DNA polymerases and PCR

Question 1

What are the two characteristics of DNA replication.

Answer 1

• DNA Replication is bidirectional: two replication forks

- DNA replication is semidiscontinuous: continuous in the leading strand, discontinuous in the lagging strand.

Question 2

What are the requirements for DNA polymerase activity? State the function of each material.

Answer 2

• Template strand: Basis for heredity
• dNTP mix: contain nitrogenous bases, building blocks for DNA synthesis
• Mg2+: Promotes reaction
• Primer: Initiates DNA polymerase elongation.

Question 3

Which type of DNA polymerase possesses 5’ to 3’ exonuclease activity? What is nick translation?

Answer 3

DNA pol I, Nick translation is the process where DNA polymerase I moves the nick on the non-template strand along the DNA.

Question 4

What are the components of a PCR reaction?

Answer 4

• Template DNA
• Primers
• Thermostable polymerases
• dNTPs
• Thermocycler
• Buffer containing Mg2+

Question 5

Why does taq polymerase have activity at such high temperatures?

Answer 5

Taq polymerase is isolated from the bacteria Thermus aquaticus, which is heat resistant and found on hot springs. The enzymes that this bacteria are thermostable to ensure the survival of the bacteria at high temps.

Question 6

Describe the 3 steps of PCR amplification.

Answer 6

• Denaturation: The DNA are subjected to high temperatures, which breaks the hydrogen bonds and denatures the DNA into single strands. (94oC)
• Hybridization/Annealing: Thermocycler reduces the temperature to the annealing temperature, the primer binds to the DNA strand.
• Extension: Thermocycler raises the temperature again to the near-optimum temperature of taq polymerase so that it can begin the synthesis of DNA. (74oC)

Question 7

What is the main drawback of using taq polymerase?

Answer 7

Taq polymerase lacks 3’-5’ proofreading activity. Use Pfu DNA polymerase instead of, or with taq polymerase. Pfu DNA polymerase have 3’-5’ proofreading activity.

Question 8

What are the steps involved in the entire gene cloning process?

Answer 8

Cell lysis and isolation of plasmid /// Purification of isolated plasmid /// PCR /// Gel electrophoresis /// Gel purification //// Introduce cut sites by adding adapter /// Cut by using restriction enzymes /// Insert gene into vector /// Transformation into host cells /// Screening.