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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