D1.1 DNA replication

What is DNA replication?

The production of new strands of DNA with base sequences identical to the existing strands

What is DNA replication required for ?

Reproduction - parents must replicate their DNA to reproduce
Growth and tissue replacement - Cell division is needed to replace lost tissues and in order for this to happen DNA needs to be copied

Define semi-conservative replication

Replication where the two nucleotide strands of DNA separate, each serving as a template for the synthesis of a new strand. Both DNA molecules will contain one new and one original strand

Explain the role of complementary base pairs

It ensures that the 2 resulting DNA molecules are identical in their base sequences to the parent molecules

Why must DNA strands be separated prior to replication ?

So that each new DNA molecule has a template strand from the original molecule

What are replisomes ?

functional subunits involved in DNA replication

What is helicase?

A ring shaped protein that unzips the two DNA strands so they can act as a template

How does helicase unwind DNA?

breaks hydrogen bonds between the bases pulling one base through its hole while the other passes to the side of it

What does DNA polymerase do?

Assembles new strands of DNA using the original strands

It moves along the template strands and adds one nucleotide at a time by creating a covalent bond between the sugar of the strand and the phosphate group of the nucleotide once hydrogen bonds have formed between base pairs

What do replisomes contain?

separate DNA polymerase for each strand

What is PCR?

polymerase chain reaction

- Automated method of DNA replication

How does PCR work ?

It follows a cycle of steps repeatedly doubling the amount of DNA each time. The steps in a PCR cycle are triggered by changes in temperature so a PCR is known as a thermal cycler

What is DNA amplification?

The process of producing large quantities of DNA from a specific base sequence

What is a primer ?

A short segment of DNA that binds to DNA at the point where the selected base sequence for replication begins

What do primers do ?

If added at the start of cycling they select which sequences are copied

What is the optimum length of DNA for amplification ?

100 base pairs

What is needed for PCR ?

Taq DNA polymerase and DNA nucleotides which each of the four bases

Where did Taq DNA polymerase evolve from ?

evolved in bacterium Thermus aquaticus which lives in hot springs

What is the first stage of PCR ?

Melting
Heating to 95 degrees for 30 -60 seconds which breaks the hydrogen bonds that holds DNA strands together. It does not break the covalent bonds within the individual strands of DNA. Taq Polymerase is not denatured by these temperatures.

What's the second stage of PCR ?

Annealing:
Cooling to 54 degrees for 30-60 seconds allows primers to bind. Different primers are needed for the 2 separate strands. Large excess amounts of primer is used to ensure rapid binding and prevent the strands joining again

What is the 3rd stage of PCR ?

Elongation:
Heating to 72 degrees provides the optimum working temperature for Taq DNA Polymerase which binds to the single strand of DNA adjacent to the primer. In 30-60 seconds it assembles new DNA strands.

What can thermal cyclers amplify ?

multiple samples simultaneously in array of wells

What can gel electrophoresis be used for?

Separate DNA molecules by length

What is the layout of gel electrophoresis ?

In the gel close to the end are holes called wells. The gel is placed in a shallow tank with electrodes at both ends. An electrolyte solution is poured over the gel to cover it. DNA samples are pipetted into each well

What is applied across the electrodes ?

A voltage is applied across the electrodes to create an electric field which allows charged molecules to move through the gel.

Outline the process of Electrophoresis

After voltage is applied the charged molecule moves through the gel. The gel is orientated in a way that the wells are close to the negative cathode. The gel consists of a mesh of filaments that resists the movement of molecules.

Why do DNA fragments separate during electrophoresis?

Small molecules move faster than larger ones in a given amount of time so electrophoresis can be used to separate DNA fragments according to their length.

What happens once enough time has elapsed in electrophoresis ?

ONce enough time has elapsed for the smallest DNA molecules to be close to the anode the voltage is switched off and gel is removed from the tank. It's treated with a dye to make DNA visible.

What movement does DNA undergo in electrophoresis ?

It moves from the wells towards the anode in parallel lines. DNA molecules of the same length will have moved the same distance so they form a visible band

How can you tell how many lengths of DNA were in the sample ?

The number of bands indicates how many different lengths of DNA were in the sample.

What is a ladder in electrophoresis ?

The right hand lane

A mixture of fragments ( of a known length ) are placed in the right hand well. This is then used to estimate the lengths of bands in other lanes on the gel.

What is PCR and gel electrophoresis used for ?

Paternity tests and testing for viral infections ( coronavirus )

What is the first 2 stages of using PCR to test for viral infections ?

A swab is taken of the nose and throat and rinsed in a saline solution to produce a liquid sample.
RNA in the sample is converted to DNA using enzyme reverse transcriptase

What are the last 2 stages of using PCR to test for viral infections ?

PCR is used to amplify specific base sequences that are markers for the virus being tested for.
As PCR progresses fluorescent markers are attached to any DNA produced. Fluorescent levels are monitored and if it rises above a target level the test is positive.

Advantages of PCR testing

- It is very sensitive ( One molecule of RNA is amplified to produce billions of molecules of DNA ) so tiny amounts are detected

Disadvantages of PCR testing

- It requires materials and equipment that are expensive and are usually only available in certain laboratories
- Results may not be immediately available due to time taken for thermal cycling

What are short tandem repeats ?

Sequences of between 2 - 7 bases that repeat

What are the stages required to produce a DNA profile ?

A sample of DNA is produced.
Selected tandem repeats are copied by PCR ( minimum of 13 some use more )
DNA produced is separated using gel electrophoresis based on fragment length and number of repeats
Patterns of bands on electrophoresis are unique to individuals and is their DNA profile.

How are paternity tests analysed ?

DNA profiles of the mother, father and child are made. If any bands from the child's profile do not occur in the father/mothers profile another person has to be the father

Why are paternity tests carried out ?

- Men sometimes claim that they are not the father of a child to avoid paying costs
- Women may have had multiple partners
- A child may wish to prove a deceased man is their father

What do terminal nucleotides have ?

a sugar or phosphate group that has not formed covalent bonds with another nucleotide

What is the 3' end of DNA polymerase ?

The 3rd carbon of the sugar is available for linkage

What is the 5' end of DNA polymerase ?

phosphate group of terminal nucleotide is available ( links to 5th carbon atom of sugar )

What joins nucleotides together?

covalent bonds between sugars and phosphate

What does DNA polymerase do ?

It adds free nucleotides to existing DNA strands. It cannot initiate the process of assembling complementary bases to the template strand

What direction does DNA polymerase work ?

5' phosphate of the free nucleotide to the 3' deoxyribose of the elongating strand

Compare the pace and direction of replication on the leading and lagging strands

Leading: continuous therefore quicker and completed in a 5' - 3' direction
Lagging: Completed in fragments as more of the molecule is unzipped which makes the process slow ( still in 5' - 3' but antiparallel )

Why is replication different on the leading and lagging strands ?

The leading strand is a continuous formation of a DNA molecule as the template gets unzipped by helicase but the lagging strand gets copied in fragments as it gets unzipped.
This is because both template strands are antiparallel meaning that one goes in the 5' - 3' direction whilst the other goes 3' - 5'. Replication always occurs 5' - 3'.

Outline the formation of Okazaki fragments on the lagging strand

DNA polymerase adds nucleotides moving away from the replication fork in a series of lengths as more of the template strand is exposed.

Explain the need for RNA primers in replication

RNA primers initiate the creation of a new strand joining around 10 nucleotides

Compare the number of RNA primers on the leading and lagging strands

On the leading strand it is only needed once but on the lagging strand it is needed every time a new fragment is started

What is the function of DNA primase ?

DNA primase generates RNA primer on each of the template strands

What is the function of DNA polymerase III ?

It binds to the template strand on the 3' side of RNA primer and assembles a chain of nucleotides with complementary bases to the template strand. It adds nucleotides in a 5' - 3' direction until the end on template strand or reaches RNA primer.

- Also proofreads each nucleotide after adding

What is the function of DNA Polymerase I ?

Lagging strand: To remove the RNA primers and replace them with DNA detaches once it reaches the next Okazaki fragment and leaves a gap where the covalent bond is missing.

What's an exonuclease?

Enzyme that can break bonds between nucleotides and a polymerase

What is the function of DNA proofreading ?

Corrects errors made in order to prevent mutations

Outline the process of DNA proofreading by DNA polymerase III

When an incorrect base has been recognised it excises the incorrect nucleotide moves back along the template strand and inserts the correct one.

What's the function of DNA ligase ?

covalently bonds gaps between Okazaki fragments to form a continuous strand

Why is there gaps between adjacent Okazaki fragments on the lagging strand ?

Because DNA polymerase cannot make 3' to 5' links between nucleotides