Recombinant DNA and Genetic Engineering

1. Recombinant DNA and Genetic Engineering Chapter 16

2. Impacts, Issues:Golden Rice or Frankenfood? • Scientists created transgenic rice (Golden Rice) as a vitamin A supplement for undernourished nations; is the benefit worth the risk in these gene-manipulated food sources?

3. 16.1 Cloning DNA • Process to add genes to food or other cell types is simple in principle • Researchers cut up DNA from different sources, then paste the resulting fragments together • Cloning vectors can carry foreign DNA into host cells

4. Cut and Paste for New DNA Combos • Restriction enzymes • Bacterial enzymes that cut DNA wherever a specific nucleotide sequence occurs • Single-stranded DNA tails produced by the same restriction enzyme base-pair together • DNA ligase bonds “sticky ends” together • Recombinant DNA • Composed of DNA from two or more organisms

5. Making Recombinant DNA This is especially useful to introduce genes into a sequence in research.

6. DNA Cloning • DNA cut into fragments by restriction enzymes is inserted into cloning vectors (plasmids) cut with the same enzyme • Cloning vectors with foreign DNA are placed in host cells, which divide and produce many clones, each with a copy of the foreign DNA

7. Cloning Vectors

8. DNA Cloning

9. cDNA Cloning • Complementary DNA (cDNA) • DNA made from an mRNA template • Reverse transcriptase transcribes mRNA to DNA, forming a hybrid molecule • DNA polymerase builds a double-stranded DNA molecule that can be cloned • Especially useful to obtain DNA without introns.

10. cDNA Cloning by Reverse Transcriptase

11. 16.1 Key Concepts DNA Cloning by Lab and Plasmids • Researchers routinely make recombinant DNA by cutting and pasting together DNA from different species • Plasmids and other vectors can carry foreign DNA into host cells

12. Genomes and DNA Libraries • Genome • The entire set of genetic material of an organism • DNA libraries are sets of cells containing various cloned DNA fragments • Genomic libraries (all DNA in a genome) • cDNA libraries (all active genes in a cell)

13. Probes Used for ID of DNA • Probe • A fragment of DNA labeled with a tracer • Used to find a specific clone carrying DNA of interest in a library of many clones • Nucleic acid hybridization • Base pairing between DNA from different sources • A probe hybridizes with the targeted gene

14. Big-Time DNA Amplification: PCR • Polymerase chain reaction (PCR) • A cycled reaction that uses a heat-tolerant form of DNA polymerase (Taq polymerase) to produce billions of copies of a DNA fragment • This is how a single drop of blood at a crime scene can become expanded to enough to make necessary tests and still be available for future testing if needed

15. PCR in Overview • DNA to be copied is mixed with DNA polymerase, nucleotides and primers that base-pair with certain DNA sequences • Cycles of high and low temperatures break and reform hydrogen bonds between DNA strands, doubling the amount of DNA in each cycle

16. PCR Steps to More

17. 16.2 Key Concepts Needles in Haystacks • Researchers manipulate targeted genes by isolating and making many copies of particular DNA fragments

18. 16.3 DNA Sequencing • DNA sequencing reveals the order of nucleotide bases in a fragment of DNA

19. DNA Sequencing • DNA is synthesized with normal nucleotides and dideoxynucleotides tagged with different colors • When a tagged base is added, DNA synthesis stops; fragments of all lengths are made • Electrophoresis separates the fragments of DNA, each ending with a tagged base, by length • Order of colored bases is the sequence of DNA • Finished sequence is basis for comparison

20. DNA Sequencing

21. 16.4 DNA Fingerprinting • One individual can be distinguished from all others on the basis of DNA “fingerprints” • Confidence here in results is extremely high, in the usually stated range of one in many millions

22. DNA Fingerprints • DNA fingerprint • A unique array of DNA sequences used to identify individuals • Short tandem repeats (STRs) • Many copies of the same 2- to 10-base-pair sequences in a series along a chromosome • Types and numbers of STRs vary greatly among individuals

23. Creating DNA Fingerprints • PCR is used to amplify DNA from regions of several chromosomes that have STRs • Electrophoresis is used to separate the fragments and create a unique DNA fingerprint • DNA fingerprints have many applications • Legal cases, forensics, population studies

24. DNA Fingerprints: Forensics Case Example You are on the jury. You are shown this prepared comparison of DNA fingerprints, with ID as shown. See if you can match suspect with sample from the crime scene.

25. 16.3-16.4 Key ConceptsDeciphering DNA Fragments • Sequencing reveals the linear order of nucleotides in a fragment of DNA • A DNA fingerprint is an individual’s unique array of DNA sequences

26. 16.5 Studying Genomes • Comparing the sequence of our genome with that of other species is giving us insights into how the human body works • You already know of 98 percent same human sequences with that of chimpanzes • How about 49 percent the same between a banana and a human?

27. The Human Genome Project • Automated DNA sequencing and PCR allowed human genome projects to sequence the 3 billion bases in the human genome • 28,976 genes have been identified, but not all of their products or functions are known • As of 2010, distinct gene numbers down to about 23,000 by best estimates from work

28. Sequencing the Human Genome Computers have greatly speeded process up and also increased accuracy.

29. Genomics is a Growing Application • Genomics: The study of genomes • Structural genomics • Comparative genomics • Analysis of the human genome yields new information about genes and how they work • Applications in medicine and other fields • Example:APOA5 mutations and triglycerides

30. DNA Chips Have a Future • DNA chips • Microarrays of many different DNA samples arranged on a glass plate • Used to compare patterns of gene expression among cells of different types or under different conditions • May be used to screen for genetic abnormalities, pathogens, or cancer

31. 16.6 Genetic Engineering • Genetic engineering • A laboratory process by which deliberate changes are introduced into an individual’s genome • Today’s most common genetically modified organisms are bacteria and yeast • Are used in research, medicine, and industry • Example: production of human insulin

32. GMOs – Now and Later • Genetically modified organisms (GMOs) • Individuals containing modified genes from the same species or a different species • Future will have major control problems as the developer of GMO usually patents process/result • Transgenic organisms • Individuals containing genes transferred from a different species (also GMOs) • Example: Bacteria with jellyfish genes

33. 16.7 Designer Plants by GM • Genetically engineered crop plants are widespread in the United States • But can their designed change(s) “jump” to other plant life or end up incorporated in animals eating the modified plants?

34. The Ti Plasmid – a GMO Mechanism • Ti plasmid • Plasmid of bacteria Agrobacterium tumefaciens • Contains tumor-inducing (Ti) genes • Used as a vector to transfer foreign or modified genes into plants, including some food crops

35. Ti Plasmid Transfer Steps

36. Genetically Engineered Plants • Crop plants are genetically modified to produce more food at lower cost • Resistance to disease or herbicides • Increased yield • Plants that make pesticides (Bt protein gene) • Drought resistance

37. GMO Controversies • 73 GMO crops are approved for use in US, with hundreds more pending • Corn, sorghum, cotton, soy, canola, alfalfa • Big problem of just a few companies doing nearly all the research and manufacturing – can lead to a monopoly problem in future • Facts and controversy – real life • In crops engineered for herbicide resistance, weeds are becoming resistant to herbicides • Engineered genes are spreading into wild plants and nonengineered crops

38. Some Genetically Modified Plants

39. 16.8 Biotech Barnyards • Animals that would be impossible to produce by traditional breeding methods are being created by genetic engineering • This can be really good for endangered animals • Genetically engineered animals are used in research, medicine, and industry

40. Of Mice and Men • 1982: The first transgenic animals – mice with genes for rat growth hormone

41. Examples of Transgenic Animals • Genetically modified animals are used as models of many human diseases • Mice used in knockout experiments • Genetically modified animals make proteins with medical and industrial applications • Goats and rabbits that make human proteins • Farms animals with desirable characteristics

42. Some Genetically Modified Animals That silly-looking featherless chicken is easily the most commercially viable possibility shown here. It would eliminate a costly part of chicken processing and could enable very warm climate poultry farms.

43. Knockout Cells and Organ Factories • Transgenic pigs with human proteins are a potential source of organs and tissues for transplants in humans • May prevent rejection by immune system • Xenotransplantation • Transplantation of a tissue or organ from one species to another • Pig heart valves used for many years.

44. 16.10 Modified Humans? • We as a society continue to work our way through the ethical implications of applying new DNA technologies • The manipulation of individual genomes continues even as we are weighing the risks and benefits of this research

45. Gene Therapy – Helping the Individual • Gene therapy • Transfer of recombinant DNA into body cells to correct a genetic defect or treat a disease • Viral vectors or lipid clusters insert an unmutated gene into an individual’s chromosomes • Examples: Cystic fibrosis, SCID-X1

46. Getting Better by Gene Therapy • 1998: A viral vector was used to insert unmutated IL2RG genes into boys with severe combined immunodeficiency disease (SCID-X1) – most recovered immune function

47. Getting Worse by Gene Therapy • No one can predict where a virus-injected gene will insert into a chromosome – several boys from the SCID-X1 study developed cancer • In other studies, severe allergic reactions to the viral vector itself have resulted in death

48. Getting Perfect Over Time • Eugenic engineering • Engineering humans for particular desirable traits, not associated with treatment of disorders

49. 16.6-16.10 Key ConceptsUsing the New Technologies of GM • Genetic engineering, the directed modification of an organism’s genes, is now used in research, and it is being tested in medical applications • Many questions must be answered about the ethics and consequences of manipulating the human genome – some of these can be answered by our government but many will remain answerable only by the individual as he or she agrees to their usage personally.