Polymerase chain reaction
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Transcript of Polymerase chain reaction
Polymerase chain reaction: introductory elements
Relevant definitions
• Nucleic acids• Nucleotides• Nucleobases (nitrogenous bases)• Base pairing• Polymerase• Primers• Denaturation• Annealing• Extension (elongation)
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)
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)
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)
RNA and DNA
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
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
Primer
• strand of nucleic acid that serves as a starting point for DNA synthesis under the action of a polymerase
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
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”
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
Strip with 8 PCR tubes containing reaction mix
PCR thermal cycler
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
PCR: annealing and extension
PCR – exponential amplification
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
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