D1.1 DNA Replication

Polymerase Chain Reaction (PCR)

PCR is a gene technology that allows us to make billions of copies of DNA molecules so that we can see them. 

95^O is used to denature the DNA strand and break the hydrogen bonds.

Cooled to below 60^O to allow the RNA primers to bind to the template strand. 

Heated above 70^O so that Taq Polymerase (which has not denatured) adds the complementary nucleotides to the new strand rapidly. 

Gel Electrophoresis

Gel electrophoresis is a technique used to separate DNA fragments according to their size by passing an electric current through a gel.

1. DNA is prepared: DNA fragments may be produced using restriction enzymes or PCR.

2. DNA is loaded into wells: Samples are placed into small wells in an agarose gel.

3. Electric current is applied: DNA is negatively charged due to its phosphate groups, so it moves towards the positive electrode (anode).

4. DNA fragments separate: Smaller fragments move faster and travel further through the gel because they experience less resistance.

5. Bands are visualised: DNA is stained with a fluorescent dye, allowing bands to be seen under suitable light.

6. Results are compared: The positions of the bands are compared with a DNA ladder containing fragments of known sizes.

DNA Replication

When Helicase binds to a strand of DNA it starts to break the hydrogen bonds in a 3’ to 5’ direction on the lagging strand. 

The replication fork is followed by polymerase III which adds nucleotides to the primers. Primers are sections of RNA that acts as a starting point for free nucleotides to be added. 

So the new nucleotides are added continuously in a 5’ to 3’ direction.  

This strand is known as the leading strand. 

However, due to the antiparallel nature of DNA the complementary strand is running the wrong way! 

This is called the lagging strand.

Lagging Strand

On the lagging strand the nucleotides can only be added in a 5’ to 3’ direction but the template strand is also running 5’ to 3’. 

So multiple primers are added and short sections of nucleotides fill in between in a 5’ to 3’ direction.

These discontinuous fragments are known  as Okazaki Fragments. Once a fragment is complete the primers are replaced with nucleotides by Polymerase I. 

Ligase then joins all the okazaki fragments by forming covalent bonds where the primers used to be to the previous fragment

DNA Proofreading

In addition to adding free nucleotides to the 3’ terminus of a newly created strand Polymerase III can simultaneously check for errors. This is known as DNA proofreading.

If the newly added base is mismatched it is simply removed by polymerase III and replaced with the correct one. 

Enzymes in DNA replication

Helicase - Breaks the Hydrogen Bonds between the complementary bases in the original template DNA in a 3’ to 5’ direction. 

Primase - Adds RNA Primers to allow Polymerase III to start adding nucleotides. 

Polymerase III - adds nucleotides in a 5’ to 3’ direction and removes any mismatched 3’ terminal errors. 

Polymerase I - removes the RNA Primers and fills in the gaps with nucleotides. 

Ligase - forms covalent bonds in the DNA backbones after complementary base pairing.