DNA Technology

1. DNA Technology Chapter 13 Lab Biology 1A Chapter 15 Lab biology 2b Chapter 13 Honors Biology 4A

2. DNA Technology • Technology used to help with genetic engineering  helps us: • 1. identify genes for specific traits • 2. transfer genes for a specific trait from one organism to another • 3. cure disease • 4. treat genetic disorders • 5. improve food crops

3. How can you get a desired trait without directly manipulating the organisms DNA? • HYBRIDIZATION; crossing organisms of different traits to produce a hardier product ex: mule • INBREEDING/SELECTIVE BREEDING; maintain the present genes by breeding only within the population ex: pedigree animals • INDUCING MUTATIONS; radiation, chemicals  polyploidy (3N or 4N) plants resulted  larger and hardier

4. Now let us manipulate the genes by altering the organisms DNA • DNA Technology: sci. involved in the ability to manipulate genes/DNA • Cure disease • treat genetic disorders • Improve crops

5. Tools: • DNA extraction • Restriction enzymes • Gel electrophoresis • DNA ligase • Polymerase chain rxn. (PCR)

6. Method: (5 steps) • Extract gene  insulin • Cut insulin producing gene out using “restriction enzymes” • Sticky ends  create overhang • Blunts no overhangs • Cutting clone vector cut plasmid with same restriction enzyme • Ligation: donor gene is spliced into plasmid DNA, DNA ligase glues it (this forms recombinant DNA = plasmid DNA + new piece of DNA) • Plasmid returned to bacterium & reproduces using donor gene in it (this is transgenic organism = organism with foreign DNA incorporated in it’s genome) • *reproduce*

7. Restriction Enzymes • BACTERIAL ENZYMES are used to cut DNA molecule into more manageable pieces • They recognize certain sequences • Creating “single-chain” tails in DNA  called STICKY ENDS

8. Sticky Ends • Readily bind to complimentary chains of DNA therefore pieces of DNA that have been cut with the same restriction enzyme can bind together to form a new sequence of nucleotides • Recognizes  CTTAAG

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11. Cloning Vectors • Cloning vector is a carrier that is used to clone a gene and transfer it from one organism to another. • Many bacteria contain a cloning vector called a PLASMID. • PLASMID  is a ring of DNA found in a bacterium in addition to its main chromosome.

12. PROCEDURE • To be used as a cloning vector in gene transfer experiments a plasmid is isolated from a bacterium. • Using restriction enzymes the plasmid is then cut and a DONOR GENE (specific gene isolated from another organism is spliced into it) • Then the plasmid is returned to the bacterium, where it is replicated as the bacterium divides, making copies of the donor gene. • Plasmid now contains a GENE CLONE

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14. Cloning Vectors • !

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16. Transplanting genes • In some cases, plasmids are used to transfer a gene to bacteria so that the bacteria will produce a specific protein • Ex: INSULIN = protein that controls sugar metabolism • Bacteria that receives the gene for insulin will produce insulin as long as the gene is not turned off

17. Steps: • 1. ISOLATING A GENE – isolate the DNA from human cells and plasmids from the bacteria • Use restriction enzyme • Splice human DNA into plasmids to create a genomic library (set of thousands of DNA pieces from a genome that have been inserted into a cloning vector)

18. Steps cont… • 2. PRODUCING RECOMBINANT DNA = combination of DNA from 2 or more sources • Inserting a donor gene such as human gene for insulin, into a cloning vector, such as bacterial plasmid results in a recombinant DNA molecule!

19. Steps cont… • 3. CLONING DNA – the plasmid containing recomb. DNA is inserted into a host bacterium (called transgenic organism • The trans. Bact. Is placed in a nutrient medium where it can grow and reproduce.

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22. Expression of Cloned Genes • Sometimes PROMOTERS must also be transferred so the genes will be turned on. • Genes are often turned off until the proteins they code for are needed.

23. Practical uses of DNA Technology • Pharmaceutical products: insulin, HBCF (human blood clotting factor) • Genetically engineered vaccines • Increased agriculteral yields • Improving quality of produce • Slow down ripening • Enhance color • Reduce fuzz • Increase flavor • Frost resistance

24. Negatives • Allergies • Label’s don’t include all information • May create “super weeds”

25. Gene therapy • Treatment of genetic disorders • Ex: cystic fibrosis

26. DNA Technology Techniques • I. DNA Fingerprints  pattern of bands made up of specific fragments from an individual’s DNA • USED FOR: • DETECTION OF A RELATIVE • SIMILARITIES BETWEEN SPECIES

27. How do you make DNA fingerprints? • RFLP (restriction fragment length polymorphism) analysis • 1. extract DNA from specimen using restriction enzymes • 2. separate fragments of DNA using electrophoresis (separates DNA according to size and charge) • 3. placed in wells made on gel • 4. electric current run through gel

28. Continue… • 5. negative fragments migrate to positive charged end of gel but not all at same rate • 6. pores in gel allow smaller fragments to migrate faster  separating fragments by size. • 7. blotted onto filter paper.

29. Can you tell if this could be the father?

30. Accuracy of DNA Fingerprints • DNA fingerprints are very accurate • However, genetic tests can only absolutely disprove, not prove, relationship! • Courts accept 99.5% accuracy as proof of alleged paternity

31. Polymerase Chain Reaction (PCR) • Used when you only have a TINY piece of DNA • PCR can be used to quickly make many copies of selected segments of the available DNA • Use a PRIMER to initiate replication • DNA doubles every 5 minutes

32. PCR is used for: • 1. crimes • 2. diagnosing genetic disorders from embryonic cells • 3. studying ancient fragments of DNA (tiny amounts)

33. HUMAN GENOME PROJECT • 2 GOALS: • 1. determine nucleotide sequence of entire human genome (aprox 3 billion nucleotide pairs or about 100,000 genes • 2. map the location of every gene on each chromosome

34. 1996 • 1 % of 3 billion nucleotide pairs of DNA human genomes were analyzed • This allows for us to identify and determine the function of 16,000 genes!

35. Gene Therapy • Treating a genetic disorder by introducing a gene into a cell or by correcting a gene defect in a cell’s genome. • Ex: Cyctic fibrosis cause one defective gene  malfunction of one protein

36. Gene Therapy for Cyctic Fibrosis • Nasal spray carrying normal cyctic fibrosis gene to cells in nose and lungs • Must repeat treatment periodically

37. Ethical Issues • Many people worry about how personal genetic information will be used: • Insurance??? • Employment???? • Human Genome Project will undoubtedly involve ethical decisions about how society should use the information! WHAT DO YOU THINK??

38. Practical Uses of DNA Technology • 1. produce perscription drugs • Vaccine (harmless version of a virus or a bacterium) • Pathogen (disease causing agent) treated chemically or physically so that they can no longer cause disease. • Pathogen (Ag)  Antibody (Ab) • DNA tech. may produce vaccines safer than traditional ones!

39. Increasing Agricultural Yields • DNA Tech.  used to develop new strains of plants • Ex: scientists can make tomato plants toxic to hornworms and effectively protect the plant from these pests.

40. See the hornworm beginning to form at the leaves!

41. This hornworm eats and destroys the tomato plant!

42. Hornworms attack tomato plants

43. Crops that do NOT need fertilizer • Plants require NITROGEN to make proteins and nucleic acids • Most plants get their N from the soil • TRANSGENIC FOOD CROPS  contain genes for nitrogen fixation so they can grow in nitrogen POOR soil.

44. Genetically Engineered Foods • Foods may have toxic proteins or substances  causing ALLERGIES • Ex: changing the gene that codes for an enzyme to ripening in tomatoes they are able to make tomatoes ripen without becoming SOFT!!

45. Genetically Engineered Crops • Some are concerned that genetically engineered crops could spread into the wild and wipe out native plant species. • SUPERWEEDS!!!!!!!!