د.يوسف حامد

1. POLYMERASE CHAIN REACTION PCR
College of Dentistry /UOD
4.April.2017
12th Practical Lab
Yousif H.M.Sharif
MSc Biomedical Sciences
Hull University/GB

2. Learning objectives
1) What is the PCR?
2) What are PCR components?
3) How does the PCR work?
4) What are the types of PCR ? YOU must LOOK ONLINE

3. What is PCR?
PCR is a technique used in molecular biology to amplify a single copy or
a few copies of a piece of DNA across several orders of magnitude,
generating thousands to millions of copies of a particular DNA
sequence.
PCR have a variety of applications including genotyping, cloning,
mutation detection, sequencing, microarrays, forensics, and
paternity testing, microbial detection etc.

4. 2% Agarose gel electrophoresis analysis of
PCR amplification products of mecA gene
of 310 bp, extracted from S. aureus.
Lane1: negative control (no DNA template);
lane 2: positive control (mecA positive
strain ATCC 33591); lanes 3–6: methicillin-
resistant S. aureus (MRSA); lane M: DNA
molecular size marker (100 bp ladder).
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important causes
of hospital infections worldwide. High-level resistance to methicillin is caused by the
mecA gene, which encodes an alternative penicillin-binding protein, PBP 2a.PCR is a
powerful tool for microbial detection

5. Components of the reaction mixture
 Template DNA.
 Primers (forward and reverse)
 dNTPs
 Taq DNA Polymerase
 Buffer solution
 Divalent cations : Mg++ ion
 Sterile deionized water
Mineral Oil (to avoid evaporation of samples)
Master mix

6. PCR Reaction:
Water
• Water( dH20)
– The medium for all
other components.

7. PCR Reaction:
Buffer
• Water
• Buffer
– Stabilizes the DNA
polymerase, DNA, and
nucleotides
– 500 mM KCl
– 100 mM Tris-HCl, pH
8.3
– Triton X-100 or Tween

8. PCR Reaction:
Template DNA
• Water
• Buffer
• DNA template
– Contains region to
be amplified
– Any DNA desired
– Purity not required
– Should be free of
polymerase
inhibitors

9. PCR Reaction:
Primers
• Water
• Buffer
• DNA template
• Primers
– Specific for ends of
amplified region
– Forward and
Reverse
– Annealing temps
should be known
• Depends on
primer length,
GC content, etc.
– Length 15-30 nt
– Conc 0.1 – 1.0 uM
(pMol/ul)

10. PCR Reaction:
Nucleotides
• Water
• Buffer
• DNA template
• Primers
• Nucleotides
– "building blocks" for
new DNA strands.
– Activated NTP’s
– dATP, dGTP, dCTP,
dTTP
– Stored at 10mM, pH
7.0
– Add to 20-200 uM
in assay

11. PCR Reaction:
Magnesium
• Water
• Buffer
• DNA template
• Primers
• Nucleotides
• Mg++ ions
– Essential co-factor of DNA
polymerase
– Too little: Enzyme won’t
work.
– Stabilizes the DNA double-helix
– Too much: DNA extra stable,
non-specific priming, band
smearing
– Used at 0.5 to 3.5 uM in the
assay

12. PCR Reaction:
Polymerase
• Water
• Buffer
• DNA template
• Primers
• Nucleotides
• Mg++ ions
• Taq Polymerase
– The enzyme
that does the
extension
– TAQ or similar
– Heat-stable
– Approx 1 (unit)
U /
reaction(rxn)
Thermus aquaticus

13. 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

14. Thermocycler:
It is a machine that cool
and heat down the
reaction in a short period
of time

17. How Does PCR Work?
A Three-Step Process
Each step happens at a different temperature
Step 1: Denaturation
Step 2: Annealing
Step 3: Extension
1 cycle

19. How Does PCR Work?
Step 1: Denaturation
• Heat over 90ºC breaks the hydrogen bonds of DNA and
separates double-stranded molecule into two single strands
Double Stranded DNA target Denatured single strand
Denatured single strand

20. Step 2: Annealing - Primer Binding to Target
also called Hybridization
Temperature is reduced ≈ 50-65ºC
(Annealing temperature depends on primer length and G-C content. )
5’ 3’
3’ 5”
5’ 3’
Template Strand #1
Reverse Primer
Template Strand #2
Forward Primer
3’ 5’

21. Step 3: Extension
5’ 3’
3’ 5’
5’ 3’
Taq Taq
Template Strand #1
Template Strand #2
Temperature is
increased≈ 72ºC
(taq’s ideal temperature)