BIOTECHNOLOGY : LIGATION TECHNIQUES SMG

A very brief account of ligation techniques - sticky end ligation and blunt end ligation

1. BIOTECHNOLOGY : LIGATION TECHNIQUES Dr. Saji Mariam George Associate Professor Assumption College Autonomous Changanacherry

2. LIGATION TECHNIQUES STICKY END (COHESIVE END) LIGATION Cutting by certain restriction enzymes →sticky (Protruding) ends – facilitate ligation reaction – Sticky ends can base pair with each other by hydrogen bonding. When fragments generated by the same restriction enzyme from different DNA are mixed → sticky end ligation → rDNA (recombinant DNA).

3. Cut foreign DNA and plasmid by same restriction enzymes - sticky ends Mix DNA fragments and plasmid Some of the foreign DNA fragments will pair with the cut ends of the plasmid. DNA ligase seals the cut ends by phosphodiester bond - recombinant plasmid that contain foreign DNA fragment.

4. STICKY END (COHESIVE END) LIGATION

5. 2. BLUNT END LIGATION a. Ligation by using synthetic oligonucleotide linkers Linkers – small , double stranded synthetic DNA fragments that contain one or more restriction sites. Foreign DNA fragment is cut by any restriction enzyme. Linkers are attached to the blunt ends by T4 ligase Sticky ends are then produced by using suitable restriction enzymes. Mix the cleaved vector and the foreign fragment → rDNA by DNA ligase .

6. Cloning by Linkers Image: Pierce,B.2003

7. b. Addition of new sticky ends by using the enzyme terminal transferase . ( Homopolymer Tailing) Complementary sticky ends are created on blunt-end pieces of DNA The plasmid and the foreign DNA are first cut by any restriction enzyme. Terminal transferase adds any available nucleotides to the 3’ end of DNA in a template independent reaction. Mix Terminal transferase , deoxyadenosine triphosphate ( dATP ) and the plasmid →create poly(A) – single stranded tails, AAAA…., on the 3’ ends of the plasmid.

8. Mix Terminal transferase , deoxythymidine triphosphate ( dTTP ) and blunt – end foreign DNA fragments →create poly (T) single stranded tails , TTT….on their 3’ ends. Poly (A) tail of the plasmid would be complementary to the poly(T) tail of the foreign DNA → anneal the plasmid and foreign DNA together. DNA polymerase fill the missing nucleotides. DNA ligase will seal the nicks in the sugar – phosphate back bone by phosphodiester linkages.

9. Cloning by Tailing Image Pierce,B.2003

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