Molecular Biology Techniques: PCR & Gel Electrophoresis

What is the primary purpose of using oligo primers in PCR?

At what temperature does the DNA denaturation step occur in PCR?

Which factor does NOT affect the migration of nucleic acids in agarose gel electrophoresis?

What is the function of the loading buffer in agarose gel electrophoresis?

What type of DNA structure does NOT affect its migration speed through agarose gel?

Which property of agarose gel affects the pore size through which nucleic acids migrate?

Which fluorescent dye is used for staining nucleic acids in agarose gel electrophoresis?

Which step in PCR follows the annealing of the primers?

PCR (Polymerase Chain Reaction)

PCR amplifies a specific DNA region.
Primers (20-30 nucleotides long) bind to target DNA sequences.
DNA polymerase synthesizes new DNA strands using free nucleotides (dNTPs).
Cycles involve melting DNA, annealing primers, and extending strands.
Starting material can be genomic or plasmid DNA.
Steps include:
- Melting double-stranded DNA at 95°C.
- Annealing primers at 54°C.
- Adding nucleotides and polymerase to extend strands.
- Repeating cycles of melting, annealing, and extension to amplify the target region.

Agarose Gel Electrophoresis

Separates charged molecules (nucleic acids) based on size.
Factors affecting migration:
- Nucleic acid size (base pairs).
- Nucleic acid conformation (supercoiled, circular, linear).
- Base composition (different bases have different pI values).
- Voltage across the gel (higher voltage = faster migration).
- Agarose gel density (higher density = slower migration).
Monitoring: loading samples contain dyes (Xylene cyanol FF and bromophenol blue) and Ficoll (increases sample density).
Dyes mark nucleic acid sizes (4000-300 base pairs).
Staining: GelRed (non-toxic fluorescent dye) stains nucleic acids.
Imaging: UV transilluminator visualizes the separated DNA bands.

This quiz covers essential molecular biology techniques, specifically PCR and agarose gel electrophoresis. Participants will explore the principles of DNA amplification through PCR, including the roles of primers and DNA polymerase, as well as the methods for separating nucleic acids based on size using gel electrophoresis.