Recombinant DNA Technology
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
Restriction Enzymes
• Most significant advancement permitting rDNA manipulation
• Differ from other nucleases– recognize and cleave a specific DNA
sequence (Type II restriction enzymes)
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
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
Restriction Enzymes
• EcoRI5’ GAATTC 3’3’ CTTAAG 5’
• Hind III5’ AAGCTT 3’3’ TTCGAA 5’
Restriction Enzymes
• Cleave DNA to generate different “ends”– Staggered cut
• 5’ extension• 3’ extension
– Blunt end
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
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
Fig 4.6 Annealing sticky ends
Fig 4.6bAnnealing Sticky ends
DNA strands heldtogether only by basepairingNicks in strandsneed to be repaired
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
Fig 4.7 T4 DNA Ligase Mode of Action
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
Fig 4.1 Recombinant DNA Cloning Procedure
Fig 4.1 Recombinant DNA Cloning Procedure
1) Enzymaticdigestion
Fig 4.1 Recombinant DNA Cloning Procedure
2) Ligation of Target and vector DNA Ligase
Fig 4.1 Recombinant DNA Cloning Procedure
3) TransformLigated DNAinto Bacteria
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
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
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.
4362 bp
Fig 4.7 pBR322old-style general purpose plasmid
Fig 4.9 pUC19
2.68kbp
Multiple Cloning Sequence (Polylinker)
EcoRI KpnI BamHI Sal I
GAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGAC
XbaISmaISacI
Part of MCS of pUC19 and other plasmids
Plasmid Cloning Vehicles
Some plasmids (Expression Plasmids) have promoters upstream of cloning sites for expression of genetic info encoded by DNA fragment
promoter Gene
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
Plasmid Cloning Vehicles
• What prevents plasmid DNA from reforming during ligation?
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
Plasmid Cloning Vehicles
• Alkaline Phosphatase– removes 5’ PO4 from end of DNA strand– prevents formation of new phosphodiester
bond by DNA Ligase
Fig 4.9 Alkaline Phosphatase Action
Fig 4.9 Alkaline Phosphatase Action
Two nicks remain
Will be repaired inbacterial cell follow-ing transformation
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
Directional Cloning
H B H B
HB
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
Suicide Plasmids
ccdBpromoter mcs
CELL DIES
ccdBpromoter target
CELL SURVIVES