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Chapter 23 (Part 1)

Chapter 23 (Part 1). Recombinant DNA Technology. Recombinant DNA Technology. Methods for isolating, manipulating, and amplifying identifiable DNA sequences. Allows us to study the structure and function of individual genes.

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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/

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