PCR is a technique that amplifies a small amount of DNA across multiple orders of magnitude, generating thousands to millions of copies of a specific DNA sequence. It involves three main steps - denaturation to separate DNA strands, annealing where primers attach to strands, and extension where a DNA polymerase synthesizes the complementary strand. PCR is used in applications like genotyping, cloning, sequencing, and microbial detection. It requires template DNA, primers, nucleotides, DNA polymerase like Taq, buffer solution, and magnesium ions.

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)