Recombinant DNA Technology - uenf.bruenf.br/Uenf/Downloads/LBT_8017_1286564736.pdfrDNA Technology...
Transcript of Recombinant DNA Technology - uenf.bruenf.br/Uenf/Downloads/LBT_8017_1286564736.pdfrDNA Technology...
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Recombinant DNA Technology
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rDNA Technology
• Restriction Enzymes and DNA Ligase• Plasmid Cloning Vectors• Transformation of Bacteria• Creating and Screening Genomic Libraries• cDNA Library Construction• Vectors for Cloning Large Pieces of DNA• Blotting Techniques
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Restriction Enzymes
• Most significant advancement permitting rDNA manipulation
• Differ from other nucleases– recognize and cleave a specific DNA
sequence (Type II restriction enzymes)
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Restriction Enzymes
• Nomenclature– EcoRI
• E = Escherichia genus name• co = coli species name• R = strain RY12 strain or serotype• I = Roman numeral one = first enzyme
– HinDIII• Haemophilus influenza serotype d
3rd enzyme
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Restriction Enzymes
• Recognition sites– Generally 4, 6, or 8 bp in length– Most sites are palindromic
• OTTO / HANNAH / REGAL LAGER• A MAN A PLAN A CANAL PANAMA
– For REases - sequence reads the same in a 5’--->3’ direction on each strand
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Restriction Enzymes
• EcoRI5’ GAATTC 3’3’ CTTAAG 5’
• Hind III5’ AAGCTT 3’3’ TTCGAA 5’
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Restriction Enzymes
• Cleave DNA to generate different “ends”– Staggered cut
• 5’ extension• 3’ extension
– Blunt end
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Staggered Cut / 5’ or 3’ Extension
5’GAATTCCTTAAG
Eco RI5’G AATTC
CTTAA G+
5’ CTGCAGGACGTC
Pst I5’ CTGCA G
G ACGTC +
Fig 4.2 for 5’ & Fig 4.3 for Blunt
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Restriction Enzymes in DNA Cloning
• How are REases used ?– Ends are “sticky”– Complementary– Any two DNAs cut with same enzyme can
stick together through complementary base pairing
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Fig 4.6 Annealing sticky ends
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Fig 4.6bAnnealing Sticky ends
DNA strands heldtogether only by basepairingNicks in strandsneed to be repaired
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Linking Restriction Fragments
• T4 DNA Ligase– repairs nicks in DNA strands
(reforms phosphodiester bond)– uses energy from ATP– works on blunt or sticky ends
• Other enzymes used in rDNAtechnology are listed in Table 4.3
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Fig 4.7 T4 DNA Ligase Mode of Action
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rDNA Technology
• Restriction Enzymes and DNA Ligase• Plasmid Cloning Vectors• Transformation of Bacteria• Creating and Screening Genomic Libraries• cDNA Library Construction• Vectors for Cloning Large Pieces of DNA• Blotting Techniques
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Fig 4.1 Recombinant DNA Cloning Procedure
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Fig 4.1 Recombinant DNA Cloning Procedure
1) Enzymaticdigestion
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Fig 4.1 Recombinant DNA Cloning Procedure
2) Ligation of Target and vector DNA Ligase
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Fig 4.1 Recombinant DNA Cloning Procedure
3) TransformLigated DNAinto Bacteria
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Plasmid Cloning Vectors
• Recombinant DNA needs to be replicated in bacterial cell
• Self-replicating piece of DNA– termed cloning vehicle– can be plasmid or phage
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Plasmid Cloning Vectors
• Small circular piece of DNA• Exists separate from chromosome• Derived from naturally occurring plasmids
• High copy number = 10-100 copies / cell• Low copy number = 1-4 copies / cell
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Plasmid Cloning Vectors
• Derived from naturally occurring plasmids• Altered features
– small size (removal of non-essential DNA)• higher transformation efficiency
– unique restriction enzyme sites– one or more selectable markers– origin of replication (retained from original plasmid)– other features: promoters, etc.
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4362 bp
Fig 4.7 pBR322old-style general purpose plasmid
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Fig 4.9 pUC19
2.68kbp
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Multiple Cloning Sequence (Polylinker)
EcoRI KpnI BamHI Sal I
GAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGAC
XbaISmaISacI
Part of MCS of pUC19 and other plasmids
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Plasmid Cloning Vehicles
Some plasmids (Expression Plasmids) have promoters upstream of cloning sites for expression of genetic info encoded by DNA fragment
promoter Gene
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Shuttle Plasmid Cloning Vehicles
Some plasmids (Shuttle Plasmids) have origins of replication for E. coli and another organism: yeast, mammalian cells or other bacteria
promoter Gene
E. coli ori yeast orimammalian ori
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Plasmid Cloning Vehicles
• What prevents plasmid DNA from reforming during ligation?
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Plasmid Cloning Vehicles
• What prevents plasmid DNA from reforming during ligation and transforming cells as do the recombinant molecules?
• Three ways to prevent– Treat with Alkaline Phosphatase– Directional Cloning– Suicide Plasmids with ccdB gene
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Plasmid Cloning Vehicles
• Alkaline Phosphatase– removes 5’ PO4 from end of DNA strand– prevents formation of new phosphodiester
bond by DNA Ligase
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Fig 4.9 Alkaline Phosphatase Action
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Fig 4.9 Alkaline Phosphatase Action
Two nicks remain
Will be repaired inbacterial cell follow-ing transformation
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Directional Cloning
• Digest plasmid and target DNA with two different restriction enzymes– Hind III and BamHI– Ends are not compatible (can’t basepair)– Plasmid won’t re-circularize unless target
DNA has inserted
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Directional Cloning
H B H B
HB
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Suicide Plasmids
• contain the ccdB gene• encodes a protein which inhibits gyrase,
which is essential for replication • located on plasmid downstream of
cloning sites• Cloning of target DNA prevents
expression of ccdB gene - cell survives
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Suicide Plasmids
ccdBpromoter mcs
CELL DIES
ccdBpromoter target
CELL SURVIVES