Question

• If you wanted to insert a gene into a plasmid, why would it be helpful to know the plasmid’s restriction enzymes cleavage sites?

Answer 1

To insert a gene into a plasmid , the plasmid has to cleaved by a restriction enzyme. Plasmids are circular , extrachromosomsal , double stranded ,self replicating sequences. These circular DNA should be opened to insert the foreign DNA. To open it , it has to be cut by a restriction enzyme.

Restriction enzymes cleaves any DNA on specific sites called restriction sites or recognition sequence. These are special type of sequence which will have four to eight bases arranged in a palindrome. A palindrome is a sequence arranged in the same order when read from 5' - 3' direction on both the strands. Eg 5' GAATTC 3'

3' CTTAAG 5'

DNA strands containing such sequences when cut with restriction will result in short single stranded overhangings as the enzyme cuts between the same bases on both the strands.

5' G +. AATTC3'

3'CTTAA +. G 5'

If the desired gene is also cut using the same enzyme , the overhangings will be the same and complementary to each other. Being complementary , the gene of interest and plasmid will form hydrogen bonds and thus the gene gets inserted into the plasmid.

Each restriction enzyme cleaves on a specific restriction site. The restriction site or recognition sequence identified and cleaved by one enzyme cannot be identified and cleaved by another enzyme. For eg. The recognition sequence given above is the sequence recognised and cleaved by a restriction enzyme named EcoRI , which cannot be cleaved by any other restriction enzyme.

So , if we know the cleavage site contained in a plasmid we can decide which enzyme is to be used for the cleavage.

The same enzyme can be used to cleave the gene of interest and the plasmid for the success of inserting the gene of interest into the plasmid.