Text of 2012 recombinant dna methods and technology(1)
1.RECOMBINANT DNAMETHODS ANDTECHNOLOGY Matthew George, Jr., Ph.D.
2. Recombinant DNA and Genetic Engineering A recombinant DNA molecule is produced by splicingtogether DNA from different sources. It is usually done forone of the following reasons: To clone large amounts of DNA for the studies of geneorganization; structure; sequencing; and gene regulation To genetically engineer cells in an organism to confer newdesired characteristics (i.e., gene therapy, and correctgenetic defects) To produce the product(s) of spliced genes in bacterial cells(i.e. insulin, human growth hormone) Alter the genetic characteristics of fruits and vegetables To produce organisms (i.e. Dolly, cows and mice fortherapeutic purposes)STEM CELL RESEARCH: to produce new tissues and organs 3. Recombinant DNA Product Effect? 4. THE UTILITY OF RECOMBINANT DNA TECHNOLOGY 5. RESTRICTION ENDONUCLEASES 6. BASIC CLONING STRATEGY 7. EcoRI Restriction Endonuclease 8. EcoRI Recombinant 9. Vector Characteristics 10. Basic components of a plasmid cloning vector thatcan replicate within an E. coli cell Molecular Cell Biology, 7th Edition, Lodish et al. 11. CLONING VECTORS 12. Structure of pBR322 - a common cloning vector derived from a naturally occurring plasmidhas unique restriction enzyme cleavage sites for insertion of foreign DNA (100bp to 10kb) has antibiotic resistance genes for selection of transformants containing the plasmid (insertion of DNA at these sites will result in the bacteria becoming sensitive to that antibiotic)has origin of DNA replication (ori) for propagation in E. coliFig. 7.7 13. Yeast Artificial Chromosome (YAC)Normal ends ofeukaryotic chromosomes 14. DNA cloning in a plasmid vector permits amplification of a DNA fragment*See Devlin, fig. 7.9; *Molecular Cell Biology 7th Edition, Lodish et al. 15. Cloning a segment of DNA into a plasmid vect orHuman DNA cut with PstIPstI PampR tetR pBR322 ampR, tetR P P combineandligateP tetRpBR322 DNA cut with PstI inactivating the amp R genetetRpBR322 (human clone) tet R bacteria are transformed with the recombinant plasmid colonies that grow in tetracycline, but not in ampicillin are isolated 16. Antibiotic Selection R 17. cDNA Formation 18. A cDNA library contains representative copies of cellular mRNA sequences*See Devlins figure 7.16; *Molecular Cell Biology, 7th Edition, Lodish et al. 19. Genomic DNA Library 20. Lambda Phage Cloning 21. See fig. 7.13 22. Northern Blotting(Transfer of RNA from a gel to a solid matrix)Gives information about whether RNAs are presentin a tissue. Often used to determine whetherparticular genes are expressed. Does NOT giveinformation about whether the gene is present inother tissues. Presence of GlucokinaseHuman Liver Human Muscle Rat Brain E. coli ------ 23. Expression Vectors Fig. 7.22 24. Some eukaryotic proteins can be produced in E. coli cells from plasmid vectors containing the lac promoterMolecular Cell Biology, 7th Edition, Lodish et al. 25. Detection of Expression 26. PCR 27. The CSI Effect 28. Sanger Dideoxy SequencingFig. 7.5 29. Dideoxynucleotide 30. Transgenic animals - the gene of interest together with appropriate regulatory elements ismicroinjected into a fertilized mouse egg which is then inserted into the uterus of a fostermother mouse 31. Microarray Technology 32. siRNA: Gene Silencing 33. Thank you for your time andpatience; make us proud andgood luck!!!