recombinant dna

10/20/2009Biochem: Recombinant DNA

Recombinant DNA

Andy HowardIntroductory Biochemistry

20 October 2008


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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

Synthesis of DNA in the laboratory

Cloning Plasmids & inserts Vector techniques Libraries & probes High-throughput Expression

Fusion Proteins Protein-protein interactions


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iClicker quiz, question 1

1. How does acetylation of histones affect their charge state? (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 2. Suppose a mutation in the gene coding for histone H1 makes it fold up incorrectly. How will this mutation influence DNA organization? (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

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

Dimethoxytrityl group blocks the 5'-OH of the first nucleoside while it is linked to a solid support by the 3'-OH 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 Figure 11.29Solid phase oligonucleotide synthesis. The four-step cycle starts with the first base in nucleoside form (N-1) 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 groupsIf the base has reactive NH2

functions, as in A, G, or C, then N-benzoyl or N-isobutyryl 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

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

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. 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 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 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 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 meansf = 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 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?

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.


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iClicker quiz, question 3 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 4. The inducer used with the ptac promoter system is (a) glucose (b) glucose-6-phosphate (c) IPTG (d) ionizing radiation (e) none of the above.


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Eukaryotic expression 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

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 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,kDa

Secreted?

Affinity Ligand

-galactosidase

E.coli

116 No APTG

Protein A Staph.

31 Yes IgG

Chloramphenicolacetyltransferase

E.coli

24 Yes Chloram-phenicol

Streptavidin Strep.

13 Yes Biotin

Glut-S-transferase

E.coli

26 No Glutathione

Maltose Bind.Prot.

E.coli

40 Yes Starch

Hemoglobin Vitreo-scilla

16/32

No None


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Improving purification via expression

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

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

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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