5. Polymerase Chain Reaction (PCR)

Who invented the Polymerase Chain Reaction technique?

Kary B. Mullis in 1985.

What is the primary purpose of PCR?

To make millions of copies of a scarce sample of DNA.

Before conducting PCR, what should you consider about cycles?

The number of cycles to run.

What is the first step in the PCR process?

Denaturation.

What temperature typically causes denaturation in PCR?

Approximately 95°C.

What occurs during the denaturation step of PCR?

Double-stranded DNA separates into single-stranded DNA.

What is the role of heat in PCR?

Heat facilitates the melting of double-stranded DNA.

What is the second step in PCR?

Annealing.

What is the purpose of annealing in PCR?

To allow primers to bind to their complementary sequences on single-stranded DNA.

What temperature range is used for primer annealing during PCR?

Between 30°C and 65°C.

What is the third step in PCR?

Extension.

What does Taq polymerase do during the extension phase?

Synthesizes a new DNA strand by adding dNTPs to the template.

What are the three main steps in a PCR cycle?

Denaturation, Annealing, Extension.

What is the significance of MgCl2 in a PCR reaction?

It is important for Taq polymerase function and aids in primer annealing.

What is the typical concentration of Taq polymerase in a PCR reaction?

0.05 units/µL.

What are dNTPs in a PCR reaction?

The raw materials used by Taq polymerase to make copies of the template DNA.

What can happen if template DNA concentration is too high in PCR?

It may inhibit the activity of Taq polymerase.

What does a reagent control in PCR accomplish?

Serves to check if the master mix was contaminated.

What is a negative control in PCR?

Template DNA that does not contain the sequence of interest.

What is a positive control in PCR?

Template DNA that does contain the target sequence.

Which chemical helps to maintain the pH during PCR?

Buffer.

What might fuzzy bands in PCR results indicate?

Diffusion of PCR products or loading errors.

What is thermal cycler used for in PCR?

To perform the temperature cycles needed for PCR automatically.

How many cycles does PCR typically repeat?

Up to 35 times.

What are the applications of PCR?

Genetic disorder diagnosis, forensics, ancient DNA amplification.

What is RT-PCR used for?

To reverse transcribe RNA into cDNA for PCR amplification.

What is qPCR?

Quantitative PCR that detects product in real time.

What is the purpose of using primers in PCR?

To bind to specific sequences on the template DNA for amplification.

What can be an effect of high Taq polymerase concentration?

Non-specific amplification or decrease in product formed.

What does gradient annealing PCR determine?

The most optimal annealing temperature for primers.

What should be aimed for when designing primers?

18-30 base pairs long for specificity.

What is the role of DMSO in PCR?

Increases specificity, especially for long target sequences.

What can harbors contaminants affecting PCR?

Several chemicals such as heparin, salt, or phenol.

What factors affect the optimization of a PCR reaction?

Primer design, template concentration, MgCl2 concentration, Taq concentration, annealing temperature.

What should you do if your PCR results show no bands?

Check for Taq inhibition, pipetting errors, or no DNA added.

What can be concluded if PCR products are faint?

Suboptimal reagent concentrations or Taq activity issues.

What type of products should PCR yield by the third cycle?

The DNA fragments intended for amplification.

What is one common mistake that could lead to no bands on a control lane?

Incorrect pipetting or not adding enough master mix.

Why is it critical to avoid primer dimer formation?

It can lead to non-specific amplification.

How can one determine the concentration of DNA in a sample during qPCR?

By making a standard curve with known template concentrations.

What is the cycle threshold (C T) value in qPCR?

The cycle at which the fluorescent signal is detectable.

What role do hybridization probes play in qPCR?

They create a fluorescent signal that increases with each PCR cycle.

What is the significance of the 3’ end in primer design?

It should end in G or C for stable binding to the template DNA.

What happens if the annealing temperature is too low?

There can be non-specific primer annealing.

What is a common application for PCR in forensics?

Creating DNA fingerprints from unique genetic markers.

How can PCR technology aid in infection testing?

By designing primers that target unique genes of specific microbes.

What are common PCR inhibitors?

Heparin, salt, and a variety of proteins.