2. Genetic Engineering Biotechnology

3. HISTORY OF GENETIC ENGINEERING Before technology, humans were using the process of selective breeding to produce the type of organism they want. Creating new breeds of animals & new crops to improve our food.

4. Example: Dog Breeding = + Labradoodle Poodle Labrador Bulldog Mastiff Bullmastiff + =

5. Animal breeding

6. Breeding food plants Evolution of modern corn “ Cabbage family” descendants of the wild mustard

7. Selective Breeding Choosing individuals with the desired traits to serve as parents for the next generation.

8. Graph: Plant Height What is the result? The frequency of desired alleles increases in the population Now, suppose only the tallest plants were used to breed

9. Test Cross A special cross use to determine an unknown genotype of a dominant phenotype Cross the unknown individual with a homozygous recessive individual A?A?

10. Let’s work this out! Outcome: If the individual is homozygous dominant  100% dominant phenotype If the individual is heterozygous dominant  50% dominant phenotype  50% recessive phenotype

11. A Brave New World

12. GENETIC ENGINEERING Scientists can now use their knowledge of the structure of DNA and its chemical properties to study and change DNA molecules.

13. Remember the code is universal Since all living organisms… – use the same DNA – use the same code book – read their genes the same way

14. Can we mix genes from one organism to another? YES! Transgenic organisms contain recombinant DNA

15. GENETIC ENGINEERING!

16. Recombinant DNA- made by connecting fragments of DNA from a different source. Transgenic Organisms- Organisms that contain DNA from a different source.

17. How do we do mix genes? Genetic engineering – Isolate gene from donor DNA – cut DNA in both organisms – paste gene from one organism into other organism’s DNA – transfer recombined DNA into host organism – organism copies new gene as if it were its own – organism produces NEW protein coded for by the foreign DNA Remember: we all use the same genetic code!

18. CUTTING DNA RESTRICTION ENZYMES are proteins that act as “molecular scissors”

19. RESTRICTION ENZYMES Restriction enzymes are proteins that cut DNA Each restriction enzyme only cuts a specific nucleotide sequence in the DNA called the recognition sequence

20. RESTRICTION ENZYMES Recognition sequences are usually palindromes Same backwards and forwards Ex. Eco R1 enzyme recognizes:

21. RESTRICTION ENZYMES Cuts usually leave little single stranded fragments called STICKY ENDS

22. RESTRICTION ENZYMES If the enzyme cuts right down the middle, the ends are BLUNT

23. Each time EcoRI recognizes the sequence CTTAAG, it cuts between the G & A and then through the middle of the strands The recognition sequence for the restriction enzyme named EcoRI is CTTAAG This results in DNA fragments that have single-stranded tails called sticky ends

24. RESTRICTION ENZYMES Pieces can be glued back together using LIGASE

25. http://www.yo utube.com/wa tch?v=8rXizm LjegI

26. GENE TRANSFER During GENE TRANSFER, a gene from one organism is placed into the DNA of another organism New DNA that is created is called RECOMBINANT DNA.  Example: Human insulin Bacterial Recombinant DNA Insulin

27. BACTERIAL PLASMIDS Bacteria have small, circular DNA segments called PLASMIDS.  Usually carry “extra info” on them Plasmids can be used as a VECTOR- object that carries foreign DNA into a host cell

28. There’s more… Plasmids – small extra circles of DNA – carry extra genes that bacteria can use – can be swapped between bacteria

29. How can plasmids help us? A way to get genes into bacteria easily – insert new gene into plasmid – insert plasmid into bacteria = vector – bacteria now expresses new gene bacteria make new protein + transformed bacteria gene from other organism plasmid cut DNA recombinant plasmid vector glue DNA

30. Bacteria Bacteria are great! – one-celled organisms – reproduce by mitosis easy to grow, fast to grow – generation every ~20 minutes

31. CREATION OF RECOMBINANT DNA 1. In a lab, plasmid is extracted from bacteria 2. Insulin also extracted from human DNA **Both gene for insulin and plasmid are cut with same restriction enzyme. Insulin gene (cut from chromosome) Bacterial Plasmid

32. TRANSFORMATION 4. The gene is inserted into the plasmid by connecting sticky ends with ligase. 5. Plasmid taken up by bacteria through TRANSFORMATION. 6. Bacteria grows in Petri dish and replicates recombinant DNA insulin human insulin

33. CREATION OF INSULIN 7. As the bacteria grow and replicate, more and more bacteria are created with the human insulin gene 8. The bacteria read the gene and create insulin for us to use

34. TRANSFORMING PLANT & ANIMAL CELLS Bacterial plasmids can also be put into plant and animal cells The plasmid incorporates into the plant or animal cell’s chromosome Transformed bacteria introduce plasmids into plant/animal cells

35. TRANSGENIC ORGANISMS Because the bacteria now has DNA from two species in it, it is known as a TRANSGENIC ORGANISM.  A.K.A. GENETICALLY MODIFIED ORGANISM

36. Transforming Bacteria Recombinant DNA Gene for human growth hormone Human Cell Bacteria cell Bacterial chromosome Plasmid Sticky ends DNA recombination Bacteria cell containing gene for human growth hormone DNA insertion

37. Grow bacteria…make more grow bacteria CLONE harvest (purify) protein TRANSFORMATION transformed bacteria plasmid gene from other organism + recombinant plasmid vector

38. REAL OR FAKE!!!!!

39. 1 - REAL OR FAKE

40. 2 - REAL OR FAKE

41. 3 - REAL OR FAKE

42. 4 - REAL OR FAKE

43. 5 - REAL OR FAKE

44. 6 - REAL OR FAKE

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47. 9 - REAL OR FAKE

48. 10 -REAL OR FAKE

49. CLONING

50. CLONE – organism with the same genetic make-up (DNA) as another An exact copy

51. CLONING – STEP 1A

52. CLONING – STEP 1B

53. CLONING – STEP 2

54. CLONING – STEP 3

55. CLONING – STEP 4

56. CLONING – STEP 5

57. POLYMERASE CHAIN REACTION

58. *MANIPULATING DNA Techniques used to manipulate DNA:  DNA Extraction  Cut DNA in to smaller pieces  Identify base sequences  Make unlimited copies of DNA

59. MAKING COPIES Often at a crime scene, DNA evidence is left behind in trace (small) amounts Hair Blood Body fluids

60. MAKING COPIES The sample is so small, it cannot be used unless more of it can be made POLYMERASE CHAIN REACTION (PCR)  Process used to amplify (multiply) the amount of DNA in a given sample

61. MAKING COPIES What did the polymerase molecule do in DNA replication / Transcription?

62. MAKING COPIES PCR also allows scientists to pick a particular gene and make many copies of it Millions of copies can be made from just a few DNA strands

63. PCR STEPS PCR Supply List  DNA  Heat  Taq (DNA) polymerase  A special polymerase from a bacterium that lives at high temperatures  Primers

64. PCR STEPS Step 1  Denature (separate) the DNA by heating it up to 95°C.

65. PCR STEPS Step 2:  Reduce the temperature  Add primers that binds to the strand.

66. PCR STEPS Step 3  Add Taq (DNA) polymerase adds nucleotides to strands, producing two complementary strands.

67. PCR STEPS Step 4, 5, 6…  Repeat Every time we repeat the procedure, we double the DNA

68. GEL ELECTROPHORESIS

69. Many uses of restriction enzymes… Now that we can cut DNA with restriction enzymes… – we can cut up DNA from different people… or different organisms… and compare it – why? forensics medical diagnostics paternity evolutionary relationships and more…

70. Comparing cut up DNA How do we compare DNA fragments? – separate fragments by size How do we separate DNA fragments? – run it through a gelatin – gel electrophoresis How does a gel work?

71. GEL ELECTROPHORESIS An electric current is applied to the gel to get the DNA moving  Small molecules move faster (move towards bottom)  Big molecules move slower (stay towards top) DNA fragments are drawn to positive electrode DNA moving through gel

72. GEL ELECTROPHORESIS The gel acts like a filter by separating strands of different sizes It’s like a sponge made of Jello – lots of small holes and a “squishy” consistency

73. Gel electrophoresis A method of separating DNA in a gelatin-like material using an electrical field – DNA is negatively charged – when it’s in an electrical field it moves toward the positive side +– DNA        “swimming through Jello”

74. Gel Electrophoresis longer fragments shorter fragments power source completed gel gel DNA & restriction enzyme wells - +

75. Running a gel 12 cut DNA with restriction enzymes fragments of DNA separate out based on size 3 Stain DNA – Dye binds to DNA – fluoresces under UV light

76. ANALYZING DNA The pieces are separated, analyzed & compared to others  Each piece has its own unique weight and shape Use these properties to perform a technique called DNA FINGERPRINTING

77. DNA FINGERPRINTING Step 1) DNA is cut using restriction enzymes Step 2) Mix of DNA and enzymes are then separated by a process called GEL ELECTROPHORESIS Step 3) DNA pattern is analyzed

78. DNA FINGERPRINTING

79. CUTTING DNA Everyone has a unique DNA sequence  Rec. sequences are in different places  When a restriction enzyme cuts the DNA of two different people, it will cut it into different sized pieces Suspect #1 Suspect #2

80. DNA fingerprint Why is each person’s DNA pattern different? – sections of “junk” DNA doesn’t code for proteins made up of repeated patterns – CAT, GCC, and others – each person may have different number of repeats many sites on our 23 chromosomes with different repeat patterns GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA GCTTGTAACGGCATCATCATCATCATCATCCGGCCTACGCTT CGAACATTGCCGTAGTAGTAGTAGTAGTAGGCCGGATGCGAA

81. Allele 1 GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA repeats DNA patterns for DNA fingerprints cut sites GCTTGTAACG GCCTCATCATCATCGCCG GCCTACGCTT CGAACATTGCCG GAGTAGTAGTAGCGGCCG GATGCGAA 1 23 DNA  –+ Cut the DNA

82. Person 1 GCTTGTAACG GCCTCATCATCATTCGCCG GCCTACGCTT CGAACATTGCCG GAGTAGTAGTAAGCGGCCG GATGCGAA Differences between people cut sites DNA  –+ person 1 Person 2: more “junk” in between the genes GCTTGTAACG GCCTCATCATCATCATCATCATCCG GCCTACGCTT CGAACATTGCCG GAGTAGTAGTAGTAGTAGTAGGCCG GATGCGAA DNA fingerprint person 2 123

83. Uses: Evolutionary relationships Comparing DNA samples from different organisms to measure evolutionary relationships – + DNA  13245 12345 turtlesnakeratsquirrelfruitfly

84. Uses: Medical diagnostic Comparing normal allele to disease allele chromosome with disease-causing allele 2 chromosome with normal allele 1 – + allele 1 allele 2 DNA  Example: test for Huntington’s disease

85. Uses: Forensics Comparing DNA sample from crime scene with suspects & victim – + S1 DNA  S2S3V suspects crime scene sample

86. DNA fingerprints Comparing blood samples on defendant’s clothing to determine if it belongs to victim – DNA fingerprinting

87. RFLP / electrophoresis use in forensics 1st case successfully using DNA evidence – 1987 rape case convicting Tommie Lee Andrews “standard” semen sample from rapist blood sample from suspect

88. Electrophoresis use in forensics Evidence from murder trial – Do you think suspect is guilty? “standard” blood sample 3 from crime scene “standard” blood sample 1 from crime scene blood sample 2 from crime scene blood sample from victim 2 blood sample from victim 1 blood sample from suspect OJ Simpson N Brown R Goldman

89. Uses: Paternity Who’s the father? + DNA  childMomF1F2 –