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11/18/2008Biochem: Recombinant DNA
Recombinant DNA
Andy HowardIntroductory Biochemistry
18 November 2008
11/18/2008Biochem: Recombinant DNA
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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
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What we’ll discuss Cloning Plasmids & inserts Vector techniques Libraries & probes High-throughput Expression
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Cloning
Cloning is the process whereby DNA is copied in a controlled way to produce desired genetic results
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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 man-made
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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
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A typical plasmid
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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
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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
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Directional cloning Guarantees that the desired DNA goes in in exactly one orientation
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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
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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
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Cosmids in action(fig. 12.9)
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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
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Typical shuttle vector
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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
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Use of YACs in mice
QuickTime™ and a decompressor
are needed to see this picture.
Diagram courtesy
Expert Reviews
in Molecula
r Medicine
, 2003
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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!)
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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)
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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!
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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
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Colony hybridization illustrated
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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
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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
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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
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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
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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
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 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 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
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
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 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 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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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
QuickTime™ and a decompressor
are needed to see this picture.