Ligation Methods in rDNA technology–Part 2

Ligation Methods in rDNA technology (part 1)

4) Joining with Linkers:

“Linkers are short pieces of double stranded DNA containing a restriction site. It is used to join blunt ended DNA fragments.”

In this process the linkers are attached to the blunt ends of desired DNA with a restriction enzyme, which cuts the linker to produce sticky ends. The same enzyme is used to cut the plasmid. Then the desired DNA and plasmid DNA fragments are mixed. Recombinant DNA and the linker of desired DNA.

5) Joining with Adapters:

“Adaptor is a short DNA fragment containing one sticky end and another blunt end”

The adaptor is very similar to linkers, but it differs in having one sticky end. It is used to join blunt ended foreign DNA with plasmid DNA. The adaptors can be attached to the blunt ended foreign DNA fragment with the help of DNA ligase. As a result the blunt ended foreign DNA becomes sticky ended. It can be joined with the plasmid sticky ended DNA fragments in the normal method.

6) Alkaline phosphatase method:

By treating the liberalized plasmid vector DNA with alkaline phosphatase to remove 5’-terminal phosphate groups, both recircularization and plasmid dimmer formation are prevented. In this case, circularization of the vector can occur only by insertion of non-phosphatase treated foreign DNA which provides one 5’-terminal phosphate at each join. One nick at each join remains un-ligated but after transformation of host bacteria, cellular repair mechanism reconstitutes the intact duplex.