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Recombinant DNA
• prepare foreign
gDNA (fragments)
(target) DNA
cDNA (copy of RNA)
• prepare vector (host)
• recombine target and
vector DNA
• introduce rDNA to host
• screen for DNA of
interest
Preparing gDNA
• restriction enzymes
• ‘random nuclease’
•  size fractionate
gDNA vs cDNA
• level of expression
• differential gene expression
• accessibility
• introns
• complicates gDNA analysis
• can preclude expression
• ease of preparation
• cDNA more work
Preparation of cDNA
1) Isolate mRNA
2) Synthesize DNA-RNA hybrid
• reverse transcriptase  RNA dependent
DNA polymerase
• oligo-dT primer
• random priming
3) Synthesize 2nd DNA strand
4) Add termini
1) Isolate mRNA
2) Synthesize DNA-RNA
hybrid
3) Synthesize 2nd DNA
strand
• self-priming
• replacement
synthesis
• primed synthesis
4) Add termini
1) Isolate mRNA
2) Synthesize DNA-RNA
hybrid
3) Synthesize 2nd DNA
strand
4) Add termini
• i.e., linkers
CGGAATTCCG
GCCTTAAGGC
Eco RI
Recombinant DNA Vectors
• autonomously-replicating DNA used to ‘carry’
and amplify foreign DNA within host cell
• eg: plasmids, phage/viruses, or combinations
Plasmids
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extra-chromosomal elements
1-200 kb size range
transmitted during conjugation
antibiotic resistance
low copy number vs high copy number
Useful Plasmid
Features
• Relaxed Replication
• Selectable Markers
• Streamlined
• Polylinker or MCS
• Identification of
Recombinants
• most derived from
pUC or pBR322
Multiple Cloning Site:
|SacI| |ScII|
|XbaI||SpeI||BamH||SmaI||PstI||EcRI||EcRV||HIII||ClaI|
|SalI||XhoI|
|KpnI|
GAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACC
CTCGAGGTGGCGCCACCGCCGGCGAGATCTTGATCACCTAGGGGGCCCGACGTCCTTAAGCTATAGTTCGAATAGCTATGGCAGCTGGAGCTCCCCCCCGGGCCATGG
Generic rDNA Protocol
• prepare foreign DNA
• prepare vector
• ligate foreign DNA and vector
• introduce vector into host
• screen for rDNA of interest
Ligation Reaction
• mix foreign and vector DNA in
presence of DNA ligase
• optimal ratios of vector to insert generally
1.5-2:1
• intermolecular base-pairing can occur
between compatible overhangs
DNA Ligase
• catalyzes the formation of phosphodiester
bond between 5’-PO4 and 3’-OH
• i.e., joins DNA fragments
• typically carried out at lower temperatures
(8-16o) for extended periods
Intramolecular vs. Intermolecular
Removal of 5’-PO4 Prevents
Vector Self Ligation
TERMINI
Identical
Overhangs
Blunt-end
Different
Overhangs
CLONING
REQUIREMENTS
Phosphatase treatment of
linear plasmid improves
efficiency.
High concentrations of DNA
and ligase needed.
Phosphatase treatment.
Purification of double-cut
plasmid increases
efficiency.
COMMENTS
Restriction sites at junctions preserved.
Both orientations of insert DNA possible.
Tandem copies of insert possible.
Restriction sites at junctions often
eliminated. Tandem copies of insert DNA
possible. Both orientations possible.
Restriction sites at junctions preserved.
Background of non-recombinants is low.
One possible orientation of insert. Tandem
copies unlikely.
• prepare foreign DNA
• prepare vector
• ligate target and vector
• introduce rDNA to host
• heat shock + Ca2+
• electroporation
• select for transformants
with antibiotic
• screen for rDNA of
interest
Colony Lift
Sources of Probes
• cloned genes
• synthetic
oligonucleotides
• PCR products
Identifying Recombinants
• based on interruption of a gene
• eg., lacZ gene = b-galactosidase
• intact b-galactosidase produces
blue color in presence of X-gal
• -complementation or bluewhite screening