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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 Recombinant DNA Recombination Specifically cut and join DNA Cut: digestion Join: ligation First steps for cloning Non Specific DNA breakage Mechanical shearing Chemical Non-specific enzyme Exonuclease Endonuclease Restriction Endonuclease Mg2+, ATP and SAM as cofactors Specific recognition site on dsDNA Move along 1-5 kb Type I Cut randomly on 1 strand Require 2nd enzyme to cut another strand Restriction Endonuclease Intermediate properties between types I and II Mg2+ and ATP as cofactors Specific recognition site Type III Cut both strands 24-26 bp from a recognition site Restriction Endonuclease Mg2+ Type II Specific recognition site Specific cleavage site within or near recognition site Cut both strands Fragments with defined length & sequence 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) Nomenclature EcoRI - from Escherichia coli BamHI - from Bacillus amyloliquefaciens HindIII - from Haemophilus influenzae PstI - from Providencia stuartii Sau3AI - from Staphylococcus aureus AvaI - from Anabaena variabilis Target Site of REII 4-8 bases Frequency of the cut 4-base cutter 8-base cutter Rotational symmetry palindrome Target Site of REII Palindrome Restriction Enzyme Break phosphodiester bond Produce 5’P and 3’OH ends Enzyme: homodimer 1 subunit cut 1 strand away from the axis: overhang / sticky / protruding at the axis: blunt end 5’ P and 3’ OH ends 5’ sticky end 3’ sticky end blunt end Source microorganism Enzyme Rec. Site Ends Arthrobacter luteus Alu I AGCT Blunt Bacillus amyloiquefaciens H Bam HI GGATCC Sticky Escherichia coli Eco RI GAATTC Sticky Haemophilus gallinarum Hga I GACGC(N)5 Sticky Haemophilus infulenzae Hind III AAGCTT Sticky Providencia stuartii 164 Pst I CTGCAG Sticky Nocardia otitiscaviaruns Not I GCGGCCGC Sticky Staphylococcus aureus 3A Sau 3A GATC Sticky Serratia marcesans Sma I CCCGGG Blunt Thermus aquaticus Taq I TCGA Sticky Restriction Enzyme Sequence-specific tails DNA with compatible ends: join Different sources Different enzymes Restriction Enzyme Different enzymes : Different recognition sites Produce compatible ends: Get rDNA Sau3AI 5’ NNN”GATCNNN 3’ BamHI 5’ NNNG”GATCCNNN 3’ Recombinant DNA Sau3AI 5’ NNN GATCNNN 3’ 3’ NNNCTAG NNN 5’ BamHI 5’ NNNG GATCCNNN 3’ 3’ NNNCCTAG GNNN 5’ rRNA 5’ NNN GATCCNNN 3’ 3’ NNNCTAG GNNN 5’ 5’ NNNGATCCNNN 3’ 3’ NNNCTAGGNNN 5’ recut by Sau3AI / BamHI ? Restriction Enzyme Methylation Modification of recognition site Effect on cutting BamHI *GGATCm5C *GGm6ATCC **GGATCm4C **GGATm5CC Restriction Enzyme Isoschizomer Different host same recognition site same or different cleavage site Restriction Enzyme Isoschizomer XhoI / PaeR7I 5’ NNNC”TCGAGNNN 3’ SmaI 5’ NNNCCC”GGGNNN 3’ XmaI 5’ NNNC”CCGGGNNN 3’ Restriction Enzyme Isoschizomer: methylation sensitivity HpaI (X) and MspI (/) Cm5CGG SmaI (X) and XmaI (/) CCm5CGGG Restriction Enzyme Applications Basic tool for creating rDNA Restriction Map 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 Restriction Map Restriction Map Restriction Map Ligation To ligate by Ligase E. coli ligase NAD+ as cofactor Sticky ends T4-infected E. coli ligase ATP as cofactor Blunt or sticky ends Ligation Seal nicks on dsDNA Form phosphodiester bond Require 5’ P and 3’ OH Ligation Ligation Example??? Ligation Intramolecular ligation (re)circularization Ligation For recombinant DNA increase DNA concentration increase temperature (Ti) dephosphorylation Dephosphorylation Other Enzymes DNA polymerase RNA / DNA dependent DNA polymerase Other Enzymes E. coli DNA polymerase DNA dependent DNA polymerase 5’-->3’ polymerase 5’-->3’ exonuclease 3’-->5’ exonuclease E. coli DNA Polymerase E. coli DNA Polymerase Klenow Fragment DNA polymerase: Klenow fragment 5’-->3’ polymerase no 5’-->3’ exonuclease 3’-->5’ exonuclease Active on ds DNA T4 polymerase DNA polymerase: T4 polymerase 5’-->3’ polymerase no 5’-->3’ exonuclease 3’-->5’ exonuclease Active on ss DNA Taq polymerase DNA polymerase: Taq polymerase thermostable 75-80 C PCR reaction Reverse transcriptase RNA dependent DNA polymerase Exonuclease III Kinase Phosphatase Vector Cloning vehicle Carry / Multiply specific DNA fragments Cloning site multiple cloning region / polylinker Origin of replication (ori) Selectable marker 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 Plasmid vector Carry antibiotic-resistance genes providing selectable phenotype to host Other selectable markers sugar fermentation heavy metal resistance hydrogen sulfide production enterotoxin production 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 Plasmid vector Ideal properties Low molecular weight easy to handle, separate & purify High copy number Unique restriction sites Disabled: no survival outside lab Plasmid vector Plasmid cloning Bacteriophage vector Bacterial virus: forming plaque Linear or Circular shape Cohesive ends (cos, short ss 5’ protruding) Lytic / Lysogenic cycle Phage cycle 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 Bacteriophage vector Bacteriophage vector Require certain size for maturation and packaging Engineered to be safe and have MCR Available for big DNA insert Phage packaging Phage cloning Plaque lift / Hybridization Cosmid Plasmid / Phage hybrid plasmid: ori and selectable marker phage: cos site for packaging For large insert (32-47 kb) Cosmid Cosmid cloning Cosmid cloning Yeast Artificial Chromosome Big inserts: hundreds of kb Cloning elements Yeast sequences Selectable markers MCR Yeast Artificial Chromosome YAC cloning 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
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