1. Chapter 20 Biotechnology

2. Focus of Chapter An introduction to the methods and developments in:
Recombinant DNA
Genetic Engineering
Biotechnology

3. Recombinant DNA
DNA in which genes from different sources are linked.
Ex: the “green” mice

4. Genetic Engineering
The direct manipulation of genes for practical purposes.
Ex: Using E. coli to produce human insulin.

5. Biotechnology
The use of living organisms or their components to perform practical tasks.
Ex: the use of bacteria to digest oil spills.

6. Restrictive Enzymes
Cut DNA at specific nucleotide sequences called “restriction sites”.
Used to "cut and splice" DNA.
Obtained from bacteria.
Ex. EcoRI and Hind III

8. Plasmids Used extensively in Biotechnology and Recombinant DNA.
Serve as a “vehicle” for transporting genes.
Comment – other “vehicles” are used in other methods

9. Steps for Plasmid Use
1. Get the DNA for the trait. 2. Insert DNA into the plasmid. 3. Bacterial Transformation. 4. Identification of the new trait.

11. Insertion Placing foreign DNA into a plasmid.
Open plasmid with enzymes to create “sticky ends”.
Splice the new DNA and plasmid together.

12. Transformation Placing the plasmid into a bacterial cell.
Reminder - our lab – need to know for AP testing purposes.

13. Methods
Temperature shock & salt treatment
Electric current
Injection

14. Identification Screening the altered cells for the desired gene.
Ex: Antibiotic sensitivity or the expression of a “new” trait (color, glowing etc.).

15. Example Applications
1. Insulin 2. Human Growth Hormone 3. Other Proteins

17. Comment
Gene can’t be above a certain size (12 kb) or a plasmid won’t work.
mRNA must not need splicing to remove introns.

18. DNA Sources
1. Organism - use a section of their chromosome. 2. cDNA - Complementary DNA - created copy of DNA from the mRNA transcript to avoid introns. Uses reverse transcriptase.

20. Other Vehicles
BACs – Bacteria Artificial Chromosome – handle inserts of kb
YACs – Yeast Artificial Chromosome – have a centromere and telomomere, handle inserts >300 kb
HACs – Human Artificial Chromosome

21. Nucleic Acid Probes
Used to find a specific DNA sequence in a mixture of DNA pieces

22. How Used DNA is denatured and cut to produced ss pieces.
Piece of compliment DNA is added as a “probe”. The probe has been “labeled”.
Look for where the probe goes in the DNA sample.

23. Probe Diagram

24. DNA Microarray

26. PCR Polymerase Chain Reaction
Method for making many copies of a specific segment of DNA.
Also called “DNA Amplification”.

27. PCR - Method
1. Separate strands by heating (denature the DNA). 2. Cool slightly. 3. Build new strand from primers and nucleotides. 4. Repeat.

29. Importance - PCR
Can amplify any DNA with as little as one original copy.
Very useful in a variety of techniques and tests.

30. Gel Electrophoresis Technique will be covered in lab.
Used to separate mixtures of DNA or proteins.

31. RFLP Analysis Restriction Fragment Length Polymophisms.
Method for detecting minor differences in DNA structure between individuals.
Common in DNA fingerprinting

32. Method
1. Digest DNA with restrictive enzymes. 2. Separate pieces by Gel Electrophoresis 3. Identify sequences with probes. 4. May use PCR to amplify specific variable regions of the DNA.

33. RFLP - Results Patterns of DNA markers or DNA fingerprint
Markers are inherited in a Mendelian pattern and can show relationships (Pedigree studies).

36. DNA Sequencing
Uses dideoxynucleotides which stop DNA Polymerase at a known point in DNA replication.
Builds new DNA from single strand DNA.
Produces fragments of different lengths.

38. DNA Sequencers

39. Application DNA sequence is read base by base.
By sequencing overlapping pieces of chromosomes, the entire genome of an organism can be read. (chromosome walking)

40. Genome Sequencing The DNA sequence has been “read” for the following:
4,000 bacteria species
190 archaeal species (a type of prokaryote)
180 eukaryotic species
More than 17,000 species are in progress
Includes several cancers, ancient humans and bacteria that live in our gut.

41. Cloning of Organisms Reproducing an organisms by asexual means.
Commonly done in plants.
Shows the concept of “Totipotency” – that a single cell can develop into a new organism.

42. Cloning in Plants

43. Cloning in Animals Has been done by nuclear transplantation.
Examples – Dolly
Many other vertebrates have now been cloned.

45. Dolly Picture

46. Cloned cows anyone?

47. Stem Cells
Stem Cell – an unspecialized cell that reproduce itself or differentiate into other cells.

48. Types of Stem Cells Embryonic – from an embryo.
Adult – found in various tissues of the adult body.

49. Research
Attempting to use stem cells to replace damaged cells or body parts.
If can use own stem cells, avoids tissue rejection problems Ex – grow a new bladder.

50. DNA Technology: Applications
1. Basic Research 2. Medical 3. Forensics 4. Agricultural

51. Basic Research
1. DNA and protein studies 2. Evolution 3. Gene structure and control mechanisms.

52. Assignments Read Chapter 20, Chapter 13 in Hillis
Chapter 19 – before spring break
Chapter 20 – Wed. 3/19
Exam 2 – Tuesday 3/25

53. Medical Uses
1. Diagnosis of Diseases 2. Gene Therapy 3. Vaccines 4. Pharmaceutical Products

54. Disease Identification
Knock-out mice – engineered to “knock-out” one or both copies of a gene in order to determine the function of the gene.
Very BIG study technique in medicine and disease studies.
Model organism for understanding the disease at the molecular level and for testing medicines and treatments.

55. Gene Therapy

56. PharmAnimals

57. Pharmaceutical Products
Using genetic engineering to produce medical proteins.
Ex. – Humilin, HGH, Factor VIII and Factor IX for blood clotting, clotting busting proteins etc.

58. Forensic Uses
DNA fingerprints for crime solving – used in every TV crime show
DNA identification records – standard for the military

60. Paternity Cases
Variable regions of DNA are cut out and amplified by PCR.
Often used tandem repeat regions (DNA sections that repeat).
Look at sets of tandem repeat areas with known frequencies.
Any mismatch excludes the “father”.
Consistent “matches” keen the individual as a candidate.

61. Current uses or applications
Some pedigree registrations now require a “proof” by a match of the DNA of the offspring to the sire and dam on the pedigree application.
Common in a number of animal pedigree registrations such as dogs, horses, beef etc.

62. Comments
Links suspect bodily to the crime scene, but doesn’t prove they committed the crime.
Results take MUCH longer than on TV shows.
Analysis of old evidence is reversing some sentences.

63. Agricultural Uses 1. Animals Increased milk production
Increased feed utilization
Increased meat production

64. Agricultural Uses 2. Plants Herbicide resistance
Retard spoilage of fruits
Insect resistance – BT corn
Nitrogen-Fixation ability

65. Golden Rice

66. Genetically Modified Organism or GMO
Produced by direct genetic manipulation, not traditional breeding practices.
FDA just approved sale of GMO animal products for human consumption.
Bioethics concerns

67. GMO – commercial applications

68. Future Of DNA Technology
Cloning of higher animals.
Stem Cells - growth of replacement tissues and organs.
Gene therapy to correct DNA defects. ?

69. Summary Know the basics of some of the DNA technology techniques.
Bacterial transformation lab
How Gel electrophoresis works
Restriction enzymes

70. Summary
Watch the news for DNA technology discoveries. Be able to discuss one recent event.

71. Questions?
Viruses?
Bioethics?