Question

QUESTION 11 a) The diagram below shows a typical plasmid cloning vector. There are three components ORI, amp and restriction enzyme recognition sizes. Explain the roles of each of these three components and explain why each is important for cloning genes. 6 marks] ORI Hindlll Sphl Pstl Sall Restriction Sites Xbal BamHI Smal Kpnl Sac EcoRI amp ORI role: importance: amp role: importance: Restriction sites role: importance: b) A technique called RT-PCR uses the enzyme reverse transcriptase (RT) in combination with PCR and gel electrophoresis to study expressed genes. Draw a simple flow diagram to illustrate the steps taken, from the point of obtaining mRNA from the samples until the gel electrophoresis. You should use the terms: PCR, CDNA, RT, PCR products, gel electrophoresis, mRNA. [4 marks]

Answer 1

ORI

Role: It is the specific site where replication starts.

Imp: It allows the vector and the foreign DNA to replicate in the host cell

amp^r

Role: It codes for the enzyme b-lactamase

Imp: The b-lactamase catalyzes the hydrolysis of the b-lactam ring of the ampicillin thus destroying the antibiotic providing selective growth of the cell

Restriction site

Role: Here restriction enzyme cuts the DNA

Imp: It helps in the formation of sticky ends where the foreign DNA will be attached

Flow chart for RT- PCR

Steps of RT-PCR Cell kem RNA olation RNA CDNA by Reverse trarnscirphon (RT) PCRampligicdiorn d c DNA Gel electrophoreis af PCR produets