Which of the following is the best procedure in order to position a gene in an expression vector downstream from a promoter (i.e.,perform directional cloning)?
A)Use a vector with 2 sites for the same restriction endonuclease.
B)Treat the gene with DNase I.
C)Restrict both the gene and the plasmid with 1 restriction endonuclease,and then screen all colonies for those that express the gene.
D)Restrict each end of the DNA fragment (gene)with a different restriction endonuclease,and do likewise for the plasmid.

Question

Quizplus · Accepted Answer

Restricting each end of the DNA fragment (gene) with a different restriction endonuclease and doing the same for the plasmid allows for directional cloning. This creates compatible overhangs that can be ligated together in only one orientation, ensuring that the gene is inserted downstream from the promoter in the correct orientation for expression. Option A does not guarantee directional insertion. Option B is not a relevant step for cloning. Option C can result in false-positives if colonies express the gene due to promoter activity from the vector rather than the inserted gene.

Which of the Following Is the Best Procedure in Order