1. POLYMERASE CHAIN REACTION
Chapter no: 9
Mohammad Hosein Bakhshi
2. WHO INVENTED IT ?
Dr. Kary Banks Mullis
born on December 28, 1944
Ph.D. degree in biochemistry from the University
of California, Berkeley
1983: Dr. Kary Mullis developed PCR
1993:Dr. Kary Mullis shares Nobel Prize in
Chemistry for conceiving PCR technology.
3. WHAT IS THAT?
• the automated process that allows a specific DNA chain also called
primers to be replicated over and over again to produce more copies
of that DNA.
This machines purpose is to alter the
temperature so that the primers can be heated
and cooled to perform the processes correctly
4. PURPOSE OF PCR
• To amplify a lot of double-stranded DNA molecules (fragments) with
same (identical) size and sequence by enzymatic method and
Much faster than using vectors
Only a little bit of target DNA is needed
Are to synthesize primers, we need to know the
sequence flanking the DNA segment of interest
Only applies to short DNA fragments, mostly less
than 5 kb
6. STEPS OF PCR
1. Denaturation- The ‘melting’ of DNA into separate
2. Annealing- Primers bind to the complementary
sequences on the lone strands of DNA
3. Extension- Continuation of annealing, creates
Heating up DNA
Splitting into two strands
94 degrees Celsius
Allowing to be bonded (primers)
54 degrees Celsius
Taq Enzyme creating copy of original DNA
Two new double stranded DNA formed
72 degrees Celsius
10. PCR PROGRAM
11. 1. DNA Template: DNA segment to be amplified
2. Primers: a short segment of DNA needed as starter for synthesize DNA
G+C content between 40% to 60%.
18-25 nucleotides long. Members of a primer pair should not differ in length by >3
The 3’ terminal sequence of one primer should not be complementary to any site
on the other primer.
Melting temperature (Tm):
The calculated Tm values of a primer pair should not differ by >5°C.
If possible, the 3’ base of each primer should be G or C.
REACTION MIXTURE FOR PCR
12. Wallace rule:
This equation can be used to calculate the Tm of duplexes 15-20 nucleotides in length in solvents of
high ionic strength (e.g. 1M NaCl).
Tm (in °C) = 2 (A+T) +4 (G+C)
Calculating the melting temperature
every enzyme requires some conditions in means of pH, ionic strength, present cofactors etc.
So, by adding the buffer to PCR reaction you get the optimal pH and Mg2+ is required as cofactor as by
most NTP-binding proteins
REACTION MIXTURE FOR PCR
13. Magnesium Chloride is required for Taq Polymerase to function effectively. When the enzyme
polymerase binds to the DNA strand, it requires magnesium ions with hydroxide groups to remove a
hydrogen proton from the deoxyribose of the nucleotide, in order to add the next nucleotide.
REACTION MIXTURE FOR PCR
5. Taq Polymerase:
This enzyme will be needed to synthesize DNA copies.
Building blocks for new DNA strand.
7. Distilled Water:
15. Reverse Transcriptase-PCR
RT-PCR, one of the most sensitive methods for the
detection and analysis of rare mRNA transcripts or
other RNA present in low abundance.
RNA cannot serve as a template for PCR, so it must
be first transcribed into cDNA with reverse
transcriptase from Moloney murine leukemia virus or
Avian myeloblastosis virus, and the cDNA copy is
The technique is usually initiated by mixing short (12-
18 base) polymers of thymidine (oligo dT) with
messenger RNA such that they anneal to the RNA's
polyadenylate tail. Reverse transcriptase is then
added and uses the oligo dT as a primer to
synthesize so-called first-strand cDNA. Roche Molecular Biochemicals: PCR Application Manual. RT-PCR
16. Nested PCR is a variation of the polymerase chain reaction (PCR), in that two pairs (instead of one pair) of
PCR primers are used to amplify a fragment.
The first pair of PCR primers amplify a fragment similar to a standard PCR. However, a second pair of primers
called nested primers bind inside the first PCR product fragment to allow amplification of a second PCR
product which is shorter than the first one.
Nested PCR is a very specific PCR amplification.
17. Hot Start PCR
Hot Start PCR significantly improve specificity, sensitivity and yield of PCR
Some components essential for polymerase activity is separated from the reaction
mixture until the temperature in the tubes has exceeded the optimal primer annealing
temperature usually 55-65 C ˚.
The technique may be performed manually by heating the reaction components to the
melting temperature (e.g., 95˚C) before adding the polymerase. Specialized enzyme
systems have been developed that inhibit the polymerase's activity at ambient
temperature, either by the binding of an antibody or by the presence of covalently
bound inhibitors that only dissociate after a high-temperature activation step.
18. Real-Time PCR
19. APPLICATIONS OF PCR
Molecular Identification Sequencing Genetic Engineering
Molecular Archaeology Bioinformatics Site-directed mutagenesis
Molecular Epidemiology Genomic Cloning Gene Expression Studies
Molecular Ecology Human Genome Project
Classification of organisms
Detection of pathogens
20. WHY ELECTROPHORESIS?
• To separate DNA fragments from each other
• To determine the sizes of DNA fragments
• To determine the presence or amount of DNA
• To analyze restriction digestion products
• separates molecules from each other on the basis of
• size and/or
• charge and/or
• basis of separation depends on how the sample and gel are prepared
22. MATERIAL REQUIRED FOR AGAROSE GEL ELECTROPHORESIS
Gel casting tray
Staining agent (dye)
Sample to be separate
23. Method For Electrophoresis
Add running buffer, load samples and marker
Run gel at constant voltage until band separation occurs
Pour into casting tray with comb and allow to solidify
View DNA on UV light box and show results
Prepare agarose gel
Melt, cool and add Ethidium Bromide. Mix thoroughly.
24. WHAT PERCENTAGE GEL?
Agarose Concentration in Gel (% [w/v]) Range of Separation of Linear DNA Molecules (kb)
If you add 2gr Agarose into 100mL water, it will give you 2% Agarose Gel)
If you add 4gr Agarose into 200mL water you will also have 2% Agarose Gel .
2 ul PCR
2 ul Loading
ddH2O 8 ddH2O 5