GENE CLONING

Ligation

 

- Ligase: Joining DNA molecules together.

                Catalyse formation of phosphodiester bond between 3’-OH and 5’-P end.

 

-  Three types of DNA ligase:

 

i) E.coli DNA ligase

    - require NAD+ and Mg2+ as cofactors and activators.

      - can ligate cohesive and blunt ends.

 

ii)  DNA ligase

     - ligate cohesive and blunt ends with higher efficiency.

     - require NAD+ and Mg2+ as cofactors and activators

     - activities is inhibited by Na+ and K+

 

iii) Thermostable DNA ligase

      - use  as cofactor

      - use in ligase amplication to detect mutation in mammalian DNA.

 

- Complementary sticky end ligation is more efficient than blunt end ligation.

 

- Efficiency of blunt end ligation can be increased by:

• Perform at high DNA concentration

• Blunt end are converted to sticky end by linkers and adaptors

 

- Linker:

• Synthetic blunt ended double strand DNA contain recognition sequence for   

             restriction enzyme.

• Ligated to blunt end of DNA fragment with  DNA ligase

• Digested with certain restriction enzyme to form sticky end.

 

- Adaptor:

• Synthetic DNA fragment

• Contain 1 blunt end for ligation; also have 1 performed cohesive end.

 

 

Video link: https://www.youtube.com/watch?v=TUWif0porA4

                   https://www.youtube.com/watch?v=MeMddsYl_TE