Recombinant DNA Technology

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

Cloning Into Bacterial Cells
Restriction
Endonuclease
DNA Ligase
transformation

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

Enzymes Used In Recombinant DNA Protocols

Annealing of Complementary “Sticky” Ends

T4 DNA Ligase Action

Early Cloning Vectors
• 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
• 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

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

Screening by Hybridization
Probes: DNA or RNA
100+ bp in size good
Sequence match >80% best
Stringency conditions

Production of Labeled Probes
Random Primer Method

Three Activities of E. coli DNAP I (1)
(1)
• Polymerization of dNTPs
at the 3’end of the growing
chain (1)
• 5’exonuclease removes
nucleotides from 5’end of
chain immediately
upstream of growing chain
(2)
• 3’exonuclease removes
unpaired nucleotides from
3’ end of growing chain

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

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

Screening by Immunological Assay

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

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

Inert Capacities Common Vector
Systems

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

Packaging of λ 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

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

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

Cosmid Cloning System
λ 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

High-Capacity Bacterial Vector
Systems
• 100-300 kb target size
• P1 bacterial systems
• F plasmid systems
• BACs (bacterial artificial chromosomes

Genetic Transformation of
Prokaryotes
• Chemical transformation
• Usually involves CaCl 2 and heat shock
• Transformation frequency about 1/1000
• Electroporation
• Electric field mediated membrane
permeabilization
• 10-100 times more efficient that chemical
approach
• Much better for large plasmids (100+ kb)

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

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 >10 6 bp can be transferred in this
manner
• Can be interspecies
• Tripartite mating and multiple selection

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