This document discusses genetic engineering and biotechnology. It begins by defining genetic engineering as the manipulation of genes, usually outside an organism's natural reproductive process. It then discusses chromosomes and mutations, including examples of chromosome numbers in different species. Techniques for genetic engineering are explained, such as restriction enzymes and bacterial transformation. Applications include creating transgenic bacteria, plants, and animals. Ethical issues related to genetic engineering are also reviewed.

1. Genetic Engineering and
Biotechnology- 3.5

2. Genetic Engineering
 A general term used for the manipulation
of genes.
 Usually outside organism’s natural
reproductive process.

3. Chromosomes and Mutations

4. Diploid Chromosomes of
Different Species
Parasitic Worm (Parascaris equorum) – 2
Rice (Oryza sativa) – 24
Human (Homo sapien) – 46
Chimpanzee (Pan troglodytes) – 48
Domestic Dog (Canis familiaris) - 78

5. Chromosomes
 Autoradiography – technique used to discover where
specific substances were located in cells or tissue
Cairn’s Technique
 Measured DNA molecules
– E. coli Cells grown in culture – Tritium (hydrogen) irradiated
Thymine.
– Cells were lysed (placed in a hypotonic solution) DNA released
– Solution spread on an photographic plate
– Developed and measured for length of Bacteria.
– 1,100 um (length of bacteria is 2 um)
– Showed that replication is semi conservative

6. Autoradiograph

7. Gene Mutations
 Permanent change in the sequence of
nucleotides which might result in the
production of a different protein
 Changes phenotype
– If in sex cells --- can be transmitted
– If in somatic cells – not inheritable

8. Sickle Cell Anemia – Gene
Mutation
 Base substitution
The Disease:
 Inherited disease that affects the RBC ability to
carry oxygen on hemoglobin.
 Produces abnormal hemoglobin…can be trapped in
capillaries, shorten RBC life span (4 days)
 Symptoms:
– Anemia,
– stroke,
– spleen and renal failure,
– acute chest syndrome,
(higher mortality)

9. Sickle Cell Anemia
 Base substitution – genetically carried - PS
 Occurs during protein synthesis:
Sickle Cell Anemia:
DNA
mRNA
A. A.

11. Mutagens, Oncogenes, Metastasis
 Mutagen
– Anything that causes a mutation.
– Affects structure of DNA, DNA Replication,
Nuclear division
– Examples:
 UV and X-ray exposure
 Chemicals: certain insecticides and benzene
 Hydrogen Peroxide
 Metals: Nickel
 Certain viruses

12. Mutagens, Oncogenes, Metastasis
 Oncogenes
– Genes that when expressed at a high level or are mutated,
can turn a normal cell into a tumor.
 Genes that are involved in the CELL CYCLE
 Oncogenes can prevent normal Apoptosis
 Most Oncogenes need a trigger: mutation or viral
infection to become cancerous.
 Metastasis:
– Spread of cancer from one part of the body to another.
 Primary tumor – first tumor
 Secondary tumor – cells enter blood or lymphatic system… form
another tumor.
– Breast cancer cells that travel to the lungs is still breast
cancer

13. Early
Techniques
in
Genetic
Engineering

14. Griffith’s Experiments - 1928

15. 15

16. Techniques
In
Genetic Engineering
Modern
Biotechnology

17. Transgenic Organisms
 Using tools of DNA technology, a
gene from one organism can be
inserted into cells from another
organism
 This process is called cell
transformation

18. Transgenic Bacteria
 Genes for human proteins like insulin and
blood clotting factor can be inserted into
bacteria
 Bacteria then translate the inserted gene
to produce the protein it codes for.

19. Plasmid
 Small DNA molecules found naturally in
bacteria.
 Useful for DNA transfer. WHY?
– It has a DNA sequence.
– It exists independent of the main naked DNA
– It can be “removed and manipulated
– If a plasmid containing foreign DNA gets into
a bacterial cell >>>>> replication occurs.

20. Recombinant DNA

22. Recombinant DNA
 If transformation is successful DNA is
combined (recombinant) onto the
chromosomes of the cell.
 Applications for genetic engineering:
– Modified COWS: Create human forms of
protein >>> insulin, HGH,
– Factor IX - clotting factor from sheep.
– Tomato plants – delayed ripening

23.  UNIVERSITY OF UTAH VIRTUAL LABS
 GENETICS SIMULATIONS
 Biology Library :: Dolan DNA Learning
Center

24. Restriction Enzymes

25. Restriction Enzymes –
“Cleaving” DNA
 Endonuclease: Enzymes that recognize a
particular short DNA sequence, and then
cut the DNA strand within that sequence.
 1st discovered in bacteria which use the
enzymes to cut and destroy viral DNA.
 DNA molecules are too large to be
analyzed as a whole.

27. Restriction Enzymes
 Eco RI – restriction enzyme found in E.
Coli.
 EACH RESTRICTION ENZYME
RECOGNIZES A DIFFERENT SEQUENCE OF
DNA AND WILL CUT THAT SEQUENCE
ONLY!!!

28. Restriction Enzyme List
 Restriction Enzymes******
 Enzymes Start with A - Restriction Enzymes

29. Steps to Perform
Bacterial
Transformation!
(GENE CLONING)

31. How to Extract DNA:
THE MOVIES!
DNA LAB PART 1 DNA LAB PART 2

32. 1. DNA Extraction
 SALT
 SOAP
 ALCOHOL

33. 2 – 8: Transformation-
“Cutting and Splicing”
2. Gene (Foreign DNA) cut by
specific endonucleases.
3. Plasmid(vector) removed from
bacteria
4. Plasmid cut with
endonucleases.
Both produce complementary
“sticky ends.”
5 Gene is spliced into cut plasmid
6. DNA Ligase seals pieces of
DNA.
7. Plasmid inserted back into
bacteria.
8. Bacteria given favorable
conditions to reproduce!

34. Sticky Ends

35. Restriction Enzymes
Blunt Cut
Blunt Cut:
5’ – GTTAAC – 3’
3’ – CAATTG – 5’
Blunt Ends:
5’ GTT AAC – 3’
3’ CAA TTG – 5’
Blunt ends can be attached to any other DNA that
produces blunt ends.

36. Transgenic Bacteria – the process

37. Transgenic
Animals

38. Animal Cell Transformation
 Uses:
–Insert human genes into
experimental animals (mice) to
study the gene function
–Create livestock that grow faster,
produce leaner meat, resist
disease better.

39. Animal Cell Transformation
 DNA to be inserted is
injected directly into
fertilized egg.
 Cell enzymes that
usually repair DNA or
help in recombination
insert the new DNA
into the animal’s
chromosomes.

40. A group of genetically
identical cells artificially
derived from a single
parent cell
Cloning

41. Cloning to Create a New Individual
Genetically Identical to an Existing
Organism
Transgenic (clonable) animals
include:
Sheep
Cows (dairy and beef)
Fish

42. Cloning to Create a New Individual
Genetically Identical to an Existing
Organism

43. Methods of Cloning
1. Separate undifferentiated early embryo
cells. Allow to grow and transfer to
surrogate mothers.
2. Enucleate an egg and replace it with a
nucleus from a somatic cell. Allow to
grow to embryo. Place back in mother.

44. What are the ethical issues involved with
Genetic Engineering including: therapeutic
cloning, Gene (GMO) and Organism cloning?
Is Genetic Engineering
Good?

45. Advantages
 Production of organisms with favorable qualities
 Can produce things to aid humans…insulin or
HGH, enhanced nutrients in plants.
 Farming (GMO Plants)
– Improve/Increase food production
– Crops that provide their own pest control
– Produce proteins for medicines
 Might make screening of genetic diseases easier
 Might help infertile couples
_____

46. Disadvantages
 Risk of GM crops being toxic to humans or other
organisms in the ecosystem.
 GM Pollen could affect other plants
 Risk of increased allergies caused by GM crops.
 Use of embryos for experimentation.
 Stem cell mutation – cancer
 Possible use of aging DNA.
 Possible use of cloned body parts.
 Religious reasons preventing tampering with
natural processes.

47. Other Aspects of Genetic
Engineering

49. Polymerase Chain Reaction
(PCR)
 Takes a small amount of
DNA, and makes many
copies of it (amplification)
– The more DNA available,
the more studies you can
do with it
– Frequently used for small
amounts of DNA left at
crime scenes

50. Polymerase Chain Reaction
 The double-stranded
DNA to be copied is
heated to make the
strands separate.
 DNA polymerase adds
new complementary
strands to each old
strand.
 The process is then
repeated over and over.
 DNA Restriction - a MAD
GOOD ANN'Y

51. DNA Fingerprinting
A practical use of DNA technology

52. DNA PROFILING
 A process of identifying the sequence of
nucleotide bases in a DNA segment.
 Used in Forensic Science:
– Paternity
– Family Relationship Research
– CSI –Murder/Rape – blood, hair,semen
 Techniques are reliable! Be careful of
contamination.

53. Profiling Process –Creating the
Fingerprint
 Involves
– Extraction – DNA
 Satellite DNA – short sequences of DNA
 Varies between individuals – no 2 the same
– PCR
– Cut with Endonucleases
– Gel Electrophoreses – to separate
– Compare Banding Patterns

55. Gel Electrophoresis
 Means of separating, by size, the DNA
fragments produced by restriction enzyme
cuts.
 Compares genes of different individuals or
organisms.
 Identifies the sequence of DNA fragments
by labeling some nucleotides.
 DNA Restriction - a MAD GOOD ANN'Y

56. Gel Electrophoresis
1.DNA fragments placed into wells in gel slab
2.Electric voltage is applied to gel.
3.DNA (negatively charged) migrates to (+) end of gel.
4.Smaller the fragment, faster and farther it moves.

57. DNA Fingerprinting
 Remember DNA is negatively
charged: PO4
-
 Gels Separate DNA by:
–SIZE
–CHARGE

58. DNA BANDING PATTERNS

59. PATERNITY TESTING

60. Human Genome

61. Human Genome Project
 Sequence the complete human genome.
Three Outcomes:
 Identifies the base sequence of all
genotypes
 Treatment and prevention of genetic
disease
 Early screening of zygotes and embryos
 Human Genome Map – it’s ‘anny time!

62. Genome – The Future is Now!

63. Genome Size in Different Species
SPECIES GENOME SIZE
(MILLIONS OF BASE
PAIRS)
NUMBER OF GENES
(APPX.)
T2 BACTERIOPHAGE 0.17 300
E. COLI 4.6 4,377
DROSOPHILA
MELANOGASTER
(FRUIT FLY)
130 17,000
HOMO SAPIENS
(HUMAN)
3,200 19,000 – 20,000
PARIS JAPONICA
(JAPANESE CANOPY
PLANT)
150,000 UNKNOWN

64. Gene Therapy

65. Gene Therapy
 Replaces a defective gene with a “normal” one
for that condition.
 Cystic Fibrosis – uses a nasal spray containing
normal gene in cold virus
 Severe Combined Immunodeficiency Disease
(SCID) – compromises T and B cell production in
newborns
 Replaces a gene to trigger the production of enzyme ADA
(adenosine deaminase)
 Uses virus to deliver gene

66. Gene Therapy

67. Genetic Screening
 Used to identify genotypes for possible
genetic diseases;
Includes:
 Karyotyping – amniocentesis. CVS.
 Newborns for potential diseases - PKU
 Pedigree Chart – traits in family history

68. Genetic Modification
OVERALL CONCERNS
 Limit variations
 Introductions of new organisms into an
ecosystem.
 Genes on some plasmids can promote
resistance to antibiotics.
 Genetically altered bacteria being released

69. END

70. Amniocentesis, CVS, and InVitro
Fertilization
 Amniocentesis –
 Used to determine occurrence of Down’s Syndrome.
 Cells taken from fluid in the Amniotic Sac – after 3 mos.
 Produce a KARYOTYPE
 Chorionic Villi Sampling
 Blood taken from chorion – after 4 weeks
 Produces KARYOTYPE – screens for genetic diseases
 IVF – Invitro Fertilization
 Zygotes or embryo can be screened. Ones with abnormalities
removed.

71. Amniocentesis Chorionic Villi Sampling

72. Oswald T. Avery -1944
Bacterial Transformation
Work centered around trying to find a
method to determine the molecule
responsible for bacterial transformation.
• Repeated the mouse studies but first
separated the components of the dead
virulent bacteria before adding it to
the mice containing avirulent bacteria
 only the mice injected with DNA
died
Therefore – the DNA contains the
information necessary to turn harmless
bacteria into killer bacteria

73. Hershey-Chase Experiments