www.le.ac.uk making recombinant dna dna structure and plasmids dna restriction and ligation
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www.le.ac.uk
Making Recombinant DNA
DNA structure and PlasmidsDNA Restriction and Ligation
www.le.ac.uk/genie
DNA structure recap
AT
CG
• 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!!
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
• Restriction (cutting the plasmid)– Restriction Endonucleases
Some terminology:
DNA insert
Plasmid
Cut site• Ligation (insertion of DNA insert and rejoining the pieces)– Ligase enzyme
• 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
• 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’
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
Joining -Ligation
5’…nnnnG-3’ 5’-AATTCnnnn…3’3’…nnnnCTTAA-5’ 3’-Gnnnn…5’
5’…nnnnGAATTCnnnn…3’3’…nnnnCTTAAGnnnn…5’
LigationJoining “sticky ends”: mix & ligate
Plasmid DNA DNA insert
Basic Cloning I Foreign DNA to be inserted
join/ligate
Antibiotic resistance gene
Plasmid vector
Recombinant DNA molecule
ABR
ABR
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
pUC19- a classic cloning vector
SelectionAntibiotic resistance gene
Maintenance:Origin of replication
Cloning:Restriction sites
Blue/ white selection for presence of insert
Restriction enzyme mapping
Plasmidone site / one cut
Full length
Plasmid Y
X
Z
A + B = Full length
B
Plasmidtwo sites / two cuts
AA
B
X Z + Y+
Y Z + X+
Z X + Y+