2. •Fidelity refers to the accuracy of a DNA polymerase in
incorporating the correct complementary nucleotide during
PCR amplification.
•During each PCR cycle, the polymerase extends the primer
by adding nucleotides complementary to the template DNA.
•High fidelity ensures minimal errors, resulting in accurate
copies of the target DNA sequence
3. Factors Affecting Fidelity
•DNA polymerase: Enzymes with proofreading activity
(e.g., Pfu DNA polymerase) possess higher fidelity.
•Reaction conditions: High temperatures, long extension
times, or unbalanced dNTP concentrations can decrease
fidelity.
•Template quality: Damaged templates can lead to errors
during copying by the polymerase.
•dNTP concentration: Low dNTP concentration can
increase the chance of incorporating incorrect nucleotides.
4. • Ensures accurate amplification of the target DNA
sequence.
• Minimizes formation of errors and mutations in the
amplified product.
• Crucial for applications like DNA cloning,
sequencing, and diagnostics.
• Inaccurate PCR products can lead to
misinterpretations and unreliable results.
5. Utilize high-fidelity DNA polymerases with proofreading activity.
Optimize reaction conditions: Maintain optimal temperature,
extension time, and dNTP concentration.
Use high-quality template DNA: Minimize template damage and
contamination.
Minimize PCR cycles: Reduce the number of amplification cycles
to limit potential errors.
6. CONCLUSION
• Utilize high-fidelity DNA polymerases with proofreading activity.
• Optimize reaction conditions: Maintain optimal temperature,
extension time, and dNTP concentration.
• Use high-quality template DNA: Minimize template damage and
contamination.
• Minimize PCR cycles: Reduce the number of amplification cycles to
limit potential errors.
8. What is Processivity in PCR?
• Processivity refers to the ability of a DNA polymerase to synthesize
a long DNA strand continuously before dissociating from the
template DNA.
• In PCR, processivity determines the length of the amplified DNA
product in each cycle.
• Highly processive enzymes generate longer DNA fragments in a
single binding event.
9. Importance of Processivity in PCR
•Enables amplification of longer DNA targets.
•Reduces the number of PCR cycles required for sufficient
amplification.
•Minimizes formation of short, incomplete DNA fragments.
•Contributes to higher yield of the desired PCR product.
10. • DNA polymerase: Certain DNA polymerases naturally exhibit
higher processivity.
• Reaction conditions: Factors like temperature, dNTP
concentration, and ionic strength can influence processivity.
• Template quality: Damaged templates can hinder processivity by
causing the polymerase to stall.
• dNTP concentration: Both excessively high and low dNTP
concentrations can negatively impact processivity
11. • [Imagen A scientist wearing gloves carefully pipetting a clear
liquid from a microcentrifuge tube into a well of a PCR plate]
• Utilize highly processive DNA polymerases specifically designed
for PCR.
• Optimize reaction conditions: Maintain optimal temperature,
dNTP concentration, and reaction buffer composition.
• Minimize template damage: Utilize high-quality template DNA
and proper handling techniques.
