Recombinant DNA Isolation / Digestion / Fractionation Purification of the target fragment Cloning into vectors / Ligation Transformation of host cell

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Recombinant DNA Isolation / Digestion / Fractionation Purification of the target fragment Cloning into vectors / Ligation Transformation of host cell / Selection Replication / Analysis Expression of DNA Slide 2 Recombination Specifically cut and join DNA Cut: digestion Join: ligation First steps for cloning Recombinant DNA Slide 3 Non Specific DNA breakage Mechanical shearing Chemical Non-specific enzyme Exonuclease Endonuclease Slide 4 Restriction Endonuclease Mg 2+, ATP and SAM as cofactors Specific recognition site on dsDNA Move along 1-5 kb Cut randomly on 1 strand Require 2nd enzyme to cut another strand Type I Slide 5 Restriction Endonuclease Intermediate properties between types I and II Mg 2+ and ATP as cofactors Specific recognition site Cut both strands 24-26 bp from a recognition site Type III Slide 6 Restriction Endonuclease Mg 2+ Specific recognition site Specific cleavage site within or near recognition site Cut both strands Fragments with defined length & sequence Type II Slide 7 Nomenclature 3 Letters (italic) host genus in upper case host species in lower case strain or type (non italic) restriction or modification system (Roman numeral) Slide 8 EcoRI - from Escherichia coli BamHI - from Bacillus amyloliquefaciens HindIII - from Haemophilus influenzae PstI - from Providencia stuartii Sau3AI - from Staphylococcus aureus AvaI - from Anabaena variabilis Nomenclature Slide 9 Target Site of REII 4-8 bases Frequency of the cut 4-base cutter 8-base cutter Rotational symmetry palindrome Slide 10 Palindrome Target Site of REII Slide 11 Restriction Enzyme Break phosphodiester bond Produce 5P and 3OH ends Enzyme: homodimer 1 subunit cut 1 strand away from the axis: overhang / sticky / protruding at the axis: blunt end Slide 12 5 P and 3 OH ends Slide 13 5 sticky end Slide 14 3 sticky end Slide 15 blunt end Slide 16 Source microorganism Enzyme Rec. Site Ends Arthrobacter luteus Alu I AG CTBlunt Bacillus amyloiquefaciens H Bam HI G GATCCSticky Escherichia coli Eco RI G AATTCSticky Haemophilus gallinarum Hga I GACGC(N) 5 Sticky Haemophilus infulenzae Hind III A AGCTTSticky Providencia stuartii 164 Pst I CTGCA GSticky Nocardia otitiscaviaruns Not I GC GGCCGCSticky Staphylococcus aureus 3A Sau 3A GATCSticky Serratia marcesans Sma I CCC GGGBlunt Thermus aquaticus Taq I T CGASticky Slide 17 Restriction Enzyme Sequence-specific tails DNA with compatible ends: join Different sources Different enzymes Slide 18 Restriction Enzyme Different enzymes : Different recognition sites Produce compatible ends: Get rDNA Sau3AI5 NNNGATCNNN 3 BamHI5 NNNGGATCCNNN 3 Slide 19 Recombinant DNA Sau3AI5 NNN GATCNNN 3 3 NNNCTAG NNN 5 BamHI5 NNNGGATCCNNN 3 3 NNNCCTAG GNNN 5 rRNA5 NNN GATCCNNN 3 3 NNNCTAG GNNN 5 5 NNNGATCCNNN 3 3 NNNCTAGGNNN 5 recut by Sau3AI / BamHI ? Slide 20 Restriction Enzyme Methylation Modification of recognition site Effect on cutting BamHI*GGATC m5 C *GG m6 ATCC **GGATC m4 C **GGAT m5 CC Slide 21 Restriction Enzyme Isoschizomer Different host same recognition site same or different cleavage site Slide 22 Restriction Enzyme Isoschizomer XhoI / PaeR7I 5 NNNCTCGAGNNN 3 SmaI5 NNNCCCGGGNNN 3 XmaI5 NNNCCCGGGNNN 3 Slide 23 Restriction Enzyme Isoschizomer: methylation sensitivity HpaI ( X ) and MspI ( / ) C m5 CGG SmaI ( X ) and XmaI ( / ) CC m5 CGGG Slide 24 Restriction Enzyme Basic tool for creating rDNA Restriction Map Applications Slide 25 Restriction Map Locations of recognition sites on DNA Highly specific fragments Digestion with 6-8 base cutter Size fractionation on gel Different patterns from different enzymes Single and Double digests Slide 26 Restriction Map Slide 27 Slide 28 Slide 29 Ligation E. coli ligase NAD+ as cofactor Sticky ends To ligate by Ligase T4-infected E. coli ligase ATP as cofactor Blunt or sticky ends Slide 30 Ligation Seal nicks on dsDNA Form phosphodiester bond Require 5 P and 3 OH Slide 31 Ligation Slide 32 Example??? Slide 33 Ligation Intramolecular ligation (re)circularization Slide 34 Ligation For recombinant DNA increase DNA concentration increase temperature (Ti) dephosphorylation Slide 35 Dephosphorylation Slide 36 Other Enzymes DNA polymerase RNA / DNA dependent DNA polymerase Slide 37 Other Enzymes E. coli DNA polymerase DNA dependent DNA polymerase 5-->3 polymerase 5-->3 exonuclease 3-->5 exonuclease Slide 38 E. coli DNA Polymerase Slide 39 Slide 40 DNA polymerase: Klenow fragment 5-->3 polymerase no 5-->3 exonuclease 3-->5 exonuclease Active on ds DNA Klenow Fragment Slide 41 T4 polymerase DNA polymerase: T4 polymerase 5-->3 polymerase no 5-->3 exonuclease 3-->5 exonuclease Active on ss DNA Slide 42 Taq polymerase DNA polymerase: Taq polymerase thermostable 75-80 C PCR reaction Slide 43 Reverse transcriptase RNA dependent DNA polymerase Slide 44 Exonuclease III Slide 45 Kinase Slide 46 Phosphatase Slide 47 Vector Cloning vehicle Carry / Multiply specific DNA fragments Cloning site multiple cloning region / polylinker Origin of replication (ori) Selectable marker Slide 48 Plasmid vector Bacterial minichromosome (2-20 kb) not linked to main chromosome Autonomous replication (replicon) Mostly ds circular form Some with linear form (actinomycete / spirochete) Take up 100 bp 10 kb Slide 49 Plasmid vector Carry antibiotic-resistance genes providing selectable phenotype to host Other selectable markers sugar fermentation heavy metal resistance hydrogen sulfide production enterotoxin production Slide 50 Plasmid vector Insertional inactivation when MCR in selectable-marker gene High copy number plasmid replicate 10-200 copies per bacterial cycle Low copy number plasmid one or a few copies Slide 51 Plasmid vector Ideal properties Low molecular weight easy to handle, separate & purify High copy number Unique restriction sites Disabled: no survival outside lab Slide 52 Plasmid vector Slide 53 Plasmid cloning Slide 54 Bacteriophage vector Bacterial virus: forming plaque Linear or Circular shape Cohesive ends (cos, short ss 5 protruding) Lytic / Lysogenic cycle Slide 55 Phage cycle Slide 56 Bacteriophage vector Central section of phage DNA For integration into host chromosome Not necessary for replication Replaced by foreign insert Left and Right arms: easily isolated Essential for replication and packaging Slide 57 Bacteriophage vector Slide 58 Require certain size for maturation and packaging Engineered to be safe and have MCR Available for big DNA insert Slide 59 Phage packaging Slide 60 Phage cloning Slide 61 Plaque lift / Hybridization Slide 62 Cosmid Plasmid / Phage hybrid plasmid: ori and selectable marker phage: cos site for packaging For large insert (32-47 kb) Slide 63 Cosmid Slide 64 Cosmid cloning Slide 65 Slide 66 Yeast Artificial Chromosome Big inserts: hundreds of kb Cloning elements Yeast sequences Selectable markers MCR Slide 67 Yeast Artificial Chromosome Slide 68 YAC cloning Slide 69 Expression vector Available for screening of gene product of cDNA insert MCR within transcription regions between promoter and terminator Bidirectional cloning for correct orientation Foreign or Fusion proteins