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Chapter 23 (Part 1)

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  1. Chapter 23 (Part 1) Recombinant DNA Technology

  2. Recombinant DNA Technology • Methods for isolating, manipulating, and amplifying identifiable DNA sequences. • Allows us to study the structure and function of individual genes. • Allows for the directed genetic manipulation of organism (modify gene function, insert novel genes)

  3. Cloning • Clone: a collection of molecules or cells, all identical to an original molecule or cell • To "clone a gene" is to make many copies of it - for example, in a population of bacteria • Gene can be an exact copy of a natural gene • Gene can be an altered version of a natural gene • Recombinant DNA technology makes it possible • Allows for in vitro manipulation of a individual gene

  4. Tools Needed for Cloning(Think of it as a cutting and pasting process) • cDNA or genomic library (source of DNA to cut) • Plasmid (where you want to paste it) • Restriction enzymes (scissors) • DNA ligase (paste) • E. coli (biological machine needed to amplify DNA)

  5. Plasmids • Naturally occurring extrachromosomal DNA • Self replicating circular double stranded DNA molecules that have their own origin of replication • Usually present in multiple copies per cell • Plasmids can be cleaved by restriction enzymes, leaving sticky ends • Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid

  6. Cloning Vector Required features • Origin of replication • Selectable marker • Screenable marker for recombinant molecules • Cloning sites

  7. Restriction Enzymes • Bacteria protect themselves from attack by viruses and other bacteria using a restriction/modification system. • Allows bacteria to recognize and destroy foreign DNA • Bacteria contain DNA methylases that modify their chromosomal DNA at specific sequences. • Also contain restriction endonucleases that recognize and cleave these same sequences when they are not methylated

  8. AAGATGCGAATTCGTACA AAGATGCGAATTCGTACA DNA methylase DNA methylase * * AAGATGCGAATTCGTACA AAGATGCGAATTCGTACA Restriction endonuclease Restriction endonuclease * * AAGATGCG AATTCGTACA AAGATGCGAATTCGTACA Restriction Modification System

  9. Restriction Enzymes • Type I – Contain methylase and endonulcease fuctions. Require ATP for hydrolysis and S-adenosylmethionine for methylation • Type II – contain only endonulcease function,. Does not require ATP for hydrolysis. • Both types recognize palindrome sequences (sequences that read the same if read forward or backwards – e.g. “BOB” or “DEED”

  10. Type II Restriction Enzymes • Names use 3-letter italicized code: • 1st letter - genus; 2nd,3rd - species • Following letter denotes strain • EcoRI is the first restriction enzyme found in the R strain of E. coli

  11. 5’ ATGCGAATTCCGGTT 3’ 3’ TACGCTTAAGGCCTT 5’ EcoR1 5’-ATGCG-3’ 5’-AATTCCGGTT-3’ 3’-TACGCTTAA-5’ 3’-GGCCTT-5’ 5’ ATGCGATATCCGGTT 3’ 3’ TACGCTATAGGCCTT 5’ EcoRV 5’-ATGCGAT-3’ 5’-ATCCGGTT-3’ 3’-TACGCTA-5’ 3’-TAGGCCTT-5’ Sticky-end cutter Blunt-end cutter

  12. Restriction Enzymes • Restriction enzymes can recognize specific 4 base, 6 base, 8 base sequences. • The probability that a given piece of DNA will contain a specific restriction site is = n4 • n = the number of bases in the restriction site • So for a 6 base cutter (64), you would expect to find your site every ~1300 base pairs. So in a 10,000 bp fragment there is likely to by 7 or 8 restriction sites corresponding to your enzyme. • You can characterize DNA fragments using gel electrophoresis

  13. T4 DNA Ligase

  14. Transformation • All of the previous steps were performed in vitro. • We have generated a very small amount of a recombinant plasmid • Need to amplify in bacteria to get enough to work with. • Transformation – process to mobilize DNA into bacterial host • Select for transformed bacteria on specific antibiotic that corresponds to the antibiotic resistance gene present on the plasmid

  15. How to produce a recombinant protein 0.1 to 1% of cellular protein 10 to 70% of cellular protein

  16. Cloning a gene from a DNA libraries • Any particular gene may represent a tiny, tiny fraction of the DNA in a given cell • Can't isolate it directly • Trick is to find the fragment or fragments in the library that contains the desired gene

  17. cDNA

  18. cDNA Library cDNA

  19. Library Screening • DNA probe hydridization • Requires that you know the protein or amino acid sequence of the gene of interest. • Need to denature (make single stranded) and immobilize the DNA from each clone of the library to a filter (nitrocellulose or nylon) • Make a labeled single stranded DNA/RNA probe (can use radioactive of fluorescent analogous of specific nucleotide triphosphates) • Labeled single stranded DNA/RNA fragments will base pair (hydridize) with the target DNA on the filter • Identify clones that are labeled.

  20. DNA hydridization screening for specific gene • Requires that you know something about the gene sequence • Can get sequence information form purified protein

  21. Now that we have the gene, what do we do with it? • We could use it make a lot of protein in a microbial protein expression system • We could use it to genetically manipulate organisms • We could use it as a diagnostic tool

  22. Why use recombinant Proteins? • Proteins are often only available in small amounts in a given tissue • Tissue sources may not be readily available • It is time consuming and expensive to purify protein from tissues • It is difficult to obtain absolutely pure protein

  23. Insulin • Was first purified from human pancreas from cadavers and then from pig pancreas. • Genentec expressed insulin gene in microbial host • Can grow microbes in large fermenters to produce unlimited supply of insulin.

  24. Product name  Protein type Application Company Adagen (Adenosine deaminase ) An enzyme Severe combined immunodeficiency disease (SCID) Enzon Genotropin (Recombinant growth hormone) A hormone Growth hormone deficiency (GHD) in children Pharmacia & Upjohn Humalog (Recombinant human insulin) A hormone Diabetes Eli Lilly Nabi-HB (Anti-Hepatitis B)  An antibody Hepatitis-B Nabi Novo Seven (Recombinant coagulation factor VIIa) A modified factor Hemophillia patients with inhibitors Novo Nordisk Ontak (Diphtheria toxin-interleukin-2) A fusion protein Cutaneous T-cell lymphoma (CTCL) Ligand Pharmaceuticals Roferon-A (Recombinant interferon alfa-2a) A modifier Hairy cell leukemia or AIDS-related Kaposi's sarcoma Hoffmann-La Roche

  25. Recombinant proteins are also important to research • For enzyme analysis need pure protein • For structural analysis need lots (milligram amounts) of very pure protein • Need pure proteins to make diagnostic tools such as antibodies

  26. Genetic Modification of Higher Organisms • Can introduce gene into animals and plants • These modified organism are powerful research tools to study the effect of a specific gene product on metabolism, development etc…. • Has also been used to develop improved agricultural products

  27. Genetically Engineered Salmon Is Bigger Better?

  28. http://www.agwest.sk.ca/sabic_index_tp.shtml

  29. Plant Genetic Engineering Improved Agricultural Production • Herbicide Resistance • Pest Resistance Improved Nutrition • Vitamins - Golden Rice, Vitamin E • Increase essential Amino Acid Content Chemical Synthesis • Bio-plastics • Bio-diesel • Lubricants/detergents • Rubber

  30. GMO Concerns • Ecological Concern • Potential Food Allergens • Antibiotic Resistance

  31. GMO Benefits • Lower application of herbicides and pesticides • Creation of foods with increased nutrition • Creation of bio-based alternative to petroleum based products http://www.colostate.edu/programs/lifesciences/TransgenicCrops/