DNA Replication Enzymes and Mechanisms

Colony PCR of Recombinant GFP

• Colony PCR is used to verify successful transformation of colonies.
• DNA is negatively charged due to its phosphate backbone.

DNA Structure

• DNA is a double-stranded helix.
• It's composed of nucleotides.
• Nucleotides have four bases: A, T, C, G.
• DNA has a 3' end and a 5' end.
• DNA strands are antiparallel.
• Base pairing is complementary (A with T, C with G).

DNA Replication

• Similar to PCR, but different
• DNA replicates creating two new strands to be identical from original strand.
• Helicase unzips the DNA double helix creating a replication fork.
• Primase creates an RNA primer, which is a starting point for DNA polymerase.
• DNA polymerase builds a new strand of DNA in the 5' to 3' direction.
• The leading strand synthesizes continuously, the lagging strand discontinuously (Okazaki fragments).
• Exonuclease removes RNA primers.
• DNA polymerase fills the gaps
• Ligase joins Okazaki fragments.
• Replication is semi-conservative (each new DNA molecule has one old and one new strand).
• Topoisomerase prevents supercoiling during replication.
• Leading strand synthesis starts with an RNA primer binded to the initiation site. DNA Polymerase adds nucleotides in 5'-3' direction and reads the template strand from 3'-5'
• Lagging strand is synthesized in fragments called Okazaki fragments. Exonuclease removes RNA primer and DNA ligase seals the gaps.

Three Main Events of DNA Replication

• Initiation: Topoisomerase (or gyrase) cuts and rejoins DNA to relieve tension caused by unwinding. DNA polymerase (specifically DNA pol III in prokaryotes) adds nucleotides in the 5' to 3' direction. Single-strand binding proteins prevent the separated strands from reannealing.
• Elongation: DNA polymerase synthesizes new DNA strands. Replication fork is formed. Leading strand is synthesized continuously; lagging strand is synthesized discontinuously.
• Termination: RNA primers are removed (exonuclease). DNA polymerase fills the gaps. DNA ligase joins the fragments.

Models of DNA Replication

• Semiconservative: Each new DNA molecule has one original and one new strand. This model is correct.
• Conservative: Original DNA strands form a new fusion and creates two identical daughters strands
• Dispersive: Original DNA strands are segmented and combines to form a new fragment

Polymerase Chain Reaction (PCR)

• PCR is used to amplify a specific segment of DNA.
• It's similar to DNA replication but in a test tube (in-vitro).
• Applications include species identification, cloning, forensics, and pathogen detection.
• Kary Mullis invented PCR. PCR is a technique for amplifying specific regions or sequences of DNA. This is different from replicating an entire genome
• A reaction mixture is needed for PCR, which has template DNA, primers (forward and reverse), dNTPs, PCR buffer, Taq polymerase, MgCl2, and ultrapure water.

Primers in PCR

• Forward and reverse primers are short DNA sequences.
• They are complementary to regions in the template DNA and define the region to be amplified.
• In GFP PCR, primers are designed to bind to the GFP gene to amplify only it.

Taq Polymerase

• Thermostable DNA polymerase from Thermus aquaticus.
• It withstands high temperatures needed in PCR.

PCR Thermal Conditions

• Initial Denaturation: High temperature (94-96°C) separates DNA strands.
• Amplification (cycles):
  • Denaturation: High temperature separates DNA strands.
  • Annealing: Lower temperature allows primers to bind to DNA.
  • Extension: Taq polymerase extends the primers.
• Final Extension: Ensures all fragments are extended.
• Hold: Low temperature to prevent further change.

DNA Replication vs PCR

• DNA replication is in-vivo (inside living cells); PCR is in-vitro (in a test tube).
• PCR has lower fidelity than DNA replication

Preparing the Master Mix

• Calculate volumes using C1V1 = C2V2.
• Include ultrapure water. Ensure complete coverage for each of components
• Check calculation.
• Prepare proper negative controls.

Thermal Profile or Cycling Conditions

• Initial Denaturation: 94°C for 5 minutes.
• 30 Cycles of:
  • Denaturation: 94°C for 1 minute.
  • Annealing: 50°C for 1 minute.
  • Extension: 72°C for 1 minute.
• Final Extension: 72°C for 5 minutes.
• Hold: 4-10°C (keep samples at a low, constant temperature).
• Proper materials and containers: 1.5mL microcentrifuge

Adding the Template to PCR Tube

• Use a sterile applicator stick to gently touch a colony and dissolve it into the PCR reaction mixture. Avoid putting the entire colony.
• Spin down.
• Place PCR tubes into a thermocycler/PCR machine.
• Run the protocol.
• Allow the process to finish.