3/31/2014

1. 3/31/2014 • Plasmid-(DNA ring found in bacteria) • Restriction Enzymes used to cut DNA • Recombinant DNA- DNA combination of 2 or more species • Bacteria with recombined plasmid • Next step is cloning

2. Standard 5c: Students know how genetic engineering(biotechnology) is used to produce novel biomedical and agricultural products CH 20 DNA Technology and Genomics

3. Chapter 20: Terms to Know • cDNA library • PCR • Gel electrophoresis • Southern blotting • DNA microarray assays • SNPs • RFLPs • Stem cells • Gene therapy • GMO (genetically modified organism) • Genetic engineering • Biotechnology • Recombinant DNA • Gene cloning • Restriction enzymes • Sticky ends • DNA ligase • Cloning vector • Nucleic acid hybridization • Genomic library

4. What You Must Know: • The terminology of biotechnology. • The steps in gene cloning with special attention to the biotechnology tools that make cloning possible. • The key ideas that make PCR possible. • How gel electrophoresis can be used to separate DNA fragments or protein molecules. • Information that can be determined from DNA gel results, such as fragment sizes and RFLP analysis

5. Definitions • Genetic Engineering: process of manipulating genes and genomes • Moving genes from a chromosome of one organism to a chromosome of a different organism, create “transgenic organism” that expresses new traits • Biotechnology: process of manipulating organisms or their components for the purpose of making useful products. • Genetic Testing: using biotechnological tools to look for the presence/absence of a particular sequences

6. Recombinant DNA: DNA that has been artificially made, using DNA from different sources • eg. Human gene inserted into E.coli • Gene cloning: process by which scientists can produce multiple copies of specific DNA segments that they can then work with in the lab

7. Tools of Genetic Engineering • Restriction enzymes(restriction endonucleases): used to cut strands of DNA at specific locations (restriction sites). DNA scissors. • Restriction Fragments: have at least 1 sticky end (single-stranded end) • DNA ligase: joins and seals DNA fragments. Glue • Cloning vector: carries the DNA sequence to be cloned (eg. bacterial plasmid). DNA molecules that can be modified to store and replicate other DNA sequences

8. Identifying the gene of interest http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_4.html How would you identify different genes? Use different probes

9. After genes are cloned… • A genomic library is made using bacteriophages or bacteria • Is stored as a collection of phage clones or bacteria clones

10. Foreign genome cut up with restriction enzyme or Recombinantphage DNA Bacterialclones Phageclones Recombinantplasmids (b) Phage library (a) Plasmid library Figure 20.6 • Storing Cloned Genes • 1. Genomic libraries: complete set of plamids containing cell clones • Cloned genes can also be stored in phages or large plasmids

11. Storing Cloned Genes • 2. cDNA-complementary DNA made from mRNA has no introns • DNA copies of RNA molecules produced using reverse transcriptase, contains consecutive coding sequence of gene • Increases stability, removes introns for prokaryotic expression of eukaryotes

12. Storing Cloned Genes 3.Nucleic acid hybridization: using a complementary DNA strand to attach to a gene of interest in a genomic library • Radioactive probe attaches to the desired gene and then a photographic film is used to view where the gene is located in the library. Find and track gene of interest

13. http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

14. What is Genetic Engineering Used for: • Sequencing the human genome • Genetically engineering drugs and vaccines • medical diagnostics • Evolutionary relationships • DNA fingerprinting (solving crimes) • “Improving” Crops • Geneticallymodified food (GMO)

15. Transformation • taking in other DNA, causing a change in organism.

16. Genetic Engineering begins with… Transforming Bacteria • During transformation: • a cell takes in DNA from outside the cell. • The external DNA becomes a component of the cell's DNA. • Foreign DNA is first joined to a small, circular DNA molecule known as a plasmid. • Plasmids are found naturally in some bacteria and have been very useful for DNA transfer.

17. Bacterial Plasmids • To be useful, plasmids must minimally have: • Origin of replication • Region containing many restriction sites (multiple cloning sites) • A gene/genes that enable screening of cells that have successfully take up the plasmid (usually ampicillin resistance) • There is a size limit on plasmids • Artificial chromosomes is now used to get more genes in cells (2010)

18. Transgenic Microorganisms Transgenic bacteria produce important substances useful for health and industry. Transgenic bacteria have been used to produce: • insulin • growth hormone • clotting factor

19. Transgenic Organisms • An organism described as transgenic, containsgenes from other species. • Genetically modified organisms (GMO) • Green Fluorescent protein (GFP) – fluorescent analogys

20. Transgenic Organisms • Idea: To add favorable characteristics to a plant or animal by manipulating the organisms’genes. • Plants: Make more tolerant to drought or resistantto specific pesticides

21. Transgenic animals Featherless chicken Transgenic animals have been used to study genes and to improve the food supply. • Researchers are trying to produce transgenic chickens that will be resistant to the bacterial infections that can cause food poisoning

22. cucumber mosaic virus (CMV)

24. Use of Genetic Engineering • To transfer human genes into bacteria allows the bacteria to produce human proteins • Next step how it is done to create recombinant DNA…

25. Gene Splicing = cutting genes from one organism and inserting into a new organism • Restriction Enzymes (Endonucleases) = chemical “scissors” used to cut segments of DNA. Recognizes palindromic DNA sequences and cuts both sides of DNA. (Ex: CTTAA). • Sticky Ends = ends of DNA that do NOT have complimentary bases, after the “cut”. • Blunt cuts- cut in the middle of the sequence

27. Different restriction enzymes can be used and enzyme recognize different restriction sites • Ex: EcoRIcuts at GAATTC • Restriction EZ are name from the bacteria they are isolated from

28. Step 1= remove cloning vector (plasmid) from bacteria and “cut” open with restriction enzymes. • Plasmid = DNA ring found in bacteria. • Replicates independently of the organism’s main chromosome. • Used as vectors •Cloning Vector = any DNA that is used to make copies of genes ( gene of interest)

29. Step 2 = Make recombinant DNA“Cut” out donor gene and insert (glue) into plasmid. • Donor Gene= the gene that you wish to have copied or inserted into other organism. •Ex: Insulin Gene •Ligase= enzyme that seals (glues) the sticky ends of a DNA fragment and joins 2 DNA molecules together. •Recombinant DNA= DNA that is a combination of 2 or more species

32. Recombinant DNA Host Cell DNA Target gene Modified Host Cell DNA

33. Insert Recombinant DNA into bacteria, allow bacteria to multiply (clone).Step 3: Cloning • Gene clone= exact copy of a gene. Host cell reproduces. • •Ex: This bacteria now makes Insulin!!!

35. Step 4: Screening • Cells that havereceivedthe gene of interest are recognizedand are separated from other cells that did not take up the vector. • The cells with the gene of interest will then transcribe and translate the gene of interest to make the protein coded for the gene

37. Techniques of Genetic Engineering

38. Ch 21The Genetic Basis of Development (415-420)

39. Embryonic development • Embryonic development involves • cell division (a.k.a.- mitosis), • cell differentiation-specialized structure/function • Morphogenesis- physical process that give an organism its shape • In embryonic development of most organisms • A single-celled zygote gives rise to cells of many different types, each with a different structure and corresponding function

40. Different cell types result from differential gene expression in cells with same DNA • Differences between cells in a multicellular organism • almost entirely from differences in gene expression (TRANSCRIPTION)

41. CELLS...Totipotency • Atotipotentcell • cell capable of generating a complete new organism • Cloning • using 1+ somatic cells from a multicellular organism to make another genetically identicalindividual

42. Nuclear Transplantation in Animals • In nuclear transplantation • nucleus of unfertilized egg cell or zygote is replaced with nucleus of a differentiated cell

43. Cloning • Aclone is an individual in a population of genetically identical cells, that was produced from a single cell. • In 1997, Ian Wilmut cloned a sheep called Dolly. Dolly and Bonnie

44. Cloning Organisms • Nuclear transplantation: nucleus of egg is removed and replaced with nucleus of somatic (body) cell

45. Cloning • Cloning Dolly 1.

46. Cloning • Cloning Dolly 2.

47. Cloning 3. • Cloning Dolly

48. Cloning • Cloning Dolly 4.

49. Cloning • Cloning Dolly 5.

50. Cloning • Cloning Dolly 6.