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