Download Enzyme Mechanisms - Illinois Institute of Technology

Recombinant DNA
Andy Howard
Introductory Biochemistry
18 November 2008
Biochem: Recombinant DNA
11/18/2008
Recombinant DNA

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
Biochem: Recombinant DNA
11/18/2008
Page 2 of 37
What we’ll discuss
Cloning
 Plasmids & inserts
 Vector techniques
 Libraries & probes
 High-throughput
 Expression

Biochem: Recombinant DNA
11/18/2008
Page 3 of 37
Cloning

Cloning is the process whereby DNA is
copied in a controlled way to produce
desired genetic results
Biochem: Recombinant DNA
11/18/2008
Page 4 of 37
Plasmids



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
Biochem: Recombinant DNA
11/18/2008
Page 5 of 37
How they’re used


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
Biochem: Recombinant DNA
11/18/2008
Page 6 of 37
A typical
plasmid
Biochem: Recombinant DNA
11/18/2008
Page 7 of 37
Building useful
plasmids


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
Biochem: Recombinant DNA
11/18/2008
Page 8 of 37
Inserts

Typically a place within the plasmid will
be set up so that small stretches (< 10
kbp) of desired DNA can be ligated in


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
Biochem: Recombinant DNA
11/18/2008
Page 9 of 37
Directional
cloning

Guarantees
that the
desired DNA
goes in in
exactly one
orientation
Biochem: Recombinant DNA
11/18/2008
Page 10 of 37
Use of
bacteriophage
lambda


Can handle somewhat
larger inserts (10-16
kbp)
Middle third of its
48.5-kbp chromosome
isn’t needed for
infection
Biochem: Recombinant DNA
11/18/2008
Page 11 of 37
Cosmids

14-bp sequence cos (cohesive end site):
5’-TACGGGGCGGCGACCTCGCG-3’
3’-ATGCCCCGCCGCTGGAGCGC-5’

… 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


Biochem: Recombinant DNA
11/18/2008
Page 12 of 37
Cosmids in
action
(fig. 12.9)
Biochem: Recombinant DNA
11/18/2008
Page 13 of 37
Shuttle vectors




These are plasmids that can operate
in two different organisms
Usually one prokaryote and one
eukaryote (e.g. E.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
Biochem: Recombinant DNA
11/18/2008
Page 14 of 37
Typical shuttle vector
Biochem: Recombinant DNA
11/18/2008
Page 15 of 37
Artificial chromosomes



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
Biochem: Recombinant DNA
11/18/2008
Page 16 of 37
Use of YACs in mice

QuickTime™ and a
decompressor
are needed to see this picture.
Biochem: Recombinant DNA
11/18/2008
Diagram
courtesy
Expert
Reviews
in
Molecular
Medicine,
2003
Page 17 of 37
DNA libraries




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!)
Biochem: Recombinant DNA
11/18/2008
Page 18 of 37
Probabilities




Probability P that some number of
clones, N, contains a particular
fragment representing a fragment 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)
Biochem: Recombinant DNA
11/18/2008
Page 19 of 37
What that means



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!
Biochem: Recombinant DNA
11/18/2008
Page 20 of 37
Finding relevant fragments
by colony hybridization





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
Biochem: Recombinant DNA
11/18/2008
Page 21 of 37
Colony
hybridization
illustrated
Biochem: Recombinant DNA
11/18/2008
Page 22 of 37
Making the probes





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
Biochem: Recombinant DNA
11/18/2008
Page 23 of 37
cDNA libraries



Sometimes the easiest thing to get ahold
of 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
Biochem: Recombinant DNA
11/18/2008
Page 24 of 37
Why is that useful?



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
Biochem: Recombinant DNA
11/18/2008
Page 25 of 37
Expressed sequence tags



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
Biochem: Recombinant DNA
11/18/2008
Page 26 of 37





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
Biochem: Recombinant DNA
11/18/2008
Page 27 of 37
Southern blots 2: blotting






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
Biochem: Recombinant DNA
11/18/2008
Page 28 of 37
Southern blots 3: hybridization



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
Biochem: Recombinant DNA
11/18/2008
Page 29 of 37
Southern
blots
illustrated
Biochem: Recombinant DNA
11/18/2008
Page 30 of 37
Variations on this idea



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
Biochem: Recombinant DNA
11/18/2008
Page 31 of 37
High-throughput
techniques


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
Biochem: Recombinant DNA
11/18/2008
Page 32 of 37
DNA microarrays




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
Biochem: Recombinant DNA
11/18/2008
Page 33 of 37
An
example
Biochem: Recombinant DNA
11/18/2008
Page 34 of 37
Using expression vectors



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
Biochem: Recombinant DNA
11/18/2008
Page 35 of 37
Protein expression




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!
Biochem: Recombinant DNA
11/18/2008
Page 36 of 37
QuickTime™ and a
decompressor
are needed to see this picture.
Example: ptac


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
Biochem: Recombinant DNA
11/18/2008
Page 37 of 37