Recombinant DNA Technology

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Recombinant DNA Technology

• Common General Cloning Strategy
• Target DNA from donor organism extracted, cut
  with restriction endonuclease and ligated into a
  cloning vector cut with compatible restriction
  endonuclease
• Recombinant construct transferred into host cell
• Host cells which do not take up construct are
  eliminated by selection protocol
• Host cell library screened to identify desired
  clone if necessary

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Cloning Into Bacterial Cells
Restriction Endonuclease
DNA Ligase
transformation
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DNA Cleavage By Restriction Endonucleases (1)
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DNA Cleavage By Restriction Endonucleases (2)
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Recognition Sequences of Restriction Endonucleases
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Restriction Mapping
Mapping Restriction Endonuclease Cleavage Sites
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DNA fragment sizes (in kilobase pairs) after
single and double restriction endonucleases
digestions of a plasmid
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Restriction Endonuclease Cleavage Map

• Created from single and multiple enzyme
  digestions
• Useful markers for noting gene locations and
  subcloning strategies

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Enzymes Used In Recombinant DNA Protocols
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Annealing of Complementary Sticky Ends
Annealing of Complementary Sticky Ends
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T4 DNA Ligase Action
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Early Cloning Vectors

• pBR322
• Plasmid
• Small independent replicon with selectable
  markers and useful cloning sites

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Cloning DNA Into a Plasmid Vector

• Restriction endonuclease cleave vector/target
• Phosphatase vector
• Ligate target into vector
• Transform into host cells

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Other Plasmid Cloning Vectors

• Now too many to count
• Many specialized for expression, etc.
• pUC series
• Multiple cloning sites
• Improved reporter/selection genes

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Multiple Cloning Sites

• Synthetic oligonucleotide construction
• Polymer of cutting sites
• Can be included in reporter gene coding sequence
  (e.g. lacZ)

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Antibiotics Commonly Used as Selective Agents
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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

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Partial Restriction Endonuclease Digestion of DNAs
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Partial Digestion Profile

• Collect fragments of a given target size after
  digestions for different times or using different
  restriction endonuclease concentrations
• Size fractionate and combine fractions of desired
  target size

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Genome Sizes of Various Organisms

• The number and size of library clones required to
  be screened to find a single copy gene varies
  according to the genome size of the organism to
  be studied

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Screening by Hybridization
Probes DNA or RNA 100 bp in size good Sequence
match gt80 best Stringency conditions
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Production of Labeled Probes
Random Primer Method
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Three Activities of E. coli DNAP I (1)

• Polymerization of dNTPs at the 3end of the
  growing chain (1)
• 5exonuclease removes nucleotides from 5end of
  chain immediately upstream of growing chain (2)
• 3exonuclease removes unpaired nucleotides from
  3 end of growing chain

(1)
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Three Activities of E. coli DNAP I (2)
(2)
(3)
Note that the 5exonuclease is used in nick
translation and the 3exonuclease activity is
used for the proofreading function
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Screening Colonies by Hybridization

• Nucleic acid probe
• Cells transferred to nylon membrane and lysed
• DNA binds to membrane, is denatured and probe
  hybridized
• Bound probe detected by autoradiography after
  washing membrane

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Screening by Immunological Assay
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Screening by Functional Complementation

• Requires strain unable to produce desired
  product/function
• Cloned DNAs must be in expression vector or
  include elements required for expression
• Select for restoration of lost function

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Isolation of Poly(adenylated) mRNAs
Matrix
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cDNA Synthesis

• Oligo(dT) primer
• Reverse transcriptase
• Klenow/DNAP I
• RNase H
• Degrades RNA of DNARNA hybrid
• S1 nuclease
• Degrades ss nucleic acids (unpaired loop)

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Enriching for Full Length cDNAs (1)
adapter

• 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 5end)
• Capture full length copies

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Enriching for Full Length cDNAs (2)

• RNase H degrades mRNA
• Add poly(G) to cDNA
• Primer/Adapter with oligo (C)
• DNAP I (Klenow)
• Restriction endonucleases
• Cut Vector
• DNA Ligase
• Transform

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Inert Capacities Common Vector Systems
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Bacteriophage Lambda Life Cycle

• Lysogenic phage
• Lysogeny vs. lytic cycle
• Chromosome about 50 kb
• Protein coat for efficient delivery into cells
  (E. coli)
• Packages DNA 38-52 kb with cos sites at each end
• DNA Replication is by rolling circle mechanism

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Packaging of l Chromosomes

• Natural DNA is concatemer with cos sites
  separated by about 50 kb (from rolling circle
  replication
• DNA is cleaved at cos and inserted into capsid

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Mature l Phage

• DNA packaged in protein coat
• Looks much like a lunar lander (actually has six
  tail fibers)

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Phage l cloning vector

• Internal segment deleted (now requires helper
  phage to replicate)
• Has cos sites intact
• Target DNA inserted between the two l arms (up
  to about 20 kb)
• DNA packaged in vitro
• Recombinant phage infect E. coli cells

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Cosmid Cloning System

• cos sites inserted
• into a small
• plasmid
• Target DNA ligated
• between two cosmid
• DNA molecules
• Recombinant DNA
• packaged and E. coli
• Infected as before
• Can clone DNAs up
• to 45 kb

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High-Capacity Bacterial Vector Systems

• 100-300 kb target size
• P1 bacterial systems
• F plasmid systems
• BACs (bacterial artificial chromosomes

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Genetic Transformation of Prokaryotes

• Chemical transformation
• Usually involves CaCl2 and heat shock
• Transformation frequency about 1/1000
• Electroporation
• Electric field meidated membrane permeabilization
• 10-100 times more efficient that chemical
  approach
• Much better for large plasmids (100 kb)

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Electroporation

• Cells suspended in DNA solution in cuvette
  between two electrodes
• High voltage electric field pulses administered
• DNA migrates through HVEF induced openings in
  cells

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Conjugation

• Natural system of transmitting plasmids from one
  cell/strain to another
• Requires specific DNA sequences on transferred
  plasmid and certain proteins which can be
  provided in trans
• Plasmids of gt106 bp can be transferred in this
  manner
• Can be interspecies
• Tripartite mating and multiple selection

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Tripartite Mating

• P. putida difficult to transform
• Transform mobilizable recombinant plasmid into
  E.coli
• Make culture with P. putida (wt), recombinant E.
  coli (auxotroph) and E. coli (aux) with
  conjugative mobilizable plasmid
• Recombinant plasmid transferred to P. putida