1. Chapter 20/21 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 Polymorphisms.
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.

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. 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.

41. Cloning in Plants

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

44. Dolly Picture

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

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

47. 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.

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

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

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

51. Gene Therapy

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

54. 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.

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

56. PharmAnimals

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

58. 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

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

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

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