Polymerase Chain
Reaction(PCR)
Muhammad Awais Zahoor
M.Phil. Pathology
PCR
“Polymerase chain reaction ( PCR), is a
laboratory technique used to make numerous
copies of a specific segment of target DNA.”
• Developed in 1985 by Kary Mullis
Principle
• The PCR involves the primer mediated
enzymatic amplification of DNA.
• PCR is based on using the ability of DNA
polymerase to synthesize new strand of DNA
complementary to the offered template strand.
Components of PCR
• DNA Template
• DNA Polymerase
• Oligonucleotide Primers
• Deoxynucleotide triphosphates (dNTPs)
• Buffer system
DNA Template
• The dsDNA of interest, separated from the sample.
DNA Polymerase
• Makes new strands of DNA, using existing strands as
templates
• Usually a thermostable Taq polymerase that does not rapidly
denature at high temperature (98℃)
Oligonucleotide Primers
• Short pieces of ssDNA (20-30 bp)
• Complementary to 3' ends of the sense & anti sense strands of
the target sequence.
• Deoxynucleotide triphosphates (dNTPs)
• Single units of the bases A,T,G & C (dATP, dTTP, dGTP,
dCTP)
• Provide energy for the polymerization and the building
blocks for DNA synthesis.
• Buffer system
• Includes potassium and magnesium
• Provide optimal condition for DNA denaturation &
renaturation.
• Important for polymerase activity and stability
General Procedure
All the PCR components are mixed together
and are taken through series of 3 major
cyclic reactions ( denaturation, annealing,
elongation) in self-contained thermocycler
machine.
Denaturation
• Temp. 94℃ for 1 min.
• During this dsDNA denatured to ssDNA
due to breakage of hydrogen bonds.
Annealing
• Temp. 54-60℃ for 20-40s
• Primer bind to their complementary sequence in the template DNA.
Elongation
• Temp. 72-80℃
• Polymerase enzyme adds bases to the 3' each primer, extending the
DNA sequence in the 5' to 3' direction.
• Under optimal conditions, DNA polymerase will add about 1,000
bp/minute.
The number of cycles is usually carried out 25–35 times but may vary
upon the amount of DNA input and the desired yield of PCR product.
Applications
Medical Applications:
• Genetic testing for presence of genetic disease mutations.
• Detection of disease causing genes in suspected parents
who act as carriers.
Infectious disease Applications:
• Analyzing clinical specimens for the presence of infectious
agents.
• Detection of new virulent subtypes of infectious organism.
Cont.
Forensic Applications:
• Can be used as tool in genetic fingerprinting
Research and Molecular Genetics:
• In genomic studies: PCR helps to compare the
genomes of two organisms.
• In phylogenetic analysis
• In study of gene expression analysis.
Different Types of PCR
Types of PCR ??
• Conventional •Hot-start PCR.
(Qualitative)PCR.
•Touchdown PCR.
• Multiplex PCR.
•Assembly PCR.
• Nested PCR.
• RT-PCR (Reverse
•Colony PCR.
Transcription). •Methylation-
• Real Time PCR specific PCR.
Multiplex-PCR:
•It is a special type of the PCR
used for detection of multiple
pathogens by using Multiple
primers sets each one targets a
particular pathogen.
Uses:
•This permits the simultaneous
analysis of multiple targets in a
single sample.
Nested-PCR:
• Used to increase the
specificity of DNA
amplification.
• Two sets of primers are
used in two successive
reactions.
Uses:
•Detection of pathogens that
occur with very few amount.
•Reduce contamination
RT-PCR (Reverse Transcription
PCR)
• In RT-PCR, the enzyme reverse transcriptase uses
the mRNA template to produce a
complementary ssDNA strand called cDNA.
• Next, DNA polymerase is used to convert the
single-stranded cDNA into dsDNA which can now
be used as templates for a PCR reaction.
Uses:
Detection of RNA virus like (HCV).
Reverse Transcription PCR
Real Time PCR or Quantitative –
PCR(qPCR):
Used to measure the specific amount of target DNA (or RNA)
in a sample.
• Method use fluorescent dyes, such as SYBR green, or
fluorescence-containing DNA probes such as TaqMan, to
measure the amount of amplified product as amplification
progresses.
Hot-start PCR:
• It is a technique performed manually by heating the
reaction components to the DNA melting temperature
(e.g. 95°C) before adding the polymerase.
• The hot start PCR technique decreases the
nonspecific bindings.
• By using the hot start Taq DNA polymerase, the
reaction can even be prepared at room temperature. It
increases the yield and accuracy of the results.
Touchdown PCR:
In this type the annealing temperature is gradually decreased in later
cycles.
The initial higher annealing temperature leads to greater specificity
for primer binding, while the lower temperatures permit more
efficient amplification at the end of the reaction. It increases the
quality of outcome of PCR
Assembly-PCR
•(also known as Polymerase Cycling Assembly or
PCA)
It is a method for the assembly of large
DNA oligonucleotides from shorter fragments.
It thus allows for the production of synthetic
genes and even entire synthetic genomes.
Colony PCR
Colony PCR is the modification of the conventional PCR in which the
bacterial colonies are directly used as a PCR template.
Rapid, highly specific PCR method to determine the presence or absence
of the inserted DNA into plasmid directly from the bacterial colonies.
To verify that the desired genetic construct is present, or to amplify a
portion of the construct.
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