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Recombinant DNA
Andy Howard
Introductory Biochemistry
20 October 2008
Biochem: Recombinant DNA
10/20/2009
Recombinant DNA
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Much of our current understanding of
molecular biology, and of the ways we
can use it in medicine, agriculture, and
basic biology, is derived from the kinds of
genetic manipulations that we describe
as recombinant DNA
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What we’ll discuss
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Synthesis of DNA in
the laboratory
Cloning
Plasmids & inserts
Vector techniques
Libraries & probes
High-throughput
Expression
Biochem: Recombinant DNA
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Fusion Proteins
Protein-protein
interactions
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iClicker quiz, question 1
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1. How does acetylation of histones
affect their charge state?
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(a) It makes them more positively charged
(b) It makes them less positively charged
(c) It does not change their charge state
(d) It depends on whether these are bacterial
histones or eukaryotic histones
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iClicker quiz, question 2
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2. Suppose a mutation in the gene coding for
histone H1 makes it fold up incorrectly. How will
this mutation influence DNA organization?
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(a) It will prevent formation of nucleosomes
(b) It will interfere with the beads-on-a-string
organization between nucleosomes
(c) It will interfere with higher-level organization
involving assembly of solenoids into loops
(d) All of the above
(e) None of the above
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Synthesizing nucleic acids
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Laboratory synthesis of nucleic acids
requires complex strategies
Functional groups on the monomeric
units are reactive and must be blocked
Correct phosphodiester linkages must
be made
Recovery at each step must high!
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Solid Phase
Oligonucleotide Synthesis
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Dimethoxytrityl group blocks the 5'-OH
of the first nucleoside while it is linked
to a solid support by the 3'-OH
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Step 1: Detritylation by trichloroacetic acid
exposes the 5'-OH
Step 2: In coupling reaction, second base is
added as a nucleoside phosphoramidate
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Synthesis I
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Figure 11.29
Solid phase oligonucleotide synthesis. The four-step
cycle starts with the first base in nucleoside form (N1) attached by its 3'-OH group to an insoluble, inert
resin or matrix, typically either controlled pore glass
(CPG) or silica beads. Its 5'-OH is blocked with a
dimethoxytrityl (DMTr) group (a).
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Blocking
groups
If the base has reactive -NH2
functions, as in A, G, or C,
then N-benzoyl or Nisobutyryl derivatives are
used to prevent their
reaction (b). In step 1, the
DMTr protecting group is
removed by trichloroacetic
acid treatment. Step 2 is
the coupling step: the
second base (N-2) is added
in the form of a nucleoside
phosphoramidite derivative
whose 5'-OH bears a DMTr
blocking group so it cannot
polymerize with itself (c).
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Solid Phase Synthesis
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Step 3: capping with acetic anhydride
blocks unreacted 5’-OHs of N-1 from further
reaction
Step 4: Phosphite linkage between N-1 and
N-2 is reactive and is oxidized by aqueous
iodine to form the desired, and more stable,
phosphate group
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Activation of the
phosphoramidate
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Cloning
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Cloning is the process whereby DNA is
copied in a controlled way to produce
desired genetic results
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Plasmids
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Small (typically < 10 kbp), usually
circular segments of DNA that get
replicated along with the organism’s
chromosome(s)
Bacterial plasmids have a defined
origin of replication and segments
defining specific genes
Some are natural; others are manmade
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How they’re used
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Typical man-made plasmid includes a gene
that codes for an enzyme that renders the
bacterium resistant to a specific antibiotic,
along with whatever other genetic materials
the experimenter or clinician wishes to
incorporate
Thus the cells that have replicated the
plasmid will be antibiotic-resistant; surviving
colonies will be guaranteed (?) to contain the
desired plasmid in all its glory
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A typical
plasmid
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Building useful
plasmids
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Take starting plasmid and
cleave it with a restriction
enzyme at a specific site
Add foreign DNA that has
been tailored to fit into that
plasmid
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Inserts
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Typically a place within the plasmid will
be set up so that small stretches (< 10
kbp) of desired DNA can be ligated in
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With sticky ends: high specificity, but you do
get self-annealing of the plasmid and of the
insert, so those have to be eliminated
With blunt ends: require more artisanry:
T4 phage ligase can rejoin ends without
stickiness; but it’s chaotic
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Directional
cloning
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Guarantees
that the
desired DNA
goes in in
exactly one
orientation
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Use of
bacteriophage
lambda
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Can handle somewhat
larger inserts (10-16
kbp)
Middle third of its
48.5-kbp chromosome
isn’t needed for
infection
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Cosmids
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14-bp sequence cos (cohesive end site):
5’-TACGGGGCGGCGACCTCGCG-3’
3’-ATGCCCCGCCGCTGGAGCGC-5’
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… one of these at each end
Must be 36 kbp < separation < 51 kbp
apart
In practice we can use these for inserts
up to 40 kbp in size
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Cosmids in
action
(fig. 12.9)
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Shuttle vectors
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These are plasmids that can operate
in two different organisms
Usually one prokaryote and one
eukaryote (e.g. Escherichia coli and
Saccharomyces cerevisiae)
Separate origins for each host
This allows us to clone the vector in a
bacterial host and then express it in a
eukaryotic setting
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Typical shuttle vector
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Artificial chromosomes
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Huge chunks (2 megabp!) can be
propagated in yeast with artificial
chromosomes
These can be manipulated in the yeast
setting or transferred to transgenic mice
in a living animal
YACs need origin, a centromere, and
telomeres
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Use of YACs in mice
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QuickTime™ and a
decompressor
are needed to see this picture.
Biochem: Recombinant DNA
10/20/2009
Diagram
courtesy
Expert
Reviews
in
Molecular
Medicine,
2003
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DNA libraries
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Set of cloned fragments that make up an
organism’s DNA
We can isolate genes from these
Most common approach to creating these is
shotgun cloning, in which we digest the total
DNA and then clone fragments into vectors
Goal is that >= 1 clone will contain at least part
of the gene of interest (might have been clipped
by the restriction enzyme!)
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Probabilities
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Probability P that some number of
clones, N, contains a particular
fragment representing a fraction f of
the genome:
P = 1 - (1 - f)N
Therefore 1-P = (1-f)N
Thus ln(1-P) = ln{(1-f)N} = Nln(1-f)
Therefore N = ln(1-P) / ln(1-f)
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What that means
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The value f is pretty small, so the
denominator is only slightly negative;
whereas we want the numerator to be ery
negative, since that corresponds to a high
value of P.
10 kbp fragments in E.coli means
f = 10/4640 = 0.0022,
so for P = 0.99, we need N=1.4*106
We’d do better with larger f values!
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Finding relevant fragments
by colony hybridization
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Plate out a library of fragments and grow
colonies or plaques
Soak those onto a flexible absorbent disc
Disc is treated with high-pH to dissociate bound
DNA duplexes; placed in a sealed bag with a
radiolabeled probe
If they hybridize, radioactivity will stick to disc
The hits can be recovered from the master plate
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Colony
hybridization
illustrated
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Making the probes
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Sometimes we have at least part of the gene
sequence and can fish for it
Other times we know the amino acid sequence
and can work backward, but with degeneracy (64
codons, 20 aa’s)
Typically use at least 17mers to guarantee that
the don’t get random association
Probes derived from a different species are
heterologous
With big eukaryotic genes we may have to look
for pieces of the gene, not the whole thing
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cDNA libraries
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Sometimes the easiest thing to obtain
are mRNA templates associated with a
particular function
Reverse transcriptase can make a
complementary (cDNA) molecule from
such an mRNA template
A library of cDNAs can be assembled
from a collection of mRNA templates
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Why is that useful?
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The mRNAs will be unique to the cell
type from which they’re derived
Often they’re also unique to the
functional role that tissue is playing
at the time
Therefore finding that collection of
DNA tells us about cellular activity
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Expressed sequence tags
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An EST is a short (~200 base) sequence
derived from a cDNA
Represents part of a gene that is being
expressed
Labeled ESTs can be mounted on a gene
chip and used to identify cells that are
expressing a particular class of mRNAs
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Southern blots I:
fractionation
Tool for identifying a particular DNA
fragment out of a vast population thereof
Exploits sequence specificity for
identification
Developed by E.M.Southern in 1975
Begins with electrophoretic fractionation
of fragments (mobility  1/mass)
Polyacrylamide gels ok 25-2000 bp;
agarose better for larger fragments
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Southern blots 2: blotting
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Gel soaked in base to denature duplexes
pH readjusted to neutral
Sheet of absorbent material placed atop the gel
Salt solution is drawn across the gel, perp to the
electrophoretic direction, in various ways to carry
the DNA onto the sheet
Sheet is dried in an oven to tightly attach the DNA
to it
Incubate sheet with protein or detergent to
saturate remaining DNA binding sites on sheet so
we don’t get nonspecific binding
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Southern blots 3:
hybridization
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Labeled probe and sheet placed in
sealed bag
If probe attaches, label will appear at
that point on the sheet via annealing
or hybridization
Label detected by autoradiography
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Southern
blots
illustrated
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Variations on this idea
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RNA can be used as the probe:
that’s called a Northern blot
Proteins can be substituted by using
an antibody as a probe and a
collection of protein fragments as the
analytes; that’s called a Western blot
Ha ha
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High-throughput
techniques
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Eagerness to provide rapid, easy-to-use
applications of these approaches has led
to considerable research on ways to
make these techniques work fast and
automatically
This high-throughput approach enables
many experiments per unit time or per
dollar
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DNA microarrays
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Thousands of oligonucleotides
immobilized on a substrate
Synthesis by solid-phase
phosphoramidite chemistry
Typically 25-base oligos
Can be used in cDNA projects to look at
expression patterns
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An
example
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Using expression vectors
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We often want to do something with
cloned inserts in expression vectors, viz.
make RNA or even protein from it
RNA: stick an efficient promoter next to
the cloning site; vector DNA transcribed
in vitro using SP6 RNA polymerase
This can be used as a way of making
radiolabeled RNA
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Protein expression
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Making (eukaryotic) proteins in bacteria via
cDNA means we don’t have to worry about
introns
Expression vector must have signals for
transcription and translation
Sequence must start with AUG and include a
ribosome binding site
Strong promoters can coax the bug into
expressing 30% of E.coli’s protein output to be
the one protein we want!
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QuickTime™ and a
decompressor
are needed to see this picture.
Example: ptac
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This is a fusion of lac promoter (lactose
metabolism) with trp promoter
(tryptophan biosynthesis)
Promoter doesn’t get turned on until an
inducer (isopropyl--thiogalactoside,
IPTG) is introduced
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iClicker quiz, question 3
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Probe systems employing RNA are called
(a) Southern blots
(b) Northern blots
(c) Western blots
(d) Eastern blots
(e) None of the above
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iClicker quiz, question 4
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4. The inducer used with the ptac
promoter system is
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(a) glucose
(b) glucose-6-phosphate
(c) IPTG
(d) ionizing radiation
(e) none of the above.
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Eukaryotic expression
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Sometimes we need the glycosylations and
other PTMs that eukaryotic expression enables
This is considerably more complex
Common approach is to use vectors derived
from viruses and having the vector infect cells
derived from the virus’s host
Example: baculovirus, infecting lepidopteran
cells; gene cloned just beyond promoter for
polyhedrin, which makes the viral capsid protein
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Screening libraries
with antibodies
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Often we have antibodies that react with a protein of
interest
If we set up a DNA library and introduce it into host
bacteria as in colony hybridization, we can put nylon
membranes on the plates to get replicas of the
colonies
Replicas are incubated to make protein
Cells are treated to release the protein so it binds to
the nylon membrane
If the antibody sticks to the nylon, we have a hit!
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Fusion proteins
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Sometimes it helps to co-express our protein of
interest with something that helps expression,
secretion, or behavior
We thereby make chimeric proteins, carrying
both functionalities
We have to be careful to keep the genes in
phase with one another!
Often the linker includes a sequence that is
readily cleaved by a commercial protease
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Fusion systems (table 12.2+)
Product
Origin
Mass, Secre Affinity Ligand
kDa
ted?
-galactosidase
E.coli
116
No
APTG
Protein A
Staph.
31
Yes
IgG
Chloramphenicol
acetyltransferase
E.coli
24
Yes
Chloramphenicol
Streptavidin
Strep.
13
Yes
Biotin
Glut-S-transferase E.coli
26
No
Glutathione
Maltose Bind.Prot. E.coli
40
Yes
Starch
Hemoglobin
16/32
No
None
Vitreoscilla
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Improving purification
via expression
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If we attach (usually at the N-terminal end) a his-tag
(several his, several cys) to our protein, it becomes
easier to purify:
The his tag forms a loop that will bind strongly to a
divalent cation like Ni2+
Thus we can pour our expressed protein through a
Ni2+ affinity column and it will stick, while other
proteins pass through
We elute it off by pouring through imidazole, which
completes for the Ni2+ and lets our protein fall off
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Protein-protein interactions
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One of the key changes in biochemistry over
the last two decades is augmentation of the
traditional reductionist approach with a more
emergent approach, where interactions
among components take precedence over
the properties of individual components
Protein-protein interaction studies are the
key example of this less determinedly
reductionist approach
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Two-hybrid screens
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Use one protein as bait; screen many
candidate proteins to see which one
produces a productive interaction with
that one
Thousands of partnering relationships
have been discovered this way
Some of the results are clearly
biologically relevant; others less so
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