D1.1 DNA Replication

Semi-conservative replication

Keeps half of the original DNA, and half is newly synthesized DNA

What is the role of Helicase?

Enzyme that disrupts hydrogen bonds between complementary bases to separate the strands of the DNA molecule.

What is the role of DNA Polymerase?

Synthesizes complimentary daughter strands for each parent strand - adding 1 nucleotide at a time

What direction does DNA Polymerase move?

5’ to 3’

What is the leading strand?

The strand of DNA which DNA polymerase builds towards the replication fork continuously

What is the lagging strand?

The strand of DNA which DNA polymerase builds away from the replication fork, in segments

What are Okazaki fragments?

Short segments of DNA synthesized from the lagging strand

DNA Primase

Creates a primer (short chain of nucleotides) on the template strand and provides the site where DNA polymerase can bind to and start adding nucleotides.

DNA polymerase III

Binds to template strand on the primer and adds nucleotides to build daughter strand + proofreads after each nucleotide has been added to the chain

DNA Polymerase I

Replaces RNA nucleotides with DNA nucleotides on the lagging strand, proofreads newly synthesized DNA

DNA Ligase

Joins Okazaki fragments together

PCR

Polymerase Chain Reaction

3 stages of PCR

Melting, Annealing, Elongation

PCR Melting stage

Heating the DNA (to 95ºC) until hydrogen bonds break

What enzyme is used in PCR and why?

Taq polymerase is used because it is resistant to high temperatures (will not denature)

PCR Annealing stage

Cooling to 54ºC, allow primers to bind (prevents DNA from pairing up again)

PCR Elongation Stage

Heat up to 72ºC (optimum temperature for taq polymerase), taq polymerase replicates the DNA

Purpose of Gel Electrophoresis

To separate and analyze mixtures of DNA of different lengths

Gel Electrophoresis Procedure

• Use of a tank with voltage (positive and negative on one side)

• DNA is placed into wells near the negative side

• DNA moves towards positive side (bc of slight negative charge from phosphate)

• Small fragments travel further, bigger fragments are slower