Polymerase chain reaction

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

• Generally only available in commercial or research laboratories.
• Several PCRs are available for rabbits, including:
  •  Eimeriaspp   Eimeria spp  .
  •  Toxoplasma gondii  Toxoplasma gondii  .
  •  Encephalitozoon cuniculi  Encephalitozoon cuniculi  .
  •  Treponemaspp (syphilis).
  • Calicivirus   Calicivirus  .
  • Myxoma virus   Myxoma virus  .
  •  Pasteurella multocida  Pasteurella multocida  .
  •  Campylobacterspp.
  • Methicillin-resistant Staphylococcus aureus(MRSA)  Link: aureus .

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.