1. Topic 3: Genetics
Topic 3.5
Genetic Modification and Biotechnology

2. Biotechnology
Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products

3. Ancient biotech: Selective breeding
Mating organisms to produce offspring with desired traits (has been around since ancient times)
Inbreeding= mating between closely related individuals
Hybrids– offspring of parents with different forms of traits. In plants, hybrids are often stronger or bigger.

4. Modern Techniques in Biotech…

5. Genetic Engineering/ Genetic Modification
Genetic Modification: The transfer of genes between different species (i.e. cutting out DNA from one organism and putting it into another.)
makes recombinant DNA
Recombinant DNA: DNA in which genes from 2 different organisms are linked
Transgenic organism= an organism with recombinant DNA
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6. Genetic Engineering Tools
Restriction Enzymes
DNA ligase
Vectors
Plasmids!
Viruses
Reverse Transcriptase

7. Restriction Endonucleases
aka: Restriction enzymes :
They cut up the DNA (restriction) at very specific sequences
in nature, these enzymes protect bacteria from intruding DNA
Restriction site: where the restriction enzyme cuts the DNA (a specific sequence)
Sticky end: short single-stranded extensions of restriction fragments (segments of DNA cut by restriction enzymes in a reproducible way)

8. Restriction Enzymes
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9. Vector: Something that carries foreign DNA into a cell.
DNA ligase: enzyme that joins the DNA fragments covalently (called gene splicing)
Vector: Something that carries foreign DNA into a cell.
Bacterial plasmids… small circles of DNA
viruses

10. Restriction Enzymes and Ligase

11. Bacterial plasmids in gene cloning

12. Gene Cloning Clone = genetically identical copy
Gene cloning: Making more copies of a gene
Bacterial plasmid with recombinant DNA reproduces as the bacteria reproduce.
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13. Summary of Steps for gene cloning
Isolation of cloning vector (bacterial plasmid) & DNA (gene of interest)
Insertion of gene of interest into the cloning vector (how?)
Introduction of cloning vector into cells (transformation of bacterial cells)
Cloning of cells (and foreign genes)
Identification of cell clones carrying the gene of interest
(nucleic acid hybridization– use radioactively labeled nucleic acid probe) or
look for protein product

14. Application: Universality of the Genetic Code
One key example: Production of human insulin in bacteria

15. Examples of GMO’s
Beta-carotene rice (a.k.a. Golden rice, vitamin A rice)-- daffodil and soil bacterium genes
Bt corn and other crops— Bacillus thuringiensis gene.
Terminator technologies– cause sterility of seeds.

16. Application and Skills:
Application: Assessment of the potential risks and benefits associated with genetic modification of crops.
Read pages at home tonight and take summary notes on the most salient points.
Skill: Analysis of data on risks to monarch butterflies of Bt crops. (see p and answer Data-based questions: Transgenic pollen and monarch larvae in your assignments notebook)
Watch “Future of Food” here.

17. Cloning
Clones= genetically identical organisms, derived from a single parent cell.
Many plant species and some animal species have natural methods of cloning.

18. Skill: Investigating Cloning in Plants
Design of an experiment to assess one factor affecting the rooting of stem-cuttings. (see page 197 for ideas)
Note: possible inspiration for IA.

19. Animal Cloning Methods
1. Breaking embryo up in to more than one group of cells (how identical twins form)
2. Cloning adult animals with differentiated cells
ex. Dolly the sheep
Application: Somatic cell nuclear transfer
Remove Nucleus from an egg
inject somatic cell nucleus into egg OR…
Fuse this unfertilized egg with a body cell
Somatic cell nuclear transfer

20. Ethical issues (discussion)
Reproductive cloning
Therapeutic cloning– creation of an embryo to provide stem cells

22. DNA Analysis & Genomics
PCR (polymerase chain reaction)
Gel electrophoresis
Restriction fragment analysis (RFLPs)
DNA sequencing
Human genome project

23. Polymerase chain reaction (PCR)
Replication of DNA without cells (in vitro)
Used to amplify small amount of DNA
Materials in tube: primers (single-stranded DNA), Taq polymerase, nucleotides
Heat and cool in thermal cycler
Applications: fossils, forensics, prenatal diagnosis, etc.

24. DNA Analysis
Gel electrophoresis: separates nucleic acid fragments on the basis of size creating bands of DNA consisting of pieces of the same length.
Used in DNA Profiling
Paternity tests
Forensics
Ethical issues?
Good DNA ANALYSIS COMPUTER LAB ACTIVITY=
See Activities for in class simulation to be used for forensics and paternity tests

25. How does gel electrophoresis work?
DNA is cut with restriction enzymes and added to a well.
The negative DNA is attracted to the positive electrode.
smaller pieces of DNA move faster through the pores in the gel
A unique banding pattern is observed.
The human genome is estimated to be about 3 billion base pairs long and to contain 20,000-25,000 distinct genes

26. Gel Electrophoresis
animationhttp://
good gel electrophoresis virtual lab

27. DNA Analysis (continued)
Restriction fragment length polymorphisms (RFLPs)= differences between different individuals in lengths of restriction fragments
Found especially in satellite DNA (highly repetitive non-coding sequences of DNA)
RFLP animation

28. Skill: Analysis of examples of DNA profiles.
Based on what you just learned, which lane contains the largest fragment of DNA? The shortest?
What is the fancy 4 word term used to describe the fact that people have different size restriction fragments?
LANES + -
Injection wells
DNA

29. Skill: Analysis of examples of DNA profiles.
Use the DNA gel, and your knowledge of DNA fingerprint to analyze and determine which suspect’s DNA matches the DNA at the crime scene.
DNA at crime scene
Suspect
1 2 3 - +

30. Skill: Analysis of examples of DNA profiles. (Paternity Problem)
Look at the electrophoresis gel picture on the right 
Two of the individuals are parents and one is their child.
Answer the following:
Which individual is the child?
How many bands of DNA does the child share with the mother? The father? Explain why.
Based off of the gel, what color are the mother’s eyes?
Just kidding…

31. O.J. Simpson capital murder case,1/95-9/95
Odds of blood in Ford Bronco not being R. Goldman’s:
6.5 billion to 1
Odds of blood on socks in bedroom not being N. Brown-Simpson’s:
8.5 billion to 1
Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J. Simpson:
21.5 billion to 1
Number of people on planet earth:
6.1 billion
Odds of being struck by lightning in the U.S.:
2.8 million to 1
Odds of winning the Illinois Big Game lottery:
76 million to 1
Odds of getting killed driving to the gas station to buy a lottery ticket
4.5 million to 1
Odds of seeing 3 albino deer at the same time:
85 million to 1
Odds of having quintuplets:
Odds of being struck by a meteorite:
10 trillion to 1

32. Theory of knowledge: Discuss
• The use of DNA for securing convictions in legal cases is well established, yet even universally accepted theories are overturned in the light of new evidence in science. What criteria are necessary for assessing the reliability of evidence?

33. DNA Sequencing Determination of nucleotide sequences
Genomics: the study of genomes
Genome: the whole of the genetic information of an organism.
The Human Genome Project sequenced the entire base sequence of human DNA.
Human Genome Project Animations:

34. Application: Use of nucleotides containing dideoxyribose to stop DNA replication in preparation of samples for base sequencing.
Dideoxyribonucleotides with different nitrogen bases are tagged with different colored fluorescent markers.
Since the 3’ OH is missing, condensation can’t continue
So reaction stops.
Electrophoresis can be used to separate by size and determine sequence.

35. Human Genome Project Outcomes:
Identification and sequencing of genes involved in traits and disease.
Genetic testing improved.
Greater understanding of genetics.
Ethical issues with genetic testing etc.
Public Genomes or Personal genetic Testing

36. International-mindedness
Sequencing of the human genome shows that all humans share the vast majority of their base sequences but also that there are many single nucleotide polymorphisms that contribute to human diversity.

37. Application: Comparison of the number of genes in humans with other species. (adapted from
Species
(#genes)
Reference
human
~21,000
ref
E. coli K-12
4,377
rice
46,022 to 55,615
dog
~25,000
fruit fly
13,600
mouse
~23,000
Guidance: The number of genes in a species should not be referred to as genome size as this term is used for the total amount of DNA. At least one plant and one bacterium should be included in the comparison and at least one species with more genes and one with fewer genes than a human.

38. International-mindedness
Sequencing of the rice genome involved cooperation between biologists in 10 countries.

39. Skill: Assignment as a Class
Use of a database to determine differences in the base sequence of a gene in two species.
Here is the link to the GenBank site:
Choose “Gene” from the search menu.
Enter the name of a gene plus an organism and click “Search.”
Ex. “cytochrome oxidase 1 for homo sapiens”
Click on the name of the gene that has the correct description.
Once loaded, scroll down to where it says “Genomic regions, transcripts, and products” and click on “FASTA”
Copy the entire sequence beginning with where it says “>gi”
Paste this into a text file (Notepad or Wordpad)
Repeat with all species that you want to compare and save the files
Download ClustalX and run it. ( )
File  “Load Sequences” and choose your first sequence
File  “Append Sequences” and choose your next sequence…
Under alignment menu choose “Do Complete Alignment”

40. Assignment at home
Use a database to determine differences in the base sequence of a gene in two species.
Submit your response to the Google Form Below:

41. Genome Size (million base pairs)
Application: Comparison of genome size in T2 phage, Escherichia coli, Drosophila melanogaster, Homo sapiens and Paris japonica.
Organism
Genome Size (million base pairs)
Description
T2 phage
0.18
Virus that attacks E. coli
E. coli 5
Gut bacterium
Drosophila melanogaster
140
Fruit Fly
Homo sapiens
3000
Humans
Paris japonica
150,000
Woodland Plant

42. Extra slides… cut from presentation

43. Practical DNA Technology Uses
Diagnosis of disease
Human gene therapy
Pharmaceutical products (vaccines)
Forensics
Animal husbandry (transgenic organisms)
Genetic engineering in plants
Ethical concerns?

44. Skip: Option F:Reverse Transcriptase
Catalyses the production of DNA from RNA
Why is this helpful in making recombinant DNA?
Hint: Think about introns and exons…
Ex. Bacteria that make human insulin…

45. Skip: Option F:RNA viruses
Where does Reverse Transcriptase come from?
Answer:
Retroviruses: transcribe DNA from an RNA template (RNA--->DNA)
DNA gets incorporated as provirus in host
Ex. HIV

46. Skip (option f) Gene Therapy
Germ line Therapy: reproductive cells– affects offspring
Somatic Therapy: body cells of an individual
Ex. White blood cells of SCID (severe combined immunodeficiency)
1. Blood cells are removed
2. Engineer viral DNA
3. Use of viral vector
4. Blood cells replaced, having needed gene.
Risks?