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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

pUC192686 bp

Restriction enzyme sites on a plasmid(pUC19 2686 bp)

Unique restriction enzyme sites

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+

small

large

M

A

B

X

Y

Z

Y+ZX+Z

X+Y

Restriction enzyme mapping