Polymerase chain reaction (PCR) is a technique used to amplify a single or few copies of a DNA sequence to generate thousands to millions of copies. It involves repeating cycles of denaturing DNA, annealing primers to the single strands, and extending the primers with a DNA polymerase. Real-time PCR allows quantification of the PCR product at each cycle by detecting fluorescence from DNA-binding dyes or probe hydrolysis. It has applications in diagnosing diseases, detecting gene expression, identifying pathogens, and assessing genetically modified organisms.
2. Contents
Definition
Meaning of PCR.
Purpose of PCR
Components of PCR.
Steps of PCR
Advantages
Disadvantages
REAL TIME PCR
Definition
Detection methods in real time PCR
Applications.
2
Meghana Patel APC - Anand
3. Definition :
Polymerase chain reaction is a technique used in molecular
biology to amplify a single copy or a few copies of a segment of
DNA ,generating thousands to millions of copies of a particular
DNA sequence.
3
Meghana Patel APC - Anand
4. What is meaning of PCR ?
P stands for polymerase
because the only enzyme
used in this reaction is DNA
polymerase
R stands for reaction
because the reaction
takes place in process.
C stands for chain because
the products of the 1st
reaction become
substrates of the following
one, and so on
PCR
4
Meghana Patel APC - Anand
5. Purpose:
To amplify a lot of double stranded DNA molecules with same size
and sequence by enzymatic method and cycling condition.
Developed in 1983 by Karry Mullis. In 1993 Mullis was awarded the
Nobel prize in chemistry for his work on PCR.
PCR is now considered as a basic tool for the molecular biologist.
As is a photocopier a basic requirement in an office, so is the PCR
machine in a molecular biology laboratory!
5
Meghana Patel APC - Anand
6. Components of PCR
DNA template: It is DNA segment to be amplified.
Two primers: Primers are synthetic DNA strands of about 18 to 25
nucleotides complimentary to 3’ end of template strand.
Forward primer: It is complimentary to the 3’ end of antisense
strand (3’-5’).
Reverse primer: It is complimentary to the 3’ end of sense strand
(5’-3’).
Taq polymerase: Taq polymerase adds nucleotides complimentary to
template strand and synthesis new strand of DNA. It is isolated from
Thermus aquaticus( heat resistant bacteria). It is used because of
high temperature stability.
6
Meghana Patel APC - Anand
7. Nucleotides(dNTPs): All types of nucleotides are “building
blocks’’ for new DNA strands and essential for reaction. It
includes Adenine(A), Guanine(G), Thymine(T), Cytosine(C).
Buffer solution: providing a suitable chemical environment for
optimum activity and stability of the DNA polymerase.
Divalent cations: Polymerases require free divalent cations
usually Mg+2 for activity. It act as cofactor in the catalytic
addition of dNTPs.
Monovalent ions(K+): It promotes primer annealing.
PCR Machine: a thermal cycler.
7
Meghana Patel APC - Anand
9. Steps of PCR
There are three major steps in a PCR, which are repeated for 30
or 40 cycles.
This is done on an automated cycler ,which can heat and cool the
tubes with the reaction mixture in a very short time.
The three major steps are as follows:
1) Denaturation at 94oC
2) Annealing at 540C
3) Extension at 72oC
9
Meghana Patel APC - Anand
10. Denaturation at 94oC
During the these step, the reaction mixture is heated to 94OC for
1 min, which causes separation of DNA double stranded. Now
each strand acts as template for synthesis of complimentary
strand.
The Hydrogen bonds between the two strands breaks down and
the two strands separates.
10
Meghana Patel APC - Anand
11. Annealing at 54oC
This step consist of cooling of reaction mixture after denaturation
step to 54oC, which causes annealing of primers to separated strand of
DNA.
The length and GC-content of the primer should be sufficient for
stable binding with template .Guanine pairs with cytosine with three
hydrogen bonds. Thus, higher GC content results in stronger binding.
Time taken to anneal is 45 second.
11
Meghana Patel APC - Anand
12. Extension at 72oC
Taq polymerase binds to the template DNA and starts adding
nucleotides that are complementary to the first strand.
This happens at 72oC as it is
the optimum temperature
for Taq polymerase.
12
Meghana Patel APC - Anand
13. Advantages :
Rapid and easy to perform.
Small amount of DNA is required per test.
Result obtained more quickly.
Usually not necessary to use radioactive material for PCR.
PCR can be used to detect point mutations
Making it possible to amplify DNA from degraded samples.
It is very accurate ,especially for determining various
diseases, leading to better diagnoses.
It is most specific ,sensitive.
13
Meghana Patel APC - Anand
14. Disadvantages :
Target DNA sequence must be known.
Errors made by polymerase.
High degree of operator skill required.
High equipment cost.
High sterile environment should be provided.
Chances of contamination.
14
Meghana Patel APC - Anand
15. What is the need of real time PCR ?
15
Meghana Patel APC - Anand
16. Real Time PCR
Definition:
A real time polymerase chain reaction is a laboratory technique of
molecular biology based on the polymerase chain reaction. It monitors
the amplification of a targeted DNA molecule during the PCR, i.e. in real
time, and not at its end, as in conventional PCR.
It is also called as quantitative real time polymerase chain reaction.
PRINCIPLE: The amount of the nucleic acid present into the sample is
quantified using the fluorescent dye or using the fluorescent labeled
oligos.
Real Time PCR is sensitive and reliable method for detection and
quantification of nucleic acid levels.
16
Meghana Patel APC - Anand
17. Detection methods in Real Time PCR:
(1) By using sequence specific fluorescent probes
(2) By using non specific fluorescent dyes
17
Meghana Patel APC - Anand
18. 1) By using sequence specific fluorescent probes
Fluorescent reporter probes detect only the DNA containing the probe
sequence ; therefore ,use of the reporter probe significantly increases
specificity.
It is hydrolysis probe which bear a reporter dye, often fluorescein at its 5’
end and a quencher attached to the 3’ end of the oligonucleotide.
Under normal condition, the probe remain coiled on itself bringing the
fluorescence dye near the quencher, which inhibits or quenches of
fluorescent single of the dye so it is also known as FRET ( fluorescence
resonance energy transfer).
During the annealing stage of the rtPCR both probe and primers anneal to
separated strand of DNA.
18
Meghana Patel APC - Anand
19. • As the taq-polymerase start to synthesize new DNA strand in the
extension stage, and once the polymerase reaches the probe ,its 5’-
3’exonuclease degrades the probe, physically separating the fluorescent
reporter from the quencher, resulting in an increase in fluorescence.
• The increase in PCR product is proportional to amount of fluorescence.
19
Meghana Patel APC - Anand
21. 2) By using non specific fluorescent dyes
Non specific fluorescent dye binds to all double stranded DNA product
in real time PCR, causing fluorescence of the dye.
An increase in DNA product during PCR therefore leads to an increase in
fluorescence intensity and is measured at each cycle, thus allowing DNA
concentrations to be quantified.
SYBR Green 1 ,SYBR Green 2, EVA green, LC green dyes are used in these
process.
SYBR Green is most widely used for its higher signal intensity.
The reaction is prepared as usual, with the addition of fluorescent dsDNA
dye.
The reaction is run in a real time PCR instrument, and after each the levels
of fluorescence are measured with a detector. 21
Meghana Patel APC - Anand
23. Detection :
The detection is based on fluorescence technology.
The specimen is first kept in proper well and subjected to thermal cycle
like in normal PCR.
The machine, however ,in the real time PCR is subjected to tungsten or
halogen source that lead to fluoresce the marker added to the sample and
the signal is amplified with the amplification of copy number of sample
DNA.
The emitted signal is detected by an detector and sent to computer after
conversion into digital signal that is displayed on screen.
The signal can be detected when it comes up the threshold level.
23
Meghana Patel APC - Anand
24. ct value: It is defined
as number of cycles
required for the
fluorescent signal to
cross the threshold.
24
Meghana Patel APC - Anand
25. Applications :
There are different applications for real time PCR as follows:
The use of PCR in clinical settings can be broadly divided into three
categories:
1) To amplify human genes to check for mutations.
2) To amplify microbial genes in a sample.
3) To amplify human gene from a limited sample for creating a
complete DNA profile of an individual.
25
Meghana Patel APC - Anand
26. Diagnosis of Virus
The virus contain mRNA as a genetic material so that for detection of
virus is done by coupling of real time PCR with a procedure called
reverse transcription.
In this method , RNA is first
transcribed into complementary
DNA(cDNA) by reverse transcriptase
from total mRNA. The cDNA is then
used as the template for the real
time PCR reaction.
It is also used in diagnosis of genetic
disease, cancer etc.
26
Meghana Patel APC - Anand
27. Gene Expression
It is most sensitive method for the detection and quantification of gene
expression levels.
It is used for determining how the gene expression and gene changes
over time ,such as in:
1) The response of tissue and cell cultures to an administration of a
pharmacological agent
2) response to change in environmental conditions.
27
Meghana Patel APC - Anand
28. Agriculture
Detection of phytopathogens:
The agricultural industry is constantly striving to produce plant
propagules that are free of pathogens in order to prevent economic
losses and safeguard health.
Discrimination between the DNA of the pathogen and the plant is based
on the amplification of specific sequences in ribosomal RNA gene’s
coding area by real time PCR.
28
Meghana Patel APC - Anand
29. Genetically modified organisms (GMO)
A genetically modified organism(GMO) is a living organism e.g. a plant
,whose genetic composition has been altered by means of gene technology.
The genetic modification usually involves insertion of a piece of DNA , into
the genome of the organism to be modified.
These smaller piece of DNA is usually taken from other naturally occurring
organisms.
Specific primers are that amplify the gene sequences used during the
process of engineering the vector.
29
Meghana Patel APC - Anand
30. In Gene therapy:
In gene therapy as a drug delivery system there are two important
parameter have to be analyzed :
1) The expression level of the therapeutic gene and
2) The distribution of the drug in different organs.
For the control of both parameters , real time PCR is used.
Microbiological uses:
used by microbiologists in the fields of food safety, food spoilage and
fermentation and for the microbial risk assessment of water quality.
30
Meghana Patel APC - Anand