1.1 DNA Replication

How is DNA replication semi-conservative?

During each instance of DNA replication, the new DNA double helix is a hybrid consisting of one strand of the parent DNA and one strand of newly synthesised DNA

Adenine

Pairs with thymine

Cytosine

Pairs with guanine

What type of bonds exist between complementary base pairs?

Hydrogen bonds

Why is DNA replication important?

Needed for growth, replacement of old/damaged cells and tissues, and reproduction

What is the importance of keeping one original DNA strand?

It ensures that there is genetic continuity with a high degree of accuracy between generations of cells

Steps of DNA replication

1. Helicase unwinds the 2 strands by breaking the hydrogen bonds between the complementary base pairs, exposing the bases on both strands

2. Using the strands as templates, DNA polymerase attaches free nucleotides one by one to the exposed bases, hydrogen bonds are formed between complementary base pairs

3. Two identical molecules of DNA are created

Which direction does DNA polymerase work in?

DNA polymerase will always attach to the 3’ end of the original template strand and read the strand in a 3' to 5' direction, making the new DNA strand from 5’ to 3’

Gel electrophoresis

A technique used to separate DNA fragments or other macromolecules, such as RNA and proteins, based on their size and charge

Why is DNA negatively charged?

Because of the presence of phosphate groups in nucleotides

How does gel electrophoresis work?

DNA fragments, which are negatively charged, will move towards the positive electrode (anode). Tiny pores in the agarose gel result in smaller molecules moving faster and travelling longer distances, whereas larger molecules move slowly and travel shorter distances

Preparation for gel electrophoresis

The number of DNA molecules must be amplified using PCR. Then, restriction enzymes will be used to cut the DNA molecules into fragments

Steps of gel electrophoresis

1. Wells (small rectangular holes) are cut into one end of the agarose gel plate

2. The gel is submerged in a buffer solution which provides ions that carry a current through the gel and maintains the constant pH

3. DNA samples are placed into the wells using a micropipette

4. An electrical current is applied

5. The DNA sample separates into bands of different sized fragments

Steps of polymerase chain reaction (PCR)

1. Denaturation - The DNA is heated to 95°C which breaks the hydrogen bonds between the complementary base pairs

2. Annealing - The temperature is decreased to between 50 - 60°C so that primers can anneal to the ends of the single strands of DNA. The primers serve as starting points for Taq polymerase.

3. Elongation - The temperature is increased to 72°C, which is the optimum temperature for Taq polymerase. Taq polymerase copies the strand by adding nucleotides.

4. 2 identical DNA molecules are produced

Outcome of PCR

Each cycle of PCR doubles the amount of DNA

Applications of gel electrophoresis and PCR

DNA profiling in paternity tests, DNA profiling in forensic investigations

DNA nucleotide structure

DNA nucleotides have a phosphate bonded to the 5’ carbon of the deoxyribose sugar. When a new nucleotide is added, the 5’ phosphate group of the incoming DNA nucleotide bonds to the free 3’ -OH group on the growing strand

DNA strands

They are two antiparallel strands, one strand runs from 5' to 3', while the other strand runs from 3' to 5'

Leading strand

Runs 5' to 3' towards the replication fork and is made continuously

Lagging strand

Runs 5' to 3' away from the replication fork and is made discontinuously in Okazaki fragments

Okazaki fragments

Short sequences of DNA nucleotides which are synthesised discontinuously and later linked together by DNA ligase to form a continuous complementary DNA strand

DNA primase

Generates a short RNA primer, providing an initiation point for DNA polymerase III to add the new nucleotides

DNA polymerase III

Begins DNA replication next to the RNA primer, linking nucleotides to form a new strand

DNA polymerase I

Removes the RNA primers on the leading and lagging strands and replaces it with DNA

DNA ligase

Joins up the Okazaki fragments by catalysing the formation of sugar-phosphate bonds

When do mutations occur?

When there are errors in DNA replication, usually incorrectly paired nucleotides

Proof reading of DNA polymerase III

In prokaryotes, DNA polymerase III acts as a proofreader of the new strand of DNA. When it recognises an incorrect base pair, it moves backwards and replaces it with the correct base pair

Why is DNA synthesised in a 5’ to 3’ direction?

DNA polymerases can only catalyse phosphodiester bond formation (a condensation reaction) between the 3' of the sugar of the last nucleotide and the 5' phosphate group of the incoming nucleotide