Question
Explain pBR322 with its cloning sites, enzymes with well labeled diagram.###Explain: cloning sites. (Diagram is not necessary)
✓
Answer
Cloning sites :
→ 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 with in 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.
→ For example, you can ligate a foreign DNA at the BamHI 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 tetracycline 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.
→ 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 with in 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.
→ For example, you can ligate a foreign DNA at the BamHI 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 tetracycline 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.
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