1.6 and 1.7- DNA- Biotechnology :(

Question 1

What is the role of heating and cooling in PCR?

Answer 1

To separate strands and synthesise DNA.

Question 2

What does PCR stand for?

Answer 2

Polymerase Chain Reaction

Question 3

What is the role of primers in PCR?

Answer 3

Primers are Short single-stranded DNA sequences complementary to the DNA being elongated. Primers indicate start and end.

Question 4

What is the role of free nucleotides in PCR?

Answer 4

Free nucleotides bind complementarily to the exposed bases, serving as the building block of the new DNA.

Question 5

Describe the role of heat resistant enzymes in PCR testing

Answer 5

Heat-resistant DNA polymerase made from Yellowstone, is used so that they do not completely denature during Denaturation and can be used in elongation.

Question 6

Describe the process of PCR (6 marks)

Answer 6

1. (denaturation) DNA is heated to roughly 95 degrees to separate strands
2. (Annealing) Primers are added and DNA is cooled to roughly 40 degrees to allow two primers to bind to each strand
3. Primers are short single-stranded DNA sequences complementary to DNA. Primers indicate start and end.
4. (elongation) DNA is heated to roughly 72 degrees, the optimum temperature for heat-resistant DNA polymerase.
5. Free nucleotides bind complementarily to the exposed bases, serving as the building block of the new DNA.
6. Heat resistant DNA polymerase is used so that they do not completely denature during Denaturation and can be used in elongation.

Question 7

How does electrophoresis determine the base sequence of DNA?

Answer 7

Electrophoresis compares the length of DNA segments. Can be used to determine the order of the nucleotide bases in a gene.

Question 8

Describe Electrophoresis

Answer 8

1. DNA fragments placed into wells on agarose gel.
2. An electric current applied to the gel, from negative to positive.
3. DNA fragments move towards positive end because DNA is negatively charged. Smaller fragments move furthest due to less friction/fit through pores.
4. Before the fragments of DNA reach the end of the gel, the electric current is turned off.

Question 9

What is an electropherogram?

Answer 9

A plot of DNA sequences from electrophoresis

Question 10

How do you interpret electropherograms that illustrate DNA sequences?

Answer 10

1. PCR used to multiply DNA.
2. Copies of DNA divided into four batches and all four normal nucleotides are added to each batch.
3. One modified nucleotide is added into each batch. Said nucleotides stop replication when inserted.
4. Copies of primers and DNA polymerase used in all four batches. At some point during DNA replication, DNA polymerase inserts a modified nucleotide, and the chain terminates.
5. Process repeated until there is a chain of every length with a modified nucleotide at the end.
6. Mixture from all four batches is loaded into four gel wells and undergoes electrophoresis. Process done so DNA segments only differ by 1 nucleotide. Modified nucleotides have different colours under UV light, so the sequence can be read.

Question 11

How is CRISPR-Cas9 used to edit and transfer genes?

Answer 11

1. gRNA, complementary to target gene, guides Cas9 to correct DNA location.
2. Cas9 cuts the DNA strands.
3. Cas9 either removes, edits, or inserts DNA.
4. Cell recognises DNA is damaged and repairs it.
5. Cell now expresses the altered gene and passes it on to daughter cells.