unique to a particular tissue,
• eg. Erythroblasts contain haemoglobin mRNA, liver
contains albumin mRNA, Beta cells contain pro insulin m
RNA, etc
• To estimate the size of RNA and also to analyte its size
variation
WESTERN BLOTTING
• Used to detect particular
protein in a sample.
Western Blotting Procedure
Proteins in test sample
Polyacrylamide gel Electrophoresis
Transfer of proteins from gel to Nitrocellulose membrane with electric
current
Nitrocellulose is treated with a specific antibody that can bind to the
protein to be tested (viral protein)
A 2nd antibody conjugated with an enzyme (eg. Peroxidase) is added to
detect the protein antibody complex
Peroxidase substrate is added, shows a colored product
Western Blotting
Applications
• To detect even extremely small quantity of a protein in cell
extract or biological fluid
• It is used in the detection and confirmation of viral infections
particularly AIDS
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