DNA manipulation

Question 1

protospacer

Answer 1

short sequence of DNA extracted by Cas1 and 2 from a bacteriophage

Question 2

Outline the steps of CRISPR-Cas9

Answer 2

1. Bacteriophage injects DNA, becomes a spacer in CRISPR gene
2. CRISPR spacer is transcribed with half a palindrome (repeat DNA) from either side of it, making gRNA
3. gRNA binds to Cas9, forming CRISPR-Cas9 complex
4. complex scans for DNA complementary to its gRNA and cuts it at PAM site

Question 3

Structure of gRNA

Answer 3

crRNA- spacers and repeats that are transcribed
tracrRNA- complimentary sequence to crRNA, allows it to bond to it. It also binds to cas9

Question 4

What happens to DNA after it’s been cut by CRIPSR-Cas9?

Answer 4

Cell repair DNA where additional nucleotides could be added, making the virus useless

Question 5

Describe the steps of PCR. Include temperatures and name of stage

Answer 5

Denaturation- Heated to 90-95 C, forms single stranded DNA
Annealing- 50-55 C. Primers bind to complimentary sequence at 5’
Elongation- 72 C. Taq polymerase binds to primer and synthesises new DNA toward 3’

Question 6

Anode and cathode

Answer 6

Anode- Positively charged
Cathode- Negatively charged

Question 7

Which end is DNA attracted to in gel electrophoresis and why?

Answer 7

Positive end because DNA is negatively charged

Question 8

Factors that influence gel electrophoresis

Answer 8

Voltage- Stronger=more DNA attraction to anode
gel composition- higher density= difficult for DNA to move
buffer concentration- more= more electric current is conducted
Time

Question 9

Plasmid

Answer 9

Circular loop of DNA in bacteria

Question 10

vector

Answer 10

A means of introducing foreign DNA, e.g. plasmids