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