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

  • View
    213

  • Download
    1

Embed Size (px)

Text of Www.le.ac.uk Making Recombinant DNA DNA structure and Plasmids DNA Restriction and Ligation...

Slide 1

Making Recombinant DNADNA structure and PlasmidsDNA Restriction and Ligation

www.le.ac.uk/geniewww.le.ac.uk1

DNA structure recapATCGDouble-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 togetherThe same building blocks are used in all forms of life, so

...CLONING IS POSSIBLE!!Cloning Vectors: PlasmidsCircular, 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 insertPlasmidCut siteLigation (insertion of DNA insert and rejoining the pieces)Ligase enzyme

4Enzymes that recognise and cut at specific sequences of DNAExist in vivo in bacteria for foreign DNA recognition and destruction4,5,6 and 8 base recognition

Different enzymes recognise different sequences:

Restriction EnzymesEnzymes that recognise and cut at specific sequences of DNAExist in vivo in bacteria for foreign DNA recognition and destruction4,5,6 and 8 base recognition

5'--G A A T T C--3'5- -G A A T T C- -33'--C T T A A G --5' 3- - C T T A A G- -5Restriction Enzymes5'--G A T C--3'5- -G A T C- -33'--C T A G--5' 3- -C T A G- -5 Cutting - Restriction5nnnnGAATT-Cnnnn35nnnnGAATT-Cnnnn33nnnnC-TTAAGnnnn53nnnnC-TTAAGnnnn55nnnnG-35-AATTCnnnn33nnnnCTTAA-5 3-Gnnnn5RestrictionRestrict EcoRI sites5 overhanging sticky-endsPlasmid DNADNA insert7

pUC192686 bpRestriction enzyme sites on a plasmid(pUC19 2686 bp)Unique restriction enzyme sites8Joining -Ligation5nnnnG-35-AATTCnnnn33nnnnCTTAA-5 3-Gnnnn55nnnnGAATTCnnnn33nnnnCTTAAGnnnn5LigationJoining sticky ends: mix & ligatePlasmid DNADNA insert9Basic Cloning I Foreign DNA to be insertedjoin/ligateAntibiotic resistance genePlasmid vectorRecombinant DNA moleculeABRABR10Cloning vectors: essential featuresMaintenance (replication) in host cell

Restriction enzyme sitesinsertion of foreign DNA fragmentuniquecreate RECOMBINANT DNA molecules

Method to introduce into host celltransformation

Identify recombinants

11pUC19- a classic cloning vector

SelectionAntibiotic resistance geneMaintenance:Origin of replicationCloning:Restriction sitesBlue/ white selection for presence of insert12Restriction enzyme mappingPlasmidone site / one cutFull length

PlasmidYXZ

A + B = Full length

BPlasmidtwo sites / two cutsAAB

XZ + Y

+YZ + X

+Z X + Y

+smalllargeMABXYZY+ZX+ZX+YRestriction enzyme mapping