Question

question 73
1.5 pts
assume that you are trying to insert a gene into a plasmid. someone gives you a preparation of genomic dna that has been cut with restriction enzyme x. the gene you wish to insert has sites on both ends for cutting by restriction enzyme y. you have a plasmid with a single site for y but not for x. your strategy should be to...

• insert the fragments cut with restriction enzyme x directly into the plasmid without cutting the plasmid.
• cut the plasmid with restriction enzyme x, and insert the fragments cut with restriction enzyme y into the plasmid.
• cut the dna again with restriction enzyme y, and insert these fragments into the plasmid cut with the same enzyme.
• cut the plasmid twice with restriction enzyme y, and ligate the two fragments onto the ends of the dna fragments cut with restriction enzyme x.

Explanation:

To insert a gene into a plasmid, the gene and plasmid need compatible ends (created by the same restriction enzyme for ligation). The genomic DNA is cut with X, the gene has Y sites, and the plasmid has a Y site. So we need to cut the genomic DNA (with the gene) with Y to create Y - compatible ends, then insert into the plasmid cut with Y (since same enzyme - cut ends can ligate). Let's analyze each option:

• First option: Inserting X - cut fragments into uncut plasmid won't work as plasmid has no X site and ends won't match.
• Second option: Cutting plasmid with X (it has no X site) is impossible, so this is wrong.
• Third option: Cut the DNA (with the gene) again with Y (to create Y - cut ends) and insert into plasmid cut with Y (which has a Y site). This works as same - enzyme - cut ends can ligate.
• Fourth option: Cutting plasmid twice with Y and ligating to X - cut DNA is incorrect as ends won't match (X - cut and Y - cut ends are different, and this approach is not a standard cloning strategy).

Answer:

C. cut the DNA again with restriction enzyme Y, and insert these fragments into the plasmid cut with the same enzyme.