DNA Replication Enzymes and Mechanisms

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Questions and Answers

What is the function of the enzyme helicase in DNA replication?

Marks the starting point for the construction of a new strand of DNA

Synthesizes new strands of DNA from 5' to 3'

Adds DNA nucleotides to fill in gaps between Okazaki fragments

Separates the two strands of DNA to form a replication fork (correct)

Why is the lagging strand synthesized discontinuously in DNA replication?

The lagging strand runs in the opposite direction of the leading strand, preventing continuous synthesis. (correct)

DNA polymerase can only add nucleotides in the 3' to 5' direction, which is opposite to the direction of the lagging strand.

Ligase is only able to join short fragments of DNA, leading to discontinuous synthesis on the lagging strand.

The lagging strand is made of RNA, which is not stable enough for continuous synthesis.

What is the role of a primer in DNA replication?

Provides a template for DNA polymerase to copy.

Provides a starting point for DNA polymerase to begin synthesis. (correct)

Seals the gaps between Okazaki fragments.

Unwinds the DNA double helix to create a replication fork.

Which of the following enzymes is responsible for removing RNA primers during DNA replication?

Exonuclease (A) Signup and view all the answers

What is the term used to describe the mechanism of DNA replication where each new DNA molecule contains one old strand and one new strand?

Semi-conservative (A) Signup and view all the answers

How is DNA polymerase involved in both DNA replication and the PCR process?

DNA polymerase is used in both processes to synthesize new strands of DNA. (B) Signup and view all the answers

What is the significance of the 3' and 5' ends of a DNA strand in relation to DNA replication?

DNA polymerase can only add nucleotides to the 3' end of the strand. (D) Signup and view all the answers

Why is the charge of DNA considered negative?

The presence of phosphate groups in the backbone. (A) Signup and view all the answers

What would be the consequence of removing the enzyme ligase from the DNA replication process?

The Okazaki fragments would not be joined together, resulting in a fragmented DNA molecule. (D) Signup and view all the answers

Which aspect of DNA structure enables the complementary pairing of bases?

The presence of hydrogen bonds between the bases. (B) Signup and view all the answers

During DNA replication, what is the primary function of DNA Polymerase III?

To add nucleotides to the newly synthesized DNA strand in the 5' to 3' direction. (A) Signup and view all the answers

What is the role of primers in PCR?

To initiate the synthesis of new DNA strands by providing a starting point for DNA Polymerase. (B) Signup and view all the answers

Which of the following is NOT a component of a PCR reaction mixture?

RNA template (C) Signup and view all the answers

What is the purpose of the annealing step in PCR?

To allow the primers to bind to the DNA template. (C) Signup and view all the answers

Which DNA replication model proposes that the product DNA is composed of one old strand and one new strand?

Semiconservative model (C) Signup and view all the answers

What is the main difference between DNA replication and PCR?

All of the above are correct. (D) Signup and view all the answers

What is the role of MgCl2 in a PCR reaction?

To act as a cofactor for Taq Polymerase, facilitating the incorporation of dNTPs. (B) Signup and view all the answers

Which of the following is a characteristic unique to Taq Polymerase?

It can withstand high temperatures. (C) Signup and view all the answers

What is the main purpose of the initial denaturation step in PCR?

To separate the DNA strands into single strands. (A) Signup and view all the answers

What is the correct order of steps in a typical PCR cycle?

Denaturation, Annealing, Extension (C) Signup and view all the answers

Signup and view all the answers

Study Notes

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.

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Description

Test your knowledge on the key enzymes and mechanisms involved in DNA replication. This quiz covers topics such as the roles of helicase, DNA polymerase, and the significance of RNA primers. Assess your understanding of the intricacies of this essential biological process.