2. Contents
• What is PCR?
• History of PCR
• Components of PCR
• Principles of PCR
• Instrumentation
• PCR Programme
• Advantages of PCR
• Applications of PCR
3. What isPCR?
•PCR is a technique that takes
specific sequence of DNA of small
amount and amplifies it by
enzymatic method and cycling
condition and to be used for further
testing.
•In vitro technique
4.
5.
6.
7.
8.
9.
10.
11. STEPS
• 1. Denaturation of ds DNA template
• 2. Annealing of primers
• 3. Extension of ds DNA molecules
13. Annealing
• Temperature: ~50-70℃ (dependent on the
melting temperature of the expected duplex)
• Primers bind to their complementary
sequences
5’
3’
5’ 3’
Forward primer Reverse primer
14. Extension
• Temperature: ~72℃
• DNA polymerase binds to the annealed
primers and extends DNA at the 3’ end of the
chain
Taq
5’
3’
Taq
5’
20. Basic requirements for PCR
reaction
• 1) DNA sequence of target region must
be known.
2) Primers - typically 20-30 bases in size.
These can be readily produced by
commercial companies. Can also be
prepared using a DNA synthesizer
21. Basic requirements for PCR
reaction
• 3)Heat-stable DNA polymerase
• eg : Taq polymerase which is not
inactivated by heating to 94 ℃
4) DNA thermal cycler - machine which can
be programmed to carry out heating and
cooling of samples over a number of
cycles.
25. Exampleof PCRprogramme
• Initial denaturation
• Thermo-cycle file -
• Denaturation :
• Annealing :
• Extension :
• Final extension
95C for 5 mins
30 cycles of
95C for 30 secs
55C for 30 secs
72C for 45 secs
72C for 5 mins
• Holding ( soak ) file usually
4C
26. Advantages of PCR
• Small amount of DNA is required per test
• Result obtained more quickly - usually
within 1 day for PCR
• Usually not necessary to use radioactive
material (32P) for PCR.
• PCR is much more precise in determining
the sizes of alleles - essential for some
disorders.
• PCR can be used to detect point
mutations.
27. Applications of PCR
• 1. Comparison of a normal gene with a
mutant form of the gene
• 2. Detection of low-abundance nucleic acid
sequences (HIV)
• 3. Forensic analysis of DNA samples
• 4. Prenatal diagnosis and carrier detection
of cystic fibrosis
• paternity test
28. Applications of PCR
Molecular Identification Sequencing Genetic
Engineering
Molecular Archaeology Bioinformatics Site-directed
mutagenesis
Molecular Epidemiology Genomic Cloning Gene Expression
Studies
Molecular Ecology
DNA fingerprinting
Classification of organisms
Human Genome
Project
Genotyping
Prenatal diagnosis
Mutation screening
Drug discovery Genetic
matching
Detection of pathogens
Editor's Notes
PCR is a test tube method for amplifying a selected DNA sequence
The DNA to be amplified is heated to separate the doublestranded target DNA into single strands
The separated strands are cooled and allowed to anneal to the two primers (one for each strand).there are
Typically 20–30 cycles are run during this process.
human hair, a tiny spot of blood, or a sample of semen
paternity test
restriction endonucleases which cleave double-stranded (ds) DNA into smaller, more manageable fragments,
a short region of the double helix, the nucleotide sequence on the two strands is identical if each is read in the 5→3
direction.
