1. www.le.ac.uk Making Recombinant DNA DNA structure and Plasmids DNA Restriction and Ligation www.le.ac.uk/genie

2. DNA structure recap A T C G Double-stranded helix with four “building blocks”: –Adenine (A), Thymine (T), Cytosine (C) and Guanine (G) G and C pair together and A and T pair together The same building blocks are used in all forms of life, so…...CLONING IS POSSIBLE!!

3. Cloning Vectors: Plasmids Circular, double-stranded DNA molecules. Taken in by bacteria, which make copies of them as they copy themselves. Have specific sites on their DNA where foreign DNA or a gene can be inserted

4. Restriction (cutting the plasmid) –Restriction Endonucleases Some terminology: DNA insert Plasmid Cut site Ligation (insertion of DNA insert and rejoining the pieces) –Ligase enzyme

5. Enzymes that recognise and cut at specific sequences of DNA –Exist in vivo in bacteria for foreign DNA recognition and destruction –4,5,6 and 8 base recognition Different enzymes recognise different sequences: Restriction Enzymes

6. Enzymes that recognise and cut at specific sequences of DNA –Exist in vivo in bacteria for foreign DNA recognition and destruction –4,5,6 and 8 base recognition 5'--G A A T T C--3'5’- -G A A T T C- -3’ 3'--C T T A A G --5' 3’- - C T T A A G- -5’ Restriction Enzymes 5'--G A T C--3'5’- -G A T C- -3’ 3'--C T A G--5' 3’- -C T A G- -5’

7. Cutting - Restriction 5’…nnnnG↓AATT-Cnnnn…3’5’…nnnnG↓AATT-Cnnnn…3’ 3’…nnnnC-TTAA↑Gnnnn…5’3’…nnnnC-TTAA↑Gnnnn…5’ 5’…nnnnG-3’5’-AATTCnnnn…3’ 3’…nnnnCTTAA-5’ 3’-Gnnnn…5’ Restriction Restrict EcoRI sites 5’ overhanging “sticky-ends” Plasmid DNA DNA insert

8. pUC19 2686 bp Restriction enzyme sites on a plasmid (pUC19 2686 bp) Unique restriction enzyme sites

9. Joining -Ligation 5’…nnnnG-3’5’-AATTCnnnn…3’ 3’…nnnnCTTAA-5’ 3’-Gnnnn…5’ 5’…nnnnGAATTCnnnn…3’ 3’…nnnnCTTAAGnnnn…5’ Ligation Joining “sticky ends”: mix & ligate Plasmid DNA DNA insert

10. Basic Cloning I Foreign DNA to be inserted join/ligate Antibiotic resistance gene Plasmid vector Recombinant DNA molecule AB R

11. Cloning vectors: essential features Maintenance (replication) in host cell Restriction enzyme sites –insertion of foreign DNA fragment –unique –create RECOMBINANT DNA molecules Method to introduce into host cell –transformation Identify recombinants

12. pUC19- a classic cloning vector Selection Antibiotic resistance gene Maintenance: Origin of replication Cloning: Restriction sites Blue/ white selection for presence of insert

13. Restriction enzyme mapping Plasmid one site / one cut Full length Plasmid Y X Z A + B = Full length B Plasmid two sites / two cuts A A B XZ + Y + YZ + X + ZX + Y +

14. small large M A B X Y Z Y+Z X+Z X+Y Restriction enzyme mapping