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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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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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