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Recombinant DNA

Recombinant DNA. Andy Howard Introductory Biochemistry 20 October 2008. Recombinant DNA.

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Recombinant DNA

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  1. Recombinant DNA Andy HowardIntroductory Biochemistry 20 October 2008 Biochem: Recombinant DNA

  2. 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

  3. Synthesis of DNA in the laboratory Cloning Plasmids & inserts Vector techniques Libraries & probes High-throughput Expression Fusion Proteins Protein-protein interactions What we’ll discuss Biochem: Recombinant DNA

  4. 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 Biochem: Recombinant DNA

  5. 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 Biochem: Recombinant DNA

  6. 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! Biochem: Recombinant DNA

  7. 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 Biochem: Recombinant DNA

  8. 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). Biochem: Recombinant DNA

  9. Blocking groups If 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). Biochem: Recombinant DNA

  10. 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 Biochem: Recombinant DNA

  11. Activation of the phosphoramidate Biochem: Recombinant DNA

  12. Cloning • Cloning is the process whereby DNA is copied in a controlled way to produce desired genetic results Biochem: Recombinant DNA

  13. 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 Biochem: Recombinant DNA

  14. 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

  15. A typical plasmid Biochem: Recombinant DNA

  16. 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

  17. 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

  18. Directional cloning • Guarantees that the desired DNA goes in in exactly one orientation Biochem: Recombinant DNA

  19. 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

  20. 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

  21. Cosmids in action(fig. 12.9) Biochem: Recombinant DNA

  22. 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 Biochem: Recombinant DNA

  23. Typical shuttle vector Biochem: Recombinant DNA

  24. 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

  25. Use of YACs in mice • Diagram courtesy Expert Reviews in Molecular Medicine, 2003 Biochem: Recombinant DNA

  26. 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

  27. 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) Biochem: Recombinant DNA

  28. 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! Biochem: Recombinant DNA

  29. 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

  30. Colony hybridization illustrated Biochem: Recombinant DNA

  31. 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

  32. 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 Biochem: Recombinant DNA

  33. 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

  34. 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

  35. 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

  36. 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

  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

  38. Southern blots illustrated Biochem: Recombinant DNA

  39. 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

  40. 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

  41. 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

  42. An example Biochem: Recombinant DNA

  43. 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

  44. 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

  45. 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

  46. 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 Biochem: Recombinant DNA

  47. 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. Biochem: Recombinant DNA

  48. 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 Biochem: Recombinant DNA

  49. 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! Biochem: Recombinant DNA

  50. 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 Biochem: Recombinant DNA

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