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Chapter 20: DNA Technology and Genomics

Chapter 20: DNA Technology and Genomics. Important Point:. If you are having trouble understanding lecture material: Try reading your text before attending lectures. And take the time to read it well!.

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Chapter 20: DNA Technology and Genomics

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  1. Chapter 20:DNA Technologyand Genomics

  2. Important Point: If you are having trouble understanding lecture material: Try reading your text before attending lectures. And take the time to read it well!

  3. DNA technology is the chemical manipulation of the genotypes and resulting phenotypes of organisms such that living organisms are modified • Alternatively, no-longer-living organisms or their no-longer-living parts may be analyzed chemically at the level of genotype • DNA technology has revolutionized how scientists study the genetics, biochemistry, even the ecology and evolutionary biology of organisms • Genetic engineering is the artificial manipulation of the genetic material of organisms, including the creation of novel genetic material (i.e., novel nucleotide sequences) • Biotechnology is the development of novel biological products, indeed whole industries are now devoted to the production and analysis of biological materials DNA Technology

  4. Cloning Step in Overview

  5. Clone Identification • Probing for correct DNA sequence • Probing for correct protein product • Antibiotic resistance • β-galactocidase expression • Etc. • Once you have the correct clone, then what? • Subcloning • Expression vector • Protein characterization • Protein purification • Etc. Have Transformant, then What? “The final and most difficult part of cloning a particular gene is identifying a colony containing that gene among the many thousands of colonies carrying other pieces of… DNA.” p. 388, Campbell & Reece (2005)

  6. Utilizing Cloned Genes

  7. Restriction Enzymes

  8. 5’…GAATTC…3’ 3’…CTTAAG…5’ 5’…G -OHP-AATTC…3’ 3’…CTTAA -PHO-G…5’ Restriction Endonucleases • A Restriction Endonucleases will cut both strands of a DNA duplex at a specific place • These “places” need not be directly opposite: 5’…GAATTC…3’ 3’…CTTAAG…5’ 5’…G -OHP-AATTC…3’ 3’…CTTAA -PHO-G…5’ • Note that the above enzyme is EcoRI, the first restriction endonuclease characterized

  9. Sticky Ends

  10. Most R.E. RecognitionSequences are Palindromes G^AATT-C C-TTAA^G G^GATC-C C-CTAG^G Nodeba Bob Abedon A^GATC-C T-CTAG^G GC^GGCC-GC CG-CCGG^CG

  11. More RE Enzymes

  12. DNA Ligase Upon ligation we now have recombinant DNA

  13. Ligation

  14. Cloning using Restriction Enzymes

  15. Note that plasmid is vector that carries DNA into recipient cells via transformation Transformation Other vectors include viruses (transduction) as well as otherwise inert projectiles

  16. One example of this might be done… is essentially cloning into animals Gene Therapy

  17. Recombinant Animals Alternatively, DNA can be cloned directly into the germ line

  18. Injecting DNA into Egg

  19. Many plants are easily cloned from individual body cells Cloning into Plants

  20. Selection for Specific Clones

  21. Selection: Nucleic Acid Hybridization

  22. Genomic Library

  23. Cloning allows the amplification of genotype (DNA) as well as phenotype (proteins) Polymerase Chain Reaction If all you really need is the DNA, then PCR is an easy way to amplify DNA without cloning

  24. PCR DNA Amplification

  25. PCR DNA Amplification

  26. Rather than making cDNA, one can clone directly into eukaryotic cells, which addresses concerns that bacteria do not modify proteins post-translationally to the extent that eukaryotes do Complementary (c)DNA

  27. Gel Electrophoresis

  28. Loading Gel

  29. Loaded Gel

  30. Run Gel

  31. 2-D Protein Electrophoresis

  32. 2-D Protein Electrophoresis Visualized proteins

  33. Restriction Fragment Analysis No need to know details of what gene was studied or specific restriction enzyme used

  34. Southern Blotting

  35. Genomic Mapping

  36. DNA Sequencing

  37. DNA Sequencing

  38. DNA Sequencing

  39. DNA Sequencer

  40. Shotgun Sequencing

  41. Genome Comparisons

  42. Microarray Analysis

  43. Online Tools • Buying primers (custom oligos): http://www.qiagen.com/ • 2x2 sequence comparisons: http://www2.igh.cnrs.fr/bin/align-guess.cgi • n x n sequence comparisons: http://www.genebee.msu.su/services/malign_reduced.html • Sequence manipulation: http://arbl.cvmbs.colostate.edu/molkit/manip/index.html • Sequence translation: http://arbl.cvmbs.colostate.edu/molkit/translate/index.html • Restriction Enzymes: http://rebase.neb.com/rebase/rebase.html • Restriction sites: http://www.ccsi.com/firstmarket/cutter/cut2.html • Sequence searches: http://www.ncbi.nlm.nih.gov/blast/ • Other tools: http://molbiol-tools.ca/

  44. The End

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