Canis ISSN: 2398-2942
Polymerase chain reaction
Contributor(s): Michael Day, Helen Milner, Paul Burr, SYNLAB-VPG
- Polymerase chain reaction (PCR) involves the sequential amplification of target sequences of DNA by repeated cycles of oligonucleotide primer-driven DNA synthesis.
- It is traditionally qualitative but is often now more quantitative which has revolutionized the investigation and diagnosis of health and disease at the molecular level.
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- Many laboratories now offer PCR testing. They require a good ‘uniflow’ design to avoid problems with contamination so the end result is accurate.
- The sample is first processed using commercially available extraction kits to obtain high quality DNA/RNA from a number of different sample types, whilst minimizing the presence of inhibitors that could interfere with the subsequent amplification process.
- PCR uses a pair of synthetic oligonucleotide sequences (primers) which are designed to anneal (bind) to a target region of DNA, both the 5 and 3 regions of the target organism.
- Primers, purified sample DNA and Taq DNA polymerase are combined in a suitable buffer and placed in a thermocycler.
- Thermocycling comprises typically a melting step (c. 95°) where the DNA strands separate. This is followed by an annealing/extension step (c. 60°C) when the primers attach/affix to their target sequence (if present). The polymerase then extends the primers sequentially using the single DNA strand template up to a certain size.
- When heated again, four DNA strands are now created for again affixing and extension. This doubling occurs on each heating and cooling cycle.
- This coupled cycle repeated 40-45 times, such that there is an exponential increase in the amount of DNA. This can then be measured via the increase in fluorescence measured at each cycle.
- The amount of target DNA in the original sample is related to the point at which the fluorescence value crosses a threshold detection value. This is commonly reported as the Ct value.
- In qPCR lower Ct values then indicate more target DNA was present as fewer cycles were needed to cross the threshold. If there was very little material there, more cycles would be required to cross this threshold. In this way if only minimal material is present (Ct 40), the significance of this result clinically can change and may be questioned.
- A final feature is reproducibility as both gel and qPCR are typically done in duplicate but with qPCR, only more than 10 copies of DNA per PCR are required for almost identical fluorescence graphs.
- Recent references from VetMed Resource and PubMed.
- Burnett R C, Vernau W, Modiano J F, Olver C S, Moore P F, Avery A C (2003) Diagnosis of Canine Lymphoid Neoplasia using Clonal Rearrangements of Antigen Receptor Genes. Vet Pathol 40, 32-41 PubMed doi:10.1354/vp40-1-32.
- Willoughby K (2003) The ABC of PCR. In Practice 25(3), 140-145.
- Zarlenga D S & Higgins J (2001) PCR as a diagnostic and quantitative technique in veterinary parasitology. Vet Parasitol 101, 215-230.
- German A J, Helps C R, Hall E J & Day M J (2000) Cytokine mRNA expression in mucosal biopsies from German shepherd dogs with small intestinal enteropathies. Digestive diseases and Sciences 45, 7-17.
- Vernau W & Moore P F (1999) An immunophenotypic study of canine leukaemias and preliminary assessment of clonality by polymerase chain reaction. Vet Immunol Immunopathol 69, 145-164.