Adaptors: Joining Molecules, Blunt END DNA fragments cloning

Adaptors are short synthetic oligonucleotides with preformed cohesive end at one termini and blunt end at another termini.
Blunt end of the Adaptors poses 5’ PO3 and 3’OH like natural DNA participates in the formation of phosphodiester bond with the blunt ends of DNA molecule by DNA ligase
But Cohesive end has 5’OH & 3’OH which will not involved in the phosphodiester bond formation with blunt end DNA nor with another adaptor by a ligase
1. Adaptors with desirable restriction sites are selected
2. Such adaptors and blunt end DNA fragments/cDNA molecules are ligated using T4 DNA ligase
3. Adapters are ligated to both termini of the DNA fragments.
4. Then, Cohesive end “OH” of adaptor ligated DNA are phosphorylated with PNK
5. Cohesive ends containing Vector and DNA fragments are ligated by using T4 DNA ligase.
6. Finally, Ligation mixture is transformed to screen and select the recombinant clones

[@dsanthosh1450]

Super sir

[@Dr.DNA-Primer]

Thank you