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

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Presentation on theme: "Recombinant DNA Isolation / Digestion / Fractionation Purification of the target fragment Cloning into vectors / Ligation Transformation of host cell /"— Presentation transcript:

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

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

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

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

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

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

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)

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

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

10 Palindrome Target Site of REII

11 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

12 5’ P and 3’ OH ends

13 5’ sticky end

14 3’ sticky end

15 blunt end

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

17 Restriction Enzyme Sequence-specific tails DNA with compatible ends: join Different sources Different enzymes

18 Restriction Enzyme Different enzymes : Different recognition sites Produce compatible ends: Get rDNA Sau3AI5’ NNN”GATCNNN 3’ BamHI5’ NNNG”GATCCNNN 3’


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

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

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

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

24 Restriction Enzyme Basic tool for creating rDNA Restriction Map Applications

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

26 Restriction Map



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

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

31 Ligation

32 Example???

33 Ligation Intramolecular ligation (re)circularization

34 Ligation For recombinant DNA increase DNA concentration increase temperature (Ti) dephosphorylation

35 Dephosphorylation

36 Other Enzymes DNA polymerase RNA / DNA dependent DNA polymerase

37 Other Enzymes E. coli DNA polymerase DNA dependent DNA polymerase 5’-->3’ polymerase 5’-->3’ exonuclease 3’-->5’ exonuclease

38 E. coli DNA Polymerase


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

41 T4 polymerase DNA polymerase: T4 polymerase 5’-->3’ polymerase no 5’-->3’ exonuclease 3’-->5’ exonuclease Active on ss DNA

42 Taq polymerase DNA polymerase: Taq polymerase thermostable 75-80 C PCR reaction

43 Reverse transcriptase RNA dependent DNA polymerase

44 Exonuclease III

45 Kinase

46 Phosphatase

47 Vector Cloning vehicle Carry / Multiply specific DNA fragments Cloning site multiple cloning region / polylinker Origin of replication (ori) Selectable marker

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

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

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

51 Plasmid vector Ideal properties Low molecular weight easy to handle, separate & purify High copy number Unique restriction sites Disabled: no survival outside lab

52 Plasmid vector

53 Plasmid cloning

54 Bacteriophage vector Bacterial virus: forming plaque Linear or Circular shape Cohesive ends (cos, short ss 5’ protruding) Lytic / Lysogenic cycle

55 Phage cycle

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

57 Bacteriophage vector

58 Require certain size for maturation and packaging Engineered to be safe and have MCR Available for big DNA insert

59 Phage packaging

60 Phage cloning

61 Plaque lift / Hybridization

62 Cosmid Plasmid / Phage hybrid plasmid: ori and selectable marker phage: cos site for packaging For large insert (32-47 kb)

63 Cosmid

64 Cosmid cloning


66 Yeast Artificial Chromosome Big inserts: hundreds of kb Cloning elements Yeast sequences Selectable markers MCR

67 Yeast Artificial Chromosome

68 YAC cloning

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

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