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Cloning DNA. May 4. Cloning DNA. the components vector (plasmids, bacteriophage) cutters (restriction endonucleases) joiners (ligase, topoisomerase) insert (DNA, mRNA->cDNA) host strains of E.coli. Restriction endonucleases. REs and methylases Type I, II, IIs, III enzymes

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Presentation Transcript
cloning dna1
Cloning DNA
  • the components
    • vector (plasmids, bacteriophage)
    • cutters (restriction endonucleases)
    • joiners (ligase, topoisomerase)
    • insert (DNA, mRNA->cDNA)
  • host
    • strains of E.coli
restriction endonucleases
Restriction endonucleases
  • REs and methylases
  • Type I, II, IIs, III enzymes
  • sites and ends
  • isochizomers
  • compatible ends
  • cleavage close to ends of linear DNA
  • star activity
  • heat inactivation
  • methylases
  • mcr and mrr restriction systems
overhangs and blunt ends
Overhangs and blunt ends

G

CTTAA

5’

AATTC

G

3’

5’

GAATTC

CTTAAG

EcoRI

5’

3’

5’

CTGCA

G

3’

G

ACGTC

5’

CTGCAG

GACGTC

PstI

3’

5’

CAG

GTC

CTG

GAC

5’

CAGCTG

GTCGAC

5’

PvuII

large and small sites
Large and small sites

GATC

CTAG

4 bases

MboI

GGATCC

CCTAGG

6 bases

BamHI

8 bases

GCGGCCGC

CGCCGGCG

NotI

compatible ends
Compatible ends

GATC

CTAG

GATC

MboI

CTAG

GATCC

G

G

CCTAG

GGATCC

CCTAGG

BamHI

AGATCT

TCTAGA

GATCT

A

A

TCTAG

BglII

dam and dcm methylases
Dam and Dcm methylases

RE sites effected by methylases

M

GATC

CTAG

Dam

AlwI

GGATC

TGATCA

BclI

M

BspDI

ATCGATc

XbaI

TCTAGAtc

M

CCAGG

GGTCC

GGTACCagg

Acc65I

Dcm

M

joining dna fragments
joining DNA fragments
  • ligase
  • topoisomerase
the inserts
The inserts
  • DNA
    • restriction digests of known sequences
    • shot-gun cloning
    • PCR products
    • forced cloning
    • constructing libraries
  • RNA
    • reverse transcription
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