11-09-11 Recombinant DNA Reverse genetics Synthesis of DNA probes

1. 11-09-11 Recombinant DNA • Reverse genetics • Synthesis of DNA probes • Restriction enzymes, plasmids and recombinant DNA • Genomic and cDNA libraries • Applications of DNA technology • Polymerase Chain Reaction (PCR) • DNA sequencing • DNA microarrays

2. Reverse Genetics Allows the Synthesis of Nucleic Acids from a Protein Sequence • Given a partial primary sequence of a protein of interest, reverse genetics can be used to produce full length copy of the gene for that protein • The partial primary sequence may be obtained by Edman degradation or from the known sequence from another source • The partial amino acid sequence allows synthesis of a specific nucleic acid “probe” via automated methods

3. Synthesis of DNA probes • Activated monomers are added to a growing chain that is linked to an insoluble support • The activated monomers are protonated deoxyribonucleoside 3-phosphoramidites in which all but one of the reactive groups of the nucleotide are protected by the temporary • attachment of nonreactive groups • In step 1, the 3-phosphorus atom of this incoming unit joins the growing chain to form a phosphite triester intermediate

6. In step 2, the phosphite triester is oxidized by iodine to stabilize the new linkage. • In step 3, the protecting group on the 5-OH group of the growing chain is removed by the addition of dichloroacetic acid. • The cycle is repeated until the desired polynucleotide chain is obtained • A DNA probe can be made by labeling one end of the polynucleotide chain with 32PO4 or a fluorescent tag • For example, a probe for the estrogen receptor

7. Recombinant DNA Technology • Restriction enzymes (endonucleases) split DNA into specific fragments • Restriction enzymes protect bacteria from invasion by foreign DNA from bacteriophages, etc. • Restriction sites are palindromic, containing two-fold rotational symmetry (inverted repeats)

9. Following cleavage, restriction fragments can reanneal via “sticky ends” and be ligated with ligase to form intact DNA

10. Molecular Cloning includes the isolation of DNA from a donor cell and splicing it into a vector • A vector is a DNA molecule capable of carrying a foreign DNA sequence and replicating in a host cell • Different vectors are chosen depending on the size of the donor DNA: • bacterial plasmid (15 kb) • bacteriophage l vectors (24 kb) • cosmid (50 kb) • bacterial artificial chromosomes • (300 kb) • yeast artificial chromosomes • (1,000 kb)

11. Requires a restriction enzyme and DNA ligase • The vector is then introduced into the host cell • This can be relatively easy (transformation and electroporation) or very complex (microinjection) • Transgenic animals are created by microinjection • Once introduced, each host cell replicates the recombinant DNA along with its own genome

12. Four Recombinant E. coli Cells, Each with a Different Variant of the Gene for Luciferase • Marker genes are often used to identify cells that have been transformed • Markers can confer antibiotic resistance (i.e., ampr) or allow the host cell to synthesize a nutrient that it couldn’t before transformation (e.g., LEU2 gene of yeast)

13. With colony hybridization technique, bacteria are screened using a radioactively labeled RNA or ssDNA probe complementary to that of DNA within the recombinant • Autoradiography is used to identify hybridization between the probe and the target DNA

14. Genomic Libraries are collections of clones derived from an entire chromosome or genome • Important uses include genome sequencing or isolating specific genes whose chromosome location is unknown • Produced in a process called shotgun cloning in which a genome is randomly digested and spliced into vectors

15. The library can then be introduced into E. coli cells Creation of a DNA Library Using the Shotgun Method

16. Or the library can • be introduced into • l bacteriophage

17. Eukaryotic Genes Can Be Manipulated with Considerable Precision • Complementary DNA (cDNA)prepared from mRNA can be expressed in host cells • Reverse transcriptase from retroviruses is used to make a DNA copy of mRNA • Reverse transcriptase - an RNA-directed DNA polymerase that synthesizes a DNA strand complementary to an RNA template using a DNA primer - [oligo(T)] sequence pairs with the poly(A) of mRNAs

20. cDNA Libraries cDNAs reflect the actual mRNA content of a specific cell; differs from genomic DNA http://www.maxanim.com/genetics/cDNA/cDNA.htm

21. cDNA Libraries 1. Isolate mRNA from target cell 2. Copy mRNA to cDNA using reverse transcriptase 3. Create cDNA library 4. cDNAs will provide the coding sequence for a gene rather than the actual gene sequence; comparison with sequences in the genomic library reveals introns

22. cDNA libraries can be screened using a specific DNA probe

23. cDNA Libraries Can Be Screened for Synthesized Protein • Expression vectors contain cDNA inserted behind powerful promoters with a gene segment that codes for ribosome binding • Immunological methods using an antibody probe specific for the protein of interest can be used to identify clones that specifically express the protein of interest

25. Applications for Recombinant DNA Technology 1. Recombinant proteins a. Bovine growth hormone b. Transformation of animal cells c. Therapeutic gene products 2. Genetically altered organisms a. Bacteria – ice minus bacteria, bioremediation, mining b. Plants c. Animals 3. Human gene therapy

26. The Polymerase Chain Reaction (PCR) provides large amounts of DNA of a specific sequence • Heat-stable DNA polymerase from Thermusaquaticus (Taq polymerase) is used to amplify any sequence as long as some flanking sequence is known • The reaction includes template DNA, Taq polymerase and ingredients required for a polymerization reaction

27. Step 1: High heat denatures the DNA Step 2: Cooling the reaction allows the primers to anneal Step 3: DNA synthesis occurs at an optimal temperature for polymerization This process is repeated 25–30 times depending on the protocol to amplify a single DNA fragment one billion times in less than two hours

30. http://www.maxanim.com/genetics/PCR/PCR.htm

31. The Sanger Dideoxy Method allows DNA to be Sequenced by Controlled Termination of Replication

32. Drugs that inhibit retroviral reverse transcriptase are often chain terminating nucleotides

33. http://www.dnalc.org/resources/3d/29-sanger-sequencing.html http://www.youtube.com/watch?v=oYpllbI0qF8 http://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter18/sanger_sequencing.html

35. DNA microarrays are used to analyze the expression of thousands of genes at once • Uses DNA “chips” that have thousands or hundreds of thousands of oligonucleotides or ssDNA fragments spotted on them

36. These chips are probed with fluorescent dye labeled cDNA that was reverse-transcribed from mRNA • Represents the transcriptome from the cell type used • Positions that are fluorescing and the intensity of that fluorescence reveals the gene expression pattern for the cell • Useful for comparing cells under different conditions (e.g., cancerous versus normal cells)