Restriction
Endonuclease
DNA Ligase
transformation
DNA Cleavage By Restriction
Endonucleases (1)
DNA Cleavage By Restriction
Endonucleases (2)
Recognition Sequences of Restriction
Endonucleases
Restricti
Mapping Restriction Endonuclease Cleavage Sites
on
Mappin
g
DNA fragment sizes (in kilobase pairs) after
single and double restriction endonucleases
digestions of a plasmid
Restriction Endonuclease
Cleavage Map
• pBR322
• Plasmid
Small independent
replicon with
selectable markers and
useful cloning sites
Cloning DNA Into a
Plasmid Vector
• Restriction endonuclease
cleave vector/target
• Phosphatase vector
• Ligate target into vector
• Transform into host cells
Other Plasmid Cloning Vectors
• Now too many to count
Many specialized for expression, etc.
• pUC series
– Multiple cloning sites
– Improved reporter/selection genes
Multiple Cloning Sites
• Synthetic oligonucleotide construction
• “Polymer of cutting sites”
• Can be included in reporter gene coding
sequence (e.g. lacZ’)
Antibiotics Commonly Used as Selective
Agents
Creating and Screening a
Recombinant DNA Library
• A library is a collection of subdivided
portions of a larger genetic element or
genome
• Commonly created by partial digestion of
genomic DNA with restriction
endonuclease and cloning the fragments
into vectors (plasmid, phage, etc.)
• Resultant transformed collection of cells is
called a library
Partial Restriction Endonuclease
Digestion of DNAs
Partial Digestion Profile
(2)
(3)
Matrix
cDNA
Synthesis
• Oligo(dT) primer
• Reverse transcriptase
• Klenow/DNAP I
• RNase H
Degrades RNA of
DNA:RNA hybrid
• S1 nuclease
Degrades ss nucleic
acids (unpaired
loop)
Enriching for Full
adapter
Length cDNAs (1)
• Primer has adapter (RE
cutting sequence)
• Ribose ends of mRNA are
biotinylated
• RNase I degrades ss RNA
• Only full length cDNA is
still attached to a
biotinylated mRNA
(biotin still on 5’end)
• Capture full length copies
Enriching for Full Length cDNAs (2)
Recombinant DNA
packaged and E. coli
Infected as before