1. Biotechnology Chapter 20

2. Gene technology

3. Biotechnology
Manipulation of organisms to make useful products

4. Genetic engineering Manipulation of genes Gene cloning:
Multiple copies of a single gene
Produce a specific product

5. Fig. 20-2
Cell containing gene of interest
Bacterium 1
Gene inserted into plasmid
Bacterial chromosome
Plasmid
Gene of interest
Recombinant DNA (plasmid)
DNA of chromosome 2
Plasmid put into bacterial cell
Recombinant bacterium 3
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
Copies of gene
Protein harvested
Figure 20.2 A preview of gene cloning and some uses of cloned genes 4
Basic research and various applications
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

6. Recombinant DNA 1970’s Combining genes from different sources
Even different species
Combined into single DNA
Example: Bacteria & mammal

7. Recombinant DNA Genetically modified bacteria
Mass produce beneficial chemicals
Insulin
Growth hormone
Cancer drugs
Pesticides

8. Plasmid

9. Plasmid Small separate circular DNA Replicated same as main DNA
Foreign DNA added to plasmid
Replicated along with plasmid

10. Recombinant DNA Nucleases: Enzymes that degrade DNA
Restriction endonulceases:
Restriction enzymes
Cut DNA into fragments
Specific points

11. Recombinant DNA Restriction sites: Places where DNA is cut
Short DNA sequence

12. Recombinant DNA Restriction enzyme Recognizes short sequences in DNA
Cuts at these sequences
Staggered cut
Leaves single-stranded ends
Called “sticky ends”

13. Recombinant DNA
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17. Recombinant DNA Sticky ends Enables insertion of other DNA
DNA fragments from other sources
Match ends by base pairs (complementary sequences)
DNA ligase:
Enzyme combines ends
Forms a phosphodiester bond

20. Recombinant DNA (Process)
1. Isolate gene of interest & bacterial plasmid
2. Cut DNA & plasmid into fragments
3. Mix DNA fragments with cut plasmid.
Fragment with gene of interest is inserted into the plasmid
4. Recombinant plasmid is mixed with bacteria

21. Recombinant DNA (Process)
5. Bacteria with recombinant DNA reproduce
6. Isolate bacterial clones that contain gene of interest
Producing protein of interest
7. Grow large quantities of bacteria that produce the protein

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

24. Recombinant DNA Vector: DNA molecule that carry foreign DNA
Enters & replicates in the host
Plasmids & phages are common vectors
Phages are larger than plasmid
Can handle inserts up to 40 kilobases

25. PCR Polymerase chain reaction Amplify DNA
Makes large quantities of DNA
1985

26. PCR
Heated
Denatured
DNA primer
Heat stable DNA polymerase
Makes DNA

27. molecules; 2 molecules (in white boxes) match target sequence
Fig. 20-8
TECHNIQUE 5 3
Target sequence
Genomic DNA 3 5 1
Denaturation 5 3 3 5 2
Annealing
Cycle 1 yields 2
molecules
Primers 3
Extension
New nucleo- tides
Figure 20.8 The polymerase chain reaction (PCR)
Cycle 2 yields 4
molecules
Cycle 3 yields 8
molecules; 2 molecules (in white boxes) match target sequence

28. Gel electrophoresis Study DNA Polymer (gel) Restriction fragments
Separates DNA based on charge & size
Nucleic acids negative charge (Phosphates)
Migrate towards + end (red)

29. Fig. 20-9 Figure 20.9 Gel electrophoresis TECHNIQUE
Mixture of DNA mol- ecules of different sizes
Power source –
Cathode
Anode +
Gel 1
Power source – +
Longer molecules 2
Shorter molecules
RESULTS
Figure 20.9 Gel electrophoresis

30. Fig
Normal -globin allele
Normal allele
Sickle-cell allele
175 bp
201 bp
Large fragment
DdeI
DdeI
DdeI
DdeI
Large fragment
Sickle-cell mutant -globin allele
376 bp
201 bp 175 bp
376 bp
Large fragment
DdeI
Figure Using restriction fragment analysis to distinguish the normal and sickle-cell alleles of the β-globin gene
DdeI
DdeI
(a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene
(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

31. Cloning Multicellular organisms come from a single cell.
Offspring are identical

32. Cloning 1950 Carrots Totipotent: Mature cells that undifferentiated
Give rise to any type of cells
Common in plants

33. Cloning Nuclear transplantation
Nucleus of an unfertilized/fertilized egg is removed
Replaced with nucleus of differentiated cell
Direct development of cell into tissues etc.

34. Cloning Removed nuclei from an egg Mammary cells Fused with egg cells
Dolly, 1997, identical to mammary cell donor
Died prematurely age 6
Arthritis & lung disease

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

38. Fig

39. Cloning Few develop normally Abnormalities
Epigenetic changes to the chromatin
More methylation of chromatin
Reprogram chromatin of differentiated cell

40. Stem cells Started 1998 at UW Early embryonic cells
Potential to become any type of cell
Master cell generates specialized cells
Such as muscle cells, bone cells, or blood cells

41. Stem cells Embryos Bone marrow Umbilical cord blood Blood stem cells
?? Turn skin cells into embryonic stem cells
Therapeutic cloning

42. From bone marrow in this example
Fig
Embryonic stem cells
Adult stem cells
Early human embryo at blastocyst stage (mammalian equiva- lent of blastula)
From bone marrow in this example
Cells generating all embryonic cell types
Cells generating some cell types
Cultured stem cells
Different culture conditions
Different types of differentiated cells
Liver cells
Nerve cells
Blood cells

43. Medical applications Genetic markers Detect abnormal disease SNP
Single nucleotide polymorphisms
Single base pair site where variation is found
RFLP
Restriction fragment length polymorphisms

44. Disease-causing allele
Fig
DNA T
Normal allele
SNP C
Figure Single nucleotide polymorphisms (SNPs) as genetic markers for disease-causing alleles
Disease-causing allele

45. Medical applications Gene therapy Treat genetic defects
Alters person’s genes
2 girls with rare blood disease
CF (vectors are viruses)
SCID (immune disorder)
Injected viral DNA with normal gene

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

47. Medical applications Transgenic animal
Gene from one animal is inserted into another
Goat milk protein anti-thrombin
Isolated from milk
“pharm” animals

48. Animals Transgenic animals engineered for specific traits
Genetically create a racehorse
Not have to breed
Sheep with better wool??

50. Agricultural applications
Manipulate tomatoes
Do not ripen as fast
“Flavr-Savr”
Slows down ethylene production

51. Agricultural applications
Introduce genes to plants
Enable them to “fix” nitrogen
Convert N2 to NH3
Help eliminate use fertilizers
Cut $$

52. Agricultural applications
Herbicide resistance
Plant genetically resists the herbicide
Insect resistance

53. Agricultural applications
Transgenic rice
“golden rice”
Rice with genes that code for better absorption of iron and beta carotene
First of many genetically engineered foods
Helps dietary deficiencies

54. Forensics Genetic profile: Individual genetic markers
“DNA fingerprint”
RFLP
STR
Short tandem repeats
Occur in specific regions in genome
Unique

56. Fig
(a)
This photo shows Earl Washington just before his release in 2001, after 17 years in prison.
Source of sample
STR marker 1
STR marker 2
STR marker 3
Figure STR analysis used to release an innocent man from prison
For the Discovery Video DNA Forensics, go to Animation and Video Files.
Semen on victim
17, 19
13, 16
12, 12
Earl Washington
16, 18
14, 15
11, 12
Kenneth Tinsley
17, 19
13, 16
12, 12
(b)
These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder.

57. Concerns over genetic engineering
Genetically modified foods
Harmful?
Genetically engineered gametes
Blonde and blue eyes??