Polymerase Chain Reaction

PCR
PCR DEFINITION

✓ The polymerase chain reaction (PCR) is a molecular technique

for in vitro amplification of a specific region of a DNA strand .

✓ It allows to amplify small amounts of DNA

exponentially.
• Each PCR cycle doubles the copies of a desired DNA fragment,
resulting in exponential growth
• ie. after 30 cycles, > 1 000 000 000 copies (230) are made

Exponential Amplification
Components
Essential components of PCR (Requirements)

1. Target DNA
2. Two primers
3. Taq polymerase
4. Buffer
5. Deoxy nucleotide triphosphates
6. Magnesium chloride
7. Water
8. Themal cyclers
1- Water

Medium for all components.

2- Target DNA and nucleotides

• It contains the sequence of interest to be amplified in the PCR reaction.

• Should be free of polymerase inhibitors

Reverse primer
5’ 3’
3’
5’
3’ 5’
3’ Forward primer
5’

Target sequence
3- Two primers(forward and reverse primers)
Primer is an oligonucleotide sequence – will target a specific sequence of opposite base
pairing (A-T, G-C only) of single-stranded nucleic acids

• Primers allow the amplification of a particular

DNA sequence.
• Specific for ends of amplified region
• Forward and Reverse
• Length 15-35 nt
• Annealing temperature should be known
• Depends on primer length, GC content,
Melting point, Specificity and complementary
primer sequences
4- Taq polymerase

• The enzyme that catalyzes the reaction of DNA synthesis.

• It catalyzes the polymerization of dNTPs into DNA strand
• Heat stable
• Taq's optimum temperature for activity is 75–80 °C, with a half-life of
greater than 2 hours at 92.5 °C, 40 minutes at 95 °C and 9 minutes
at 97.5 °C, and can replicate a 1000 base pair strand of DNA in less
than 10 seconds at 72 °C.
5- Buffers

• it maintains pH suitable for the activity of the enzyme.

• It creates an environment for the optimum activity of Taq
polymerase (salt).
• Sometimes it contains of Magnesium chloride which
supplies Mg divalent cations required as a cofactor.

The most commonly used buffer:

100 mM Tris-HCl, pH 8.3
500 mM KCl
DMSO
PEG 6000
Glycerol formamide
Triton X-100 or Tween
6- Mg++

• Mg joins to nucleotides to be
recognized by the polymerase enzyme.
• Essential co-factor of DNA
polymerase
• Too little: Enzyme won’t work.

• Too much: DNA extra stable, non-

specific priming, band smearing
• Used at 0.5 to 3.5 uM in the assay
7- Deoxy nucleotides

• it is the DNA building blocks required for the synthesis of new DNA
strands (growing chain).
• It consist of 4 deoxy nucleotides (dATP, dCTP, dGTP and dTTP).
• They are stored at 10mM, pH 7.0
• Add to 20-200 uM in assay
8- Themal cyclers (thermocyclers)

➢PCR reaction is carried out in thermo cyclers.

➢The machine provides a different degree of
temperature required for DNA Synthesis.
➢The basis of PCR is temperature changes and
the effect that these temperature changes on
the DNA.

Note.
Many thermo cyclers have heating lids to prevent condensation
at the top of the reaction tube. Past thermo cyclers lack heating
lid instead of which a small ball of wax/oil was placed inside the tubes .
Setting up a Typical PCR Reaction
Sterile Water 38.0 ul
10X PCR Buffer 5.0 ul
MgCl2 (50mM) 2.5 ul
dNTP’s (10mM each) 1.0 ul
PrimerFWD (25 pmol/ul) 1.0 ul
PrimerREV 1.0 ul
DNA Polymerase 0.5 ul
DNA Template 1.0 ul

Total Volume 50.0 ul

Master mix
Mixing Common Reagents Saves Time

Component 1X 20X
Sterile Water 38.0 ul 760 ul
10X PCR Buffer 5.0 ul 100 ul
MgCl2 (50mM) 2.5 ul 50 ul
dNTP’s (10mM each) 1.0 ul 20 ul
PrimerFWD (25 pmol/ul) 1.0 ul 20 ul
PrimerREV 1.0 ul 20 ul
DNA Polymerase 0.5 ul 10 ul
DNA Template 1.0 ul --

Total Volume 50.0 ul 980 ul

Aliquot
Add DNA (1ul)
49 ul
as last step
• Keep primers, dNTPs, DNA and DNA polymerase on ice.
• Add the components with the highest volume first.
• Keep track on what has been added.
• Change tips at each step.
PCR process ( Steps )

There are 3 main steps

1. Denaturing \ melting
2. Annealing
3. Elongation\ extension step
1- Denaturing \ melting

• The first step involved is heating to a temperature of 94-98 ‘C for 2-5 min
• To separate the two complementary strands
2- Annealing (hybridization)

• This step in which the hydrogen bonds are

formed between primers and DNA template.

• It is done by cooling the DNA to 50-68°C.

• The annealing temperature depends on primer

length and sequence.
3-Extension

• The step in which DNA polymerase catalyzes the building of new strand in
the 5’-3’ direction.
• It starts at the primers, using dNTPs.
• The Optimal temperature for this step is 72 C.
o
 How dose it work?
The reaction was applied in PCR machine through the following program (5 steps).
N. Steps Temp
1 Initial denaturation 95oC to
5-10 minutes
2 Denaturation 95oC for 30 sec
to denature DNA
strands. The following
steps(2---4) is
3 Annealing 56oC
repeated 20-
to anneal primers to
40 times.
template.

4 extension 72oC DNA synthesis.

5 Final extension 72 C for 7-10 minutes.

6 hold 4o C
Theoretical Yield Of PCR
Theoretical yield = 2n x y
• y = the starting number of copies
• n = the number of thermal cycles

If you start with 100 copies, how many copies are

made in 30 cycles?
2n x y
= 230 x 100
= 1,073,741,824 x 100
= 107,374,182,400
 PCR 30x
100 Melting Melting

Temperature
94 oC Extension 94 oC
Annealing 72 oC
Primers
50 50 oC

0
T i m e 3’ 5’
3’ 5’

3’ 5’
5’ 5’
3’ 5’
5’
3’ 5’
5’ 3’ 5’
5’ 3’
5’ 5’
5’ 3’

5’ 3’
5’ 3’
Amplification is done.
Number of templates
1 2 4 8 16 32 64

0 1 2 3 4 5 6
Cycles