Read the following paragraph and answer the given questions:
In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites for the commonly used restriction enzymes. Presence of more than one recognition sites within the vector will generate several fragments, which will complicate the gene cloning. The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes. The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA but can still be selected out from non-recombinant ones by plating the transformants on tetracycline containing medium. The recombinants will grow in ampicillin containing medium but not on that containing tetracycline. But, non- recombinants will grow on the medium containing both the antibiotics. In this case, one antibiotic resistance gene helps in selecting the transformants, whereas the other antibiotic resistance gene gets inactivated due to insertion of alien DNA, and helps in selection of recombinants. Origin of replication (ori) is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA. So, if one wants to recover many copies of the target DNA it should be cloned in a vector whose origin support has high copy number.
Which one of the following is not correct statements about cloning sites of vectors?
Answer & explanation
Correct answer: option 1
The correct answer is Option (1) → Presence of more than one restriction site
Note: The answer marked is Option (1). However, the question can have more than one correct answer, i.e. both Option (1) and Option (2), as explained below. A restriction enzyme cuts DNA at the very sequence it recognises. So for one enzyme, its recognition site and its restriction site are the same place, and the two options say the same thing. The passage is taken from NCERT. NTA has put lines of the NCERT text together to make it.
A cloning site is the place where a vector is cut so that alien DNA can be joined into it. The vector should be cut at one place only by the chosen enzyme. Then it opens into one linear piece, and the alien DNA joins into that single gap.
Option (1) Presence of more than one restriction site — Not a correct statement. If the chosen enzyme can cut the vector at more than one place, the vector breaks into several fragments. The pieces may join back in any order, and gene cloning gets complicated.
Option (2) Presence of more than one recognition site in the vector — Also not a correct statement. This is the passage’s own line. More than one recognition site generates several fragments, so a vector should have very few, preferably single, recognition sites for the commonly used enzymes.
Option (3) The ligation of alien DNA is carried out at a restriction site — A correct statement. The alien DNA is joined (ligated) at a restriction site present in one of the two antibiotic resistance genes. In pBR322, for example, it can be joined at the BamHI site in the tetracycline resistance gene.
Option (4) The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA — A correct statement. When the alien DNA sits inside the tetracycline resistance gene, that gene stops working. Recombinants then grow on ampicillin but not on tetracycline, which is how they are picked out.