ISSN 2398-2969      

Polymerase chain reaction

Clapis
Contributor(s):

Lesa Thompson

Anna Meredith

Synonym(s): PCR


Overview

  • Polymerase chain reaction (PCR) are used to make large numbers of copies of specific DNA fragments from tiny quantities of source material.
  • PCR is most frequently used in veterinary medicine for in vitroinfectious disease diagnostics. It can also be used for genetic identity testing and research.
  • Rapid, cheap and simple technique.

Sampling

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Tests

Methodologies

Step 1 - Denaturation

  • At high temperatures, eg 95°C - initial cycle for 2 min and then 30 sec to 1 min for later repeat cycles, double stranded DNA (dsDNA) helix is melted open to form single stranded DNS (ssDNA) templates.
  • ssDA strands are now accessible to primers.
  • Primers are short DNA molecules (oligonucleotides) defining the specific DNA sequence to be amplified.
  • Enzymatic reactions cease during this phase.

Step 2 - Annealing

  • Reaction mixer is cooled, eg 1 min between 42-65°C.
  • Primers anneal (form bonds) to complementary regions on the ssDNA, acting as starting points for polymerase enzyme.
  • Polymerase attaches the two together, hence forming dsDNA once more and copying the template.
  • Primers are selected to prime DNA synthesis in the region of interest; they joint from the 3' ends of the ssDNA.

Step 3 - Extension

  • DNA polymerase enzyme is most effective at a slightly higher temperature, eg 1 min at 68-74°C, and it synthesizes a complementary strand by reading sequence of opposing strand and extending primers by adding nucleotides (deoxynucleotide triphosphates (dNTPs)).
  • dNTPs are the building blocks for newly synthesized DNA strands. dNTP adds to 3' end of extending DNA strand, resulting in DNA synthesis in 5' to 3' direction.
  • Primers with stronger bonds, ie a better match, remain in place, extending the copied fragment. Primers without an exact match loosen bonds at this higher temperature, preventing incorrect extension.
  • DNA synthesis extends to include the target region and into the flanking region for variable distances, resulting in variable length "long fragments".

Repeat

  • Repeat steps 1-3 for a number of cycles (typically 25-45 times, depending on expected yield of PCR product). This is automated, in a cycler that rapidly heats and cools tubes of reaction mixture. During this step, primers are stimulated to bind to both original and newly synthesized sequences.
  • Polymerase enzyme extends primer sequences again.
  • Repetition of the cycles means copies are copied, only replicating the target region, with an exponential increase in number of copies of the specific sequence.
  • Thermostable DNA polymerase (most commonly TaqDNA polymerase) is not inactivated by high temperature during denaturation step.
  • Samples are usually incubated after final cycle for 5-10 min at 68-74°C, a final extension to complete protruding ends of newly synthesized PCR products.
  • The final sample is then chilled to approximately 4°C.
  • The PCR process is conducted in a specific chemical solution, the reaction buffer, providing optimal environmental conditions for the reaction. Magnesium is also present as a necessary cofactor for DNA polymerase activity.
  • Productes of the PCR reaction are separated bygel electrophoresis.
  • In some instances, amplified product is directly visualized after gel electrophoresis by staining, with ethidium bromide or silver staining, or using radioisotopes and autoradiography.

Availability

Validity

Sensitivity

  • PCR is an extremely sensitive test.
  • Optimization of the technique may include alter magnesium, concentration, primer annealing, temperature, PCR primer design, DNA quality and DNA quantity.
  • Nested PCR, utilizing two sets of primers, results in very high sensitivity.

Specificity

  • Primers define the sequence of DNA to be amplified and thus provide specificity to the reaction.
  • Distance between the two primers used in each reaction determines the length of DNA synthesized. Longer lengths of DNA will result in more specific tests.

Predictive value

  • Varies depending on test, usually high positive predictive value for validated commercial tests.

Technique (intrinsic) limitations

  • Knowledge about target DNA may not be available, precluding choice of primers or reducing optimization of protocol.
  • Contaminants may be present within the original DNA sample.
  • Critical to success of the technique are the following processes: complete denaturation of initial DNA template, optimal temperatures for annealing and extension steps, number of cycles, and final extension step.

Technician (extrinsic) limitations

  • Contaminants may be introduced during purification process.
  • Good laboratory technique is necessary to ensure successful PCR, eg DNA extraction and PCR reaction mixing/processing should be conducted in different areas, pipettes/tips should not be cross-contaminated, and solutions should be stored appropriately.
  • Performing a control reaction in the PCR process can be used to confirm absence of contamination.

Result Data

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Further Reading

Publications

Refereed papers

  • Recent references from PubMed and VetMedResource.
  • DeMay S M, Becker P M, Eidson C A, Rachlow J L, Johnson T R & Waits L P (2013) Evaluating DNA degradation rates in faecal pellets of the endangered pygmy rabbit. Molecular Ecol Res 13 (4), 654-662 PubMed.
  • Loncaric I & Künzel F (2013) Sequence type 398 meticillin-resistant Staphylococcus aureus infection in a pet rabbit. Vet Derm 24, 370-384 PubMed.
  • Wang B et al (2013) Construction and applications of rabbit hemorrhagic disease virus replicon. PLoS ONE (5), e60316 PubMed.
  • Ahmad S T, El-Samadony H A & Soliman Y A (2012) Diagnostic applications of reverse transcriptase-polymerase chain reaction, gel electrophoresis and western immunoblot for detection of rabbit hemorrhagic disease virus. Global J Molecular Sci (1), 1-10.
  • Wang X, Hao H, Qiu L, Dang R, Du E, Zhang S & Yang Z (2012) Phylogenetic analysis of rabbit hemorrhagic disease virus in China and the antigenic variation of new strains. Arch Virol 157 (8), 1523-1530 PubMed.
  • Huybens N, Houeix J, Licois D, Mainil J & Marlier D (2011) Epizootic rabbit enteropathy: Comparison of PCR-based RAPD fingerprints from virulent and non-virulent samples. Vet J 190 (3), 416-417 PubMed.
  • Oliveira U C, Fraga J S, Licois D, Pakandl M & Gruber A (2011) Development of molecular assays for the identification of the 11 Eimeriaspecies of the domestic rabbit (Oryctolagus cuniculus). Vet Parasitol 176 (23), 275-280 PubMed.
  • Sánchez P J, Jr Wendel G D, Grimprel E, Goldberg M, Hall M, Arencibia-Mireles O, Radolf J D & Norgard M V (1993) Evaluation of molecular methodologies and rabbit infectivity testing for the diagnosis of congenital syphilis and neonatal central nervous system invasion by Treponema pallidum. J Infect Dis 167 (1), 148-157 PubMed.

Other sources of information

  • Erlich H A (1989) PCR Technology: Principles and Applications for DNA Amplifications. Stockton Press, NY.

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