Cloning Vectors
Read the following passage carefully 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. The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes. For example, you can ligate a foreign DNA at the BamH I site of tetracycline resistance gene in the vector pBR322. 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 ampicillin containing medium. The transformants growing on ampicillin containing medium are then transferred on a medium containing tetracycline. 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. Therefore, alternative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate.
What will be the consequences, if we use more than one recognition site within the vector?
Answer & explanation
Correct answer: option 1
The correct answer is Option (1) → Generate several fragments that will complicate the gene cloning
A recognition site is the short DNA sequence that a restriction enzyme recognises and cuts. A cloning vector needs very few of these sites, and preferably only one. The alien DNA is joined at that site.
(1) Generate several fragments that will complicate the gene cloning — Correct. If the vector has more than one recognition site, the enzyme cuts it at several places and several fragments are formed. These fragments have to be sorted out and joined back in the right way, so the cloning becomes complicated.
(2) Generate several fragments that will make gene cloning impossible — Incorrect. The first part is right, but the effect is overstated. The extra sites make the cloning complicated.
(3) Generate single DNA fragment which will make gene cloning easy — Incorrect. A single fragment comes from a single site, not from several sites.
(4) Generate single fragment that complicates DNA ligation — Incorrect. Several sites cannot give a single fragment.
Note: The passage is taken from NCERT. NTA has put lines of the NCERT text together to make it. Most CUET (UG) questions come straight from NCERT, so study the book line by line, including figures and their titles.
NCERT Extract:
Cloning Vectors – …
Figure 9.4 E. coli cloning vector pBR322 showing restriction sites (Hind III, EcoR I, BamH I, Sal I, Pvu II, Pst I, Cla I), ori and antibiotic resistance genes (ampR and tetR). rop codes for the proteins involved in the replication of the plasmid