Unit 9 1 UNIT 9 RECOMBINANT DNA METHODS Recombinant DNA techniques have been invaluable to the study of genes and the determination of the biological function of the proteins which they encode. Recombinant DNA technology has also been used to isolate genes so that their protein products can be produced on a large scale in bacteria. The ability to produce these products has had a profound impact on medicine. For example the cloning of the insulin gene has alowed researchers to produce the protein in bacteria on a large scale, instead of isolating it from amalian sources. Other proteins of commercial and medical interest are the human growth hormone and interleukins. In this unit we wil describe the proceses whereby a gene is isolated, identified, and manipulated in the laboratory as wel as some aspects of genomics and proteomics. Asignment: Nelson & Cox, pp. 292 - 295, 303 - 338, 1145-1146. 1. Use Fig. 8-33 (p. 293) to discuss the Sanger method for DNA sequencing. a. Draw the structure of ddATP (you may use A as an abbreviation for the structure of the base). Explain how incorporation of a dideoxynucleoside wil afect subsequent polymerization by DNA polymerase. b. How many reactions are included in a typical DNA sequencing experiment. Unit 9 2 1) Describe the composition of each reaction. 2) Review the definition of "radioactive isotope" given in the glossary of the text book (p. G-13). How is radioactivity incorporated into the sequences? c. Given the sequence and primer, write the sequences that would be produced using the dideoxy analog of dATP. Do the same for the other thre dideoxy analogs. 3' AGCTATCGTGAC 5' 5' TCGA 3' d. The bands sen on the electrophoresis gel difer in length by how many nucleotides? e. Do problem 12 on p. 300. 2. In a more modern and more highly automatable version of the original Sanger method, fluorescent tags can be atached to the dideoxynucleotides to make them detectable by fluorescence measurements (Fig. 8-34, p. 294). Review the definition of "fluorescence" given in the glosary of the text book (p. G-6). Distinguish betwen radioactivity (p. G-13) and fluorescence. a. Why is it possible with this method to have only a single reaction mixture? b. After separating the fragments, how do you know which fragment stopped at which residue? c. Why does this method alow more sequence information to be derived from a single sequencing reaction? Unit 9 3 3. Currently several sequencing methods are being developed which do not separate the synthesized DNA, making the proces much les expensive. One that is commercialy available now is caled "Pyrosequencing" because it takes advantage of the Pi that is released when a dNTP is incorporated into the growing DNA strand. dNTP's are added one at a time and the Pi is detected by converting it to ATP and using the ATP to drive light formation by luciferase. In one version of this method thousands of reactions are run simultaneously on a plate and the genome of a bacterium can be sequenced in a single day by one person. Optional: To se more about this method check the following web site: http:/ww.genoseq.ucla.edu/action/view/Pyrosequencing 4. Use Fig. 9-1 (p. 304) to describe the five general procedures in DNA cloning. 5. Restriction Endonucleases (pp. 304 - 307) a. Thousands of restriction endonucleases have been discovered. Are restriction enzymes made by eukaryotes or prokaryotes? What is their function in vivo? Discuss the restriction-modification system (pp. 304 - 305). b. Select from Table 9-2 (p. 305) a restriction endonuclease that generates sticky ends and one that generates blunt ends. c. When two DNA fragments generated by the same restriction endonuclease are mixed together, the complementary ends pair and form hydrogen bonds (a proces caled annealing), DNA ligase then forms phosphodiester bonds that covalently join the fragments, and the restriction endonuclease cleavage site is restored. Use Fig. 9-2 (p. 306) to ilustrate this point. Unit 9 4 6. Vectors (pp. 307 - 310) a. Define (p. 307): 1) Plasmid 2) Transformation b. What is the purpose of each of the following: 1) the selectable marker gene (ex. antibiotic resistance gene) 2) the origin of replication (ori) c. Use Fig. 9-4 (p. 308) to ilustrate how plasmids are used to clone DNA. d. Discuss when you might use a bacteriophage, a bacterial artificial chromosome (BAC), or a YAC vector instead of a plasmid (pp. 308 - 310). 7. Use Fig. 9-8 (p. 311) to describe how hybridization is used to identify a clone with a specific DNA sequence. a. Use Fig. 9-9 (p. 311) to describe how the probe sequence can be determined if the amino acid sequence of a protein is known. Note that sometimes antibodies are alternatively used as probes to scren cDNA libraries prepared in expresion vectors for clones containing the gene encoding a specific protein. Do problem 4 on p. 340. b. Synthesis of single stranded DNA probes (pp. 294 - 295). Use Fig. 8-35 (p. 295) to describe how DNA probes of a specific sequence are synthesized to be used as a probe. (Focus on an overview of the process rather than the specific details of the chemistry.) Unit 9 5 8. If your goal is to expres a eukaryotic gene in E. coli, you wil use an expresion vector (p. 312). a. Discuss the importance of each region of the plasmid shown in Fig. 9-10 (p. 312). b. Why is it necesary to have a promoter in the expresion vector when using cDNA inserts? c. With very strong promoters, expresion vectors sometimes recruit many of the cel's transcription and translation enzymes to "over- expres" the cloned gene. Discuss why is it desirable to have a regulatable promoter in an expresion vector. 9. Define site directed mutagenesis (pp. 312 - 313). a. Use Fig. 9-11 (p. 313) to distinguish oligonucleotide directed mutagenesis (b) from the synthetic insert method (a). b. Describe the two types of progeny plasmids that would result from oligonucleotide directed mutagenesis. What wil be the ratio of one to the other? c. Suppose you want to use oligonucleotide directed mutagenesis to make a mutant gene that encodes a protein containing phenylalanine rather than tyrosine. Given the following DNA and protein sequence, write the sequence of the primer you would design to make the mutant protein. .... Cys Ser Leu Tyr 14 Gln Leu Glu.. .... TGT AGT CTC TAT CA TA GA.. Unit 9 6 10. Use Fig. 9-12 (p. 314) to describe a procedure used to tag proteins thereby facilitating their purification using afinity chromatography techniques. 11. From Genes to Genomes (pp. 315 - 317): What is the meaning of the term "library" to a molecular biologist? a. Genomic Libraries (p. 315) 1) What is a genomic library (p. 315)? 2) Describe the preparation of a genomic library (p. 315). 3) Use Fig. 9-13 (p. 315) to discuss how individual clones in a library can be ordered. What is a "contig"? hat is an "STS"? b. cDNA Libraries (pp. 316 - 317) 1) What is a cDNA library (p. 316)? 2) Use Fig. 9-14 (p. 316) to describe the synthesis of cDNA. 3) What is an "EST" (p. 316)? 4) Use Fig. 9-15 (p. 316) to discuss how fusions with the gene for gren fluorescent protein can be used to localize specific proteins within cels and organisms. 5) Use Fig. 9-15 (p. 316) to describe how fusions with epitope tags can be used to identify interacting proteins. Unit 9 7 c. Genomic and cDNA libraries compared 1) In preparing a genomic library for a given kind of animal or plant, does it mater from which type of cel you prepare the DNA? Why or why not? 2) In preparing a cDNA library, does it mater from which type of cel you prepare the mRNA? In answering this question keep in mind that there are genes (caled "housekeeping genes") that are expresed in almost al cels?for example the genes that code for glycolytic enzymes. Other genes are expresed in some cel types but not in others. Given this information: How would a cDNA library prepared from ouse liver cels difer from a cDNA library prepared from mouse thyroid cels? List thre types of DNA (i.e. not including genes for specific proteins) which would be present in a complete mouse genomic library which would not be present in a cDNA library made from mouse thyroid cels. To answer this question think of regions of the genome that are not transcribed at al and regions that are transcribed but are not expresed as proteins. 12. Polymerase Chain Reaction (PCR, pp. 317 - 318) a. What is needed in order to amplify a segment of DNA using PCR (p. 317)? b. Use Fig. 9-16 (p. 318) to discuss the selection of primers and the thre repeated steps in the proces: Heating, Cooling, and Replication. c. For each copy of the target sequence that you started with, how many copies do you expect after cycle 10 (asuming that the amplification performs perfectly in each cycle)? d. A normal enzyme is denatured above 50-60 ° C, but in each cycle of PCR the mixture is heated above 90 ° C. Explain why the use of DNA polymerase from the bacterium Thermus aquaticus (found in hot springs) made a big improvement in this procedure. Unit 9 8 e. Among its many other uses, PCR is finding important applications in early clinical diagnosis of disease. An example is in testing for the presence of viruses to which antibodies do not appear until a long time after infection. Describe how PCR could be used to detect HIV infection at an early stage, including what information about HIV would be needed to begin with. f. Do problem 5 on p. 340. g. Now that we know the sequence of many genomes, PCR wil be used to get genes for expresion and study instead of traditional cloning procedures learned earlier in this unit. Much of the work wil be caried out at a computer terminal instead of at the lab bench. In the series of steps below, computer operations are given in bold faced text, lab operations are not bold. 1) If you had the amino acid sequence of a short segment of a protein that you wanted to study from humans, how would you locate the gene in the genomic data base? 2) Once the part of the gene that encoded these amino acids was located, how would you recognize the complete ORF (open reading frame) in the data base? 3) Once the ORF is identified, PCR primers can be designed based on the sequences that flank it. 4) The primers can be mixed with cDNAs and PCR can be used to amplify this gene out of the mixture. h. In addition to isolating or amplifying specific sequences of DNA for various uses, PCR can be used to quantitate the amount of a specific DNA or RNA in a sample. Caled "Real Time PCR", this method uses a dye that fluoresces when bound to double stranded DNA to measure the amount of PCR product as the reaction is being run. From the number of cycles needed to reach a given level, one can calculate the amount of the sequence in the original sample (Not in text). Unit 9 9 13. List and describe two types of sequence variation used in DNA Fingerprinting (p. 319). a. Describe the Southern Blotting procedure as shown in Fig. 1 (p. 319). Which type of sequence variation is analyzed using Southern blots? b. Use Fig. 2 (p. 321) to describe the analylsis of STRs in DNA Fingerprinting. Do problems 8 and 11 on p. 341. 14. The Human Genome a. Use Fig. 9-17 (p. 322) to describe the strategy for determining the human genome sequence used in the International Human Genome Project. b. How does the "whole genome shotgun sequencing strategy" difer from the strategy used by the international group (p. 322)? c. Besides human, list several other genomes that have been sequenced (p. 322). d. The Human Genome (pp. 322 - 323) 1) How many genes do we actualy have (p. 323)? 2) How can we make more diferent proteins than we have genes in our genome (review Fig. 26-20, p. 1041)? 3) If only 1.1 - 1.4% of our genome encodes protein, use Fig. 9-19 (p. 323) to describe the rest. 4) What is a "SNP" (p. 323)? Unit 9 10 15. From Genomes to Proteomes (pp. 324 - 330) a. Define "proteome" (p. 324). b. List the thre main paths to investigating protein functions (p. 324). 1) Sequence or structural relationships can be studied to provide information on protein function (comparative genomics; p. 325): Define: ortholog: paralog synteny 2) Determining celular expresion paterns to determine protein function Describe 2-D gel electrophoresis (p. 325; also review Fig. 3-21, p. 91). How can 2-D gel electrophoresis be used to identify changes in gene expresion (p. 325)? How can a protein visualized on a 2-D gel be identified in the genomic database (p. 325)? What is a DNA Microaray (pp. 325 - 328) Use Fig. 9-21 (p. 326) to describe the photolithographic method for fabricating DNA microarays. Use Fig. 9-22 (p. 327) to discuss how a DNA microaray (or chip) can be used to study changes in gene expresion that acompany development. Note that the microaray is probed with two samples prepared with diferent fluorescent markers giving the relative abundance of the sequence of interest in each sample. Unit 9 11 DNA microarays can also be used to analyze sequences in DNA and wil be increasingly used for medical diagnosis (e.g. for the detection of DNA variation known to be linked to disease). How can "protein chips" be used to study gene expresion? (p. 328). 3) Detection of Protein - Protein interactions to determine celular function. Use Fig. 9-24 (p. 328) to discuss how genome sequence information can be used to deduce specific protein interactions. Discuss how an epitope tagged protein can be used to purify and identify proteins with which it interacts (p. 328). Use Fig. 9-25 (p. 329) to describe how the yeast two-hybrid system is used to identify interacting proteins. 16. Plant Biotechnology (pp. 330 - 332) a. Use Fig. 9-26 (p. 330) to show how infection of a plant with Agrobacterium tumefasciens results in tumor formation. b. Use Fig. 9-28 (p. 331) to show how Agrobacterium is used to introduce DNA into plant cels. c. List some traits introduced into plants using these methods (p. 332). 17. Use Fig. 9-32 (p. 334) to ilustrate how a retrovirus can be used to transfer DNA into mamalian cels. 18. Human Gene Therapy (Box 9-2, pp. 335 - 336) - this has been more dificult than was originaly predicted and is a very distant goal. Unit 9 12 19. List several medicaly and/or commercialy important proteins that are currently produced using recombinant DNA technologies (Table 9-4, p. 337). 20. What are micro-RNAs (review pp. 1145-1146). Explain how RNAi can be used as a gene silencing method. Jim Blankenship Microsoft Word - U09_F08.doc
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