1. Don’t forget to count your flies!

2. Biotechnology and Genetic Engineering AP Biology Chapter 20

3. Terminology Genetic engineering – direct manipulation of genetic material for practical purposes Biotechnology – use of living organisms or their components to make products for us Recombinant DNA – combining pieces of DNA from different organisms Gene cloning – making copies of DNA

4. Making recombinant DNA Plasmids (small circular pieces of DNA in bacterial cells) are used to insert pieces of foreign DNA

5. The DNA is cut using restriction enzymes

6. What are restriction enzymes? Restriction enzymes come from bacteria and recognize a particular pattern of DNA, often 4, 6 or 8 base pairs long, and then cut the DNA within this recognized sequence. Bacteria use these enzymes to kill off other competing bacteria by cutting up their DNA.

7. How do they cut? STICKY ENDS B LUNT ENDS

8. ACT GAA TTC CGG AAT GAA TTC TGA CTT AAG GCC TTA CTT AAG Where would the enzyme EcoRI cut?

9. ACT GAA TTC CGG AAT GAA TTC TGA CTT AAG GCC TTA CTT AAG There would be three pieces: one 4 bases, one 12 bases, and one 5 bases.

10. How do bacteria protect it’s own DNA from being cut by the enzymes? It methylates it’s own DNA.

11. Making recombinant DNA in plasmids http://glencoe.mcgraw- hill.com/sites/9834092339/student_view0/chapter18/steps_in_cloning_a_gene.html

12. http://www.nearingzero.net/natural/screenres/natural039.jpg

13. Bacterial plasmids often contain antibiotic resistance genes.

14. Genes can be cloned into vectors such as plasmids

15. Fig. 20-2 DNA of chromosome Cell containing gene of interest Gene inserted into plasmid Plasmid put into bacterial cell Recombinant DNA ( plasmid ) Recombinant bacterium Bacterial chromosome Bacterium Gene of interest Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Plasmid Gene of Interest Protein expressed by gene of interest Basic research and various applications Copies of gene Protein harvested Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth 2 4 1 3

16. Fig. 20-2a DNA of chromosome Cell containing gene of interest Gene inserted into plasmid Plasmid put into bacterial cell Recombinant DNA (plasmid) Recombinant bacterium Bacterial chromosome Bacterium Gene of interest Plasmid 2 1 2

17. Fig. 20-2b Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Interest Protein expressed by gene of interest Basic research and various applications Copies of gene Protein harvested Basic research on gene Basic research on protein 4 Recombinant bacterium Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth 3

18. Fig. 20-4-1 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE

19. Fig. 20-4-2 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid

20. Fig. 20-4-3 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids

21. Fig. 20-4-4 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids RESULTS Colony carrying non- recombinant plasmid with intact lacZ gene One of many bacterial clones Colony carrying recombinant plasmid with disrupted lacZ gene

22. Steps 1.Plasmid and DNA of gene of interest are isolated. 2.Both DNAs are cut with the same restriction enzyme. 3.“new” DNA is ligated into plasmid 4.Recombinant plasmids are inserted into bacterial cells. 5.Plate bacteria on agar. Bacteria will express new genes.

23. Plasmid Maps

25. Plasmid Maps Sometimes called restriction maps are graphical representation of plasmids, that show the locations of major identifiable landmarks on DNA like restriction enzyme sites, genes of interest, plasmid length etc.plasmids

27. The collection of thousands of clones of bacteria containing recombinant plasmids is called a genomic library.

28. In molecular biology, plasmid (or restriction) maps are used as a reference to engineer plasmids.molecular biology The plasmids are digested by enzymes chosen and the resulting samples are subsequently run on an electrophoresis gel.electrophoresis

30. Our experiment: to transform E.coli with pGLO plasmid containing the jellyfish gene GFP to make them have the ability to glow

32. To isolate only the cells containing the pGLO DNA, the plasmid contains the beta-lactamase gene which encodes for an ampicillin resistance (Amp r ) protein. After the transformation, the cells are grown on a solid medium called an agar plate. This medium will contain the antibiotic ampicillin. In the presence of the ampicillin, only the bacteria containing the pGLO plasmid will have the Amp r protein which will break down the antibiotic, and be able to grow. This process is called antibiotic selection.

33. GFP results in E.coli

34. This plate shows bacteria expressing six different types of flourescent proteins

35. GRP has been used as tracers to see if the plasmid has been taken up by the bacteria.

36. Expression of eukaryotic genes in prokaryotes Use an expression vector with a prokaryotic promoter upstream from the location of the gene (ie operon) Create artificial genes without introns since bacteria do not have the machinery for eliminating introns.

37. Ways to introduce new genes into bacteria. Conjugation – through tubes between bacteria Transformation – negative DNA taken up Transduction by bacteriophages or other viruses Mutation ALL of these introduce GENETIC VARIATION!

38. What about transferring DNA into eukaryotic cells Electroporation - injecting DNA into eukaryotic cells

40. Plants can have genes transferred through bacterial plasmids.

41. Storing Cloned Genes in DNA Libraries These can be also be used as vectors to transfer genes to other organisms. 1) Plasmid libraries plasmids that contain genes of interest 2) Phage library that is made using bacteriophages which store genes of interest

42. Fig. 20-5a Bacterial clones Recombinant plasmids Recombinant phage DNA or Foreign genome cut up with restriction enzyme (a) Plasmid library(b) Phage library Phage clones

43. Viruses used as vectors

44. 3)BACs (bacterial artificial chromosome) a plasmid that can carry a large DNA insert

45. Fig. 20-5 Bacterial clones Recombinant plasmids Recombinant phage DNA or Foreign genome cut up with restriction enzyme (a) Plasmid library(b) Phage library (c) A library of bacterial artificial chromosome (BAC) clones Phage clones Large plasmid Large insert with many genes BAC clone Formation of BAC Clones

46. 4) YACS Yeast artificial chromosomes that carry foreign DNA. Yeast cells have plasmids that can act as vectors.

47. 5) Complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell A cDNA library represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells http://glencoe.mcgraw- hill.com/sites/9834092339/student_view0/chapter18/cdna.html

48. Fig. 20-6-1 DNA in nucleus mRNAs in cytoplasm

49. Fig. 20-6-2 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail DNA strand Primer mRNA

50. Fig. 20-6-3 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail DNA strand Primer mRNA Degraded mRNA

51. Fig. 20-6-4 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail DNA strand Primer mRNA Degraded mRNA DNA polymerase

52. Fig. 20-6-5 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail DNA strand Primer mRNA Degraded mRNA DNA polymerase cDNA http://glencoe.mcgraw- hill.com/sites/9834092339/ student_view0/chapter18/f ish.html

53. In Summary, libraries of cloned genes can be made by Plasmids Bacteriophage viruses BACS YACS cDNA (with reverse transcriptase)

54. Other biotechniques Nucleic acid hybridization PCR Gel electrophoresis DNA fingerprinting (Southern Blotting)

55. Nucleic Acid Hybridization Used to detect specific sequences in DNA fragments Steps: 1) Cut DNA with restriction enzymes and electrophorese. 2) The DNA is denatured to produce single stranded DNA. 2) The radioactive probe will hybridize (bond) with complementary bases if present. Probes can be radioactive isotopes or flourescent dyes.

56. Looking for this sequence

57. The radioactive probe is made by determining a short segment of the protein sequence, then "back translating" to the possible short DNA sequence. The DNA sequence is radiolabeled, and applied to the blotted clones. They should hybridize only to sequences that would identify that particular piece of DNA.

58. How does this work? Protein segment gly – gly – ser – glutamic acid Look on Genetic Codon chart, find mRNA codons GGU – GGU – UCU – GAA Make a radioactive DNA (oligos)probe CCA – CCA – AGA - CTT

59. PCR Polymerase Chain Reaction Used to amplify DNA Discovered by Kary Mullis (GT grad) A Thermocycler

60. Steps of PCR? Denature DNA (94-96 C) Anneal (base pair) primers (50 – 65 C) Extend primers (72 for polymerase to work) Machines called thermocyclers do this. http://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html http://glencoe.mcgraw- hill.com/sites/9834092339/student_view0/chapter18/polymerase_chain_reactio n.html

61. Fig. 20-8b Cycle 1 yields 2 molecules Denaturation Annealing Extension Primers New nucleo- tides 3 5 3 2 53 1

62. Fig. 20-8 5 Genomic DNA TECHNIQUE Cycle 1 yields 2 molecules Denaturation Annealing Extension Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Target sequence Primers New nucleo- tides 3 3 3 3 5 5 5 1 2 3

63. In PCR, a heat-stable DNA polymerase is used, most commonly Taq Polymerase from the thermophilic microbe Thermus aquaticus. Thomas Brock discovered T. aquaticus from a hot spring at Yellowstone National Park.

64. Applications of PCR PCR has replaced cloning for many purposes, particularly the sequencing of DNA. It is faster and requires no vectors, which can mutate as they reproduce. It can be used forensically, to amplify tiny amounts of DNA from criminal evidence; or clinically, to detect DNA sequences linked to inherited disorders.

65. What is gel electrophoresis? A technique to separate DNA based on the movement of DNA fragments from neg to pos (DNA is neg). Smaller fragments travel farther. Samples are placed in gels. Gel Electrophoresis

67. DNA can be digested by restriction enzymes and the resulting samples are run on an electrophoresis gel. electrophoresis They can be compared to a reference ladder (a DNA ladder) or a sample with known sized pieces.

69. You can compare your pieces to a marker as shown below.

70. Or you can make a graph from a known sample to determine the sizes. Line of best fit

71. Southern Blotting “DNA Fingerprinting” - named for Edwin Southern - used to identify DNA fragments 1.Isolate DNA 2.Cut DNA into fragments with restriction enzymes. 3.Electrophorese. 4.Blot onto nylon membrane. 5.Apply radioactive probes. 6.Wash to remove unbonded probes. http://highered.mcgraw- hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_5.html

73. In DNA fingerprinting Single nucleotide polymorphisms (SNPs) are useful genetic markers These are single base-pair sites that vary in a population (most of our DNA is identical http://glencoe.mcgraw- hill.com/sites/9834092339/student_view0/chapter18/restriction_fragment_length_polym orphisms.html

74. Fig. 20-21 Disease-causing allele DNA SNP Normal allele T C http://glencoe.mcgraw- hill.com/sites/9834092339/student_view0/chapter18/video_quiz_- _world_trade_center_dna.html Many genetic diseases are the result of a polymorphism at a single locus.

76. When a restriction enzyme is added, SNPs result in DNA fragments with different lengths, or restriction fragment length polymorphisms (RFLP) Some polymorphisms cause disease, while some do not. Others indicate a predisposition to disease.

77. Too many abbreviations! SNP – different size pieces due to different nucleotide sequences RFLP - fragments of DNA from restriction enzyme procedure RFLP-s differ in sizes due to SNP’s in a population.

78. Hemoglobin S is missing a restriction site due to a polymorphism

79. RFLP Analysis in Paternity Cases

82. Short Tandem Repeats Even more sensitive is the use of genetic markers called short tandem repeats (STRs), which are variations in the number of repeats of specific DNA sequences

83. The Human Genome Project has shown that there are tens of thousands of STR loci in human DNA.Human Genome Project An individual inherits one copy of an STR from each parent,

84. D7S280 is one of the 13 core CODIS STR genetic loci. This DNA is found on human chromosome 7. The DNA sequence of a representative allele of this locus is shown below. The tetrameric repeat sequence of D7S280 is "gata". Different alleles of this locus have from 6 to 15 tandem repeats of the "gata" sequence. How many tetrameric repeats are present in the DNA sequence shown below? 1 aatttttgta ttttttttag agacggggtt tcaccatgtt ggtcaggctg actatggagt 61 tattttaagg ttaatatata taaagggtat gatagaacac ttgtcatagt ttagaacgaa121 ctaacgatag atagatagat agatagatag atagatagat agatagatag atagacagat181 tgatagtttt tttttatctc actaaatagt ctatagtaaa catttaatta ccaatatttg241 gtgcaattct gtcaatgagg ataaatgtgg aatcgttata attcttaaga atatatattc301 cctctgagtt tttgatacct cagattttaa ggcc

85. If the genotypes of both parents are known, we use a Punnett Square to predict the possible phenotypes of their offspring. Each child inherits one allele of a given locus from each parent. Panel (a) - At the D21S11 locus, the children of Bob Blackett and wife Anne can have four different genotypes. Son David is 28, 31. Daughter Katie is 29, 30. Panel (b) - Bob Blackett inherited the 31 allele from his mother, Norma. Therefore the 29 allele is paternal. If Bob's paternal was not 29, what would be your conclusion?

86. Huntington’s Disease can be diagnosed by the number of CAG repeats The data below shows the results of electrophoresis of PCR fragments amplified using probes for the site which has been shown to be altered in Huntington's disease. The male parent, as shown by the black box, got Huntington's disease when he was 40 years old. His children include 6 (3,5,7,8,10,11) with Huntington's disease, and the age at which the symptoms first began is shown by the number above the band from the PCR fragment. What is the prognosis for the normal children 4, 6, and 9?

87. Sanger Sequencing Used to sequence short segments of DNA Single-stranded fragments are incubated with fluorescent-tagged short segments for DNA hybridization. When fragments hybridize with the tagged nucleotide, the hybridization stops. Fragments are electrophoresed and analyzed. http://glencoe.mcgraw- hill.com/sites/9834092339/student_view0/chapter18/sang er_sequencing.html

90. Analyzing Expression of Genes Northern Blotting – using radioactive probes to look for mRNA being produced RT-PCR – Reverse transcriptase- polylmerase chain reaction – makes cDNA from mRNAs and then PCRs the DNA for electrophoresis in situ hybridization – can locate specific mRNA’s in cells

91. Micro – arrays - Isolate mRNA from cells, make cDNA using reverse transcriptase, then uses cDNA to explore collections of genomic DNA to see if they hybridize http://glencoe.mcgraw- hill.com/sites/9834092339/student_view0/chapter18/using_a_dna_microarray.html

92. Microarrays are useful in discerning gene expression in different tissues AND at different stages of development. Different brightness and colors signify rates of expression. Google Image Result for http://www.g2conline.info/content/1178/1 178_what_microarray_thumb.jpg http://www.dnalc.org/resources/3d/2 6-microarray.html

93. An example DNA Microarray Methodology Animation

94. Determining Gene Function In vitro mutagenesis – changes made to cloned gene, gene returned to cell and it “knocks out” the normal gene. Then look for abnormalities. RNA interference (RNAi) – uses RNA to block translation of mRNA and see what happens.

95. Cloning Organisms Organismal cloning – producing genetically identical individuals from a single somatic cell of a multicellular organism

96. In plants Steward demonstrated genomic equivalence in plants by growing carrot plants from differentiated root cells. Most plant cells remain totipotent, retaining the ability to give rise to a complete new organisms.

97. In Animals Briggs and all transplanted nuclei from embryonic frog cells into enucleated egg cells and produced cloned frogs Nuclear transplantation – name of process Whether normal development occurred depended on developmental age of the transplanted nucleus.

98. Fig. 20-17 EXPERIMENT Less differ- entiated cell RESULTS Frog embryo Frog egg cell UV Donor nucleus trans- planted Frog tadpole Enucleated egg cell Egg with donor nucleus activated to begin development Fully differ- entiated (intestinal) cell Donor nucleus trans- planted Most develop into tadpoles Most stop developing before tadpole stage

99. Nuclear Transplantation

100. And then Dolly came along in 1997

101. Fig. 20-18 TECHNIQUE Mammary cell donor RESULTS Surrogate mother Nucleus from mammary cell Cultured mammary cells Implanted in uterus of a third sheep Early embryo Nucleus removed Egg cell donor Embryonic development Lamb (“Dolly”) genetically identical to mammary cell donor Egg cell from ovary Cells fused Grown in culture 1 3 3 4 5 6 2

102. Why Dolly died young 6 yrs Dolly's telomeres were found to be approximately 80% of the length they should be for a sheep her age. Also there is the concern of damaged DNA being carried into the clone

103. Cloned animals do not look exactly like the transplanted nucleus due to cytoplasmic affects. Rainbow CC CC and her Surrogate mom Hi Mrs. Smith!

104. In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth Many epigenetic changes, such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development

105. Stem Cells Relatively unspecialized cells that continue to reproduce themselves and can be induced to form specialized cells Embryonic cells are more totipotent than adult stem cells http://cbm.msoe.edu/stupro/so/SOStemCellVideo2010.html

108. Therapeutic cloning – using stem cells to replace organs and tissues Reproductive cloning – using stem cells to reproduce new organisms Both raise ethical debates

109. Research points to a new direction in Stem Cell Research Induced Pluripotent Stem Cells

110. The Nobel Prize in Medicine 2012 was awarded to two biologists for their breakthroughs in the field of stem-cell research — two discoveries that happened 44 years apart. The honors go to Britain's Sir John B. Gurdon and Japan's Shinya Yamanaka for their pioneering work with the life-shaping cells, which can be reprogrammed to create any kind of tissue in the body.

111. A mouse embryo injected with cells made pluripotent through stress, tagged with a fluorescent protein. Acid bath offers easy path to stem cells; just squeezing or bathing cells in acidic conditions can readily reprogram them into an embryonic state. Acid bath offers easy path to stem cells The latest…

112. Benefits of DNA technology Medical Applications identification of human genes in which mutation plays a role in genetic diseases

113. Huntington’s Disease can be diagnosed by the number of CAG repeats The data below shows the results of electrophoresis of PCR fragments amplified using probes for the site which has been shown to be altered in Huntington's disease. The male parent, as shown by the black box, got Huntington's disease when he was 40 years old. His children include 6 (3,5,7,8,10,11) with Huntington's disease, and the age at which the symptoms first began is shown by the number above the band from the PCR fragment. What is the prognosis for the normal children 4, 6, and 9?

114. Human Gene Therapy Gene therapy is the alteration of an afflicted individual’s genes Vectors, such as viruses, are used for delivery of genes into specific types of cells, for example bone marrow It may be difficult to target cells. Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations

115. Fig. 20-22 Bone marrow Cloned gene Bone marrow cell from patient Insert RNA version of normal allele into retrovirus. Retrovirus capsid Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Inject engineered cells into patient. 1 2 3 4

116. Pharmaceutical Products Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases In particular “pharm” animals and plants can be used to produce certain products

117. Fig. 20-23

118. Forensic Evidence and Genetic Profiles An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids

119. Fig. 20-24 This photo shows Earl Washington just before his release in 2001, after 17 years in prison. These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder. (a) Semen on victim Earl Washington Source of sample Kenneth Tinsley STR marker 1 STR marker 2 STR marker 3 (b) 17, 19 16, 18 17, 19 13, 1612, 12 14, 1511, 12 13, 1612, 12

120. Environmental Cleanup Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials Biofuels make use of crops such as corn, soybeans, and cassava to replace fossil fuels

121. Genetic Engineering in Plants Agricultural scientists have endowed a number of crop plants with genes for desirable traits The Ti plasmid is the most commonly used vector for introducing new genes into plant cells

122. Is this safe? Most public concern about possible hazards centers on genetically modified (GM) organisms used as food Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives

123. Fig. 20-25 Site where restriction enzyme cuts T DNA Plant with new trait Ti plasmid Agrobacterium tumefaciens DNA with the gene of interest Recombinant Ti plasmid TECHNIQUE RESULTS

124. Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology http://ecowatch.com/2 014/10/15/bill-nye- science-guy-gmo- foods/