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Making Recombinant DNA

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

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Making Recombinant DNA

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

  2. DNA structure recap • 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… C G A T ...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 • Ligation (insertion of DNA insert and rejoining the pieces) • Ligase enzyme Cut site Plasmid

  5. Restriction Enzymes • 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:

  6. Restriction Enzymes • 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’ 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 RestrictEcoRI sites DNA insert Plasmid DNA 5’…nnnnG↓AATT-Cnnnn…3’5’…nnnnG↓AATT-Cnnnn…3’ 3’…nnnnC-TTAA↑Gnnnn…5’3’…nnnnC-TTAA↑Gnnnn…5’ Restriction 5’…nnnnG-3’5’-AATTCnnnn…3’ 3’…nnnnCTTAA-5’3’-Gnnnn…5’ 5’ overhanging “sticky-ends”

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

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

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

  11. 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 Cloning vectors: essential features

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

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

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

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