an introduction to PCR principles and applications in microbiological diagnosis; to serve as a support for students in the second year of medical school

1. Polymerase chain reaction:
introductory elements

2. Relevant definitions
• Nucleic acids
• Nucleotides
• Nucleobases (nitrogenous bases)
• Base pairing
• Polymerase
• Primers
• Denaturation
• Annealing
• Extension (elongation)

3. Nucleic acids
- long, linear macromolecules = polymers which
carry genetic information
- composed of linked nucleotides = monomers
- Each nucleotide has 3 components:
- a 5 carbon sugar = pentose:
- dezoxiribose in DNA or
- ribose in RNA
- a phosphate group
- a nitrogenous base (nucleobase)

4. Nucleobases (nitrogenous bases)
• Nitrogen containing biological compounds found in the
structure of nucleotides
• Primary nucleobases:
– Cytosine (C) (in DNA and RNA)
– Guanine (G) (idem)
– Adenine (A) (idem)
– Thymine (T) (only in DNA)
– Uracil (U) (only in RNA)

5. Base pairing
• Base pairs - formed between specific nucleobases due
to complementarity i.e.
– A with T
– C with G
• ensures the DNA double helix → folded structure of both
DNA and RNA
• DNA structure of each species depends on nucleotide
sequence = succession on DNA strand (basis of the
genetic code)

6. RNA and DNA

7. Polymerases
• DNA-, RNA-polymerase, reverse-transcriptase =
enzymes that catalyze the formation of
DNA or RNA using an existing strand of DNA or RNA as
a template

8. Semiconservative DNA replication
1. DNA strands separated
2. New complimentary DNA
strands synthesized by base
pairing
3. RESULT:
• 2 identical copies (all biological
information from ”parental”
DNA)
• ”daughter” DNA molecules are
"Half old" and "Half new“ = Half
of parental DNA is saved
(conserved) in each daughter
DNA = semi-conservative
replication

9. Primer
• strand of nucleic acid that serves as a
starting point for DNA synthesis under the
action of a polymerase

10. Polymerase chain reaction (PCR)
• Based upon semiconservative DNA replication
• Purpose in microbiological diagnosis:
– to obtain a huge number of copies of nucleic acid of a certain
microorganism (amplification) e.g. bacteria, viruses
– to detect and identify the amplified product

11. PCR – preparatory steps
1. Extract nucleic acid (NA) from biological product e.g.
nasopharyngeal exudate – bacterial / viral NA:
• cell lysis
• elution
• membrane filtration
1. Prepare ”reaction mix”:
• Specific primers (sequence depends on NA to be detected =
target NA)
• Polymerase
• Other components to favour future steps
1. Add extracted NA to ”reaction mix”

12. PCR – the cycling reactions
• Performed in thermal cyclers (PCR machines) =
instruments that employ precise temperature control and
rapid temperature changes
• Thermal block where PCR tubes are placed in
• Thermal prophile is defined:
– number of cycles
– temperature and duration for each cycle

13. Strip with 8 PCR tubes containing reaction
mix

14. PCR thermal cycler

15. PCR – the cycling reactions (30-40 cycles)
1. Denaturation (around 94 C):
• DNA double strand opens → single stranded DNA
• Annealing (around 54 - 64 C):
- Primers in the reaction mix find complementary nucleobase
sequences on each DNA strand and bind in the respective
positions (A with T; C with G)
- Extension (around 72 C):
- Polymerase in the reaction mix catalyzes the synthesis of the
2 new DNA strands

16. PCR: annealing and extension

17. PCR – exponential amplification

20. PCR: detection and identification of
amplified product
Conventional end-point
PCR:
• gel electroforesis of
amplified products
• Visualise sample
migration under UV
light
• Compare bands of
samples with bands of
positive control

21. PCR: detection and identification of
amplified product (2)
Real time PCR
• Fluorescence-based detection; compare cycle threshold
(Ct) of sample with Ct of positive control