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

Recombination Specifically cut and join DNA Cut: digestion Join: ligation First steps for cloning Recombinant DNA

Non Specific DNA breakage Mechanical shearing Chemical Non-specific enzyme Exonuclease Endonuclease

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

Restriction Endonuclease Intermediate properties between types I and II Mg 2+ and ATP as cofactors Specific recognition site Cut both strands bp from a recognition site Type III

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

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)

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

Target Site of REII 4-8 bases Frequency of the cut 4-base cutter 8-base cutter Rotational symmetry palindrome

Palindrome Target Site of REII

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 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

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 Sau3AI5’ NNN”GATCNNN 3’ BamHI5’ NNNG”GATCCNNN 3’

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 ?

Restriction Enzyme Methylation Modification of recognition site Effect on cutting BamHI*GGATC m5 C *GG m6 ATCC **GGATC m4 C **GGAT m5 CC

Restriction Enzyme Isoschizomer Different host same recognition site same or different cleavage site

Restriction Enzyme Isoschizomer XhoI / PaeR7I 5’ NNNC”TCGAGNNN 3’ SmaI5’ NNNCCC”GGGNNN 3’ XmaI5’ NNNC”CCGGGNNN 3’

Restriction Enzyme Isoschizomer: methylation sensitivity HpaI ( X ) and MspI ( / ) C m5 CGG SmaI ( X ) and XmaI ( / ) CC m5 CGGG

Restriction Enzyme Basic tool for creating rDNA Restriction Map Applications

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

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

Ligation Seal nicks on dsDNA Form phosphodiester bond Require 5’ P and 3’ OH

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

DNA polymerase: Klenow fragment 5’-->3’ polymerase no 5’-->3’ exonuclease 3’-->5’ exonuclease Active on ds DNA Klenow Fragment

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 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 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

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

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