1. GENETIC ENGINEERING and BIOTECHNOLOGY
Topic 4.4
IB Biology
Miss Werba

2. TOPIC 4 - GENETICS 4.1 CHROMOSOMES, ALLELES, GENES & MUTATIONS 4.4 4.2
MEIOSIS
4.3
THEORETICAL GENETICS
4.4
GENETIC ENGINEERING & BIOTECHNOLOGY
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3. THINGS TO COVER Polymerase Chain Reaction (PCR) Gel electrophoresis
DNA profiling
Paternity and forensic investigations
Human Genome Project
Gene transfer
Genetically modified (GM) crops & animals
Cloning
Ethics of cloning
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4. BEFORE WE BEGIN....
TOOLS OF THE TRADE

5. TOOLS OF THE TRADE: RESTRICTION ENZYMES
4.4.8
TOOLS OF THE TRADE: RESTRICTION ENZYMES
Enzymes which recognise a specific repeating base sequence of DNA
They cut at a specific point in this sequence
Create “sticky ends” with known DNA sequences that can be utilised for genetic engineering
eg. HindIII, EcoR1
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6. TOOLS OF THE TRADE: RESTRICTION ENZYMES
4.4.8
TOOLS OF THE TRADE: RESTRICTION ENZYMES
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7. TOOLS OF THE TRADE: REVERSE TRANSCRIPTASE
4.4.8
TOOLS OF THE TRADE: REVERSE TRANSCRIPTASE
It reverses transcription!
Enzymes which can cause a piece of RNA to produce a matching piece of DNA
The DNA can then be made double-stranded and used to transfer a genetic characteristic to another organism
DNA made is called copy DNA (cDNA)
MISS J WERBA - TERM 7

8. TOOLS OF THE TRADE: REVERSE TRANSCRIPTASE
4.4.8
TOOLS OF THE TRADE: REVERSE TRANSCRIPTASE
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9. TOOLS OF THE TRADE: DNA POLYMERASE
4.4.8
TOOLS OF THE TRADE: DNA POLYMERASE
Enzyme used for making multiple copies of DNA
Enzyme allows single-stranded cDNA to be made double-stranded
Also allows small DNA samples to be replicated so that they can be tested multiple times if necessary
eg. DNA from an embryo during aminocentesis, DNA from a crime scene
MISS J WERBA - TERM 9

10. TOOLS OF THE TRADE: DNA LIGASE
4.4.8
TOOLS OF THE TRADE: DNA LIGASE
Enzyme used for joining pieces of DNA which are being spliced together
ie. genetic “glue”
Allows an isolated or created piece of DNA to be joined to the DNA of a recipient organism.
MISS J WERBA - TERM 10

11. TOOLS OF THE TRADE: GENE PROBES
4.4.8
TOOLS OF THE TRADE: GENE PROBES
Single-stranded DNA used to identify genes with base sequences complementary to the probe.
Probes are made using reverse transcriptase enzymes.
Probes are radioactively labelled so that where the probe pairs with the gene, the probe and the gene can be identified.
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12. TOOLS OF THE TRADE: MARKER GENES
4.4.8
TOOLS OF THE TRADE: MARKER GENES
Genes that are close to the gene being studied that produce an obvious phenotype so it can be easily identified.
Presence of the marker indicates that the neighbouring gene has probably been inherited.
In genetic engineering, a gene is normally introduced into an organism together with its marker.
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13. 4.4.8
TOOLS OF THE TRADE
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14. GENETIC ENGINEERING and BIOTECHNOLOGY
AND NOW....
GENETIC ENGINEERING and BIOTECHNOLOGY

15. POLYMERASE CHAIN REACTION (PCR)
4.4.1
POLYMERASE CHAIN REACTION (PCR)
Polymerase Chain Reaction (PCR) is a way of producing large quantities of a specific target sequence of DNA
It is useful when only a small amount of DNA is available for testing
eg. crime scene samples of blood, semen, tissue, hair, etc.
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16. POLYMERASE CHAIN REACTION (PCR)
4.4.1
POLYMERASE CHAIN REACTION (PCR)
PCR involves a repeated procedure of 3 steps:
Denaturation: DNA is heated to separate it into 2 strands
Annealing: DNA primers attach to opposite ends of the target sequence
Elongation: DNA polymerase copies the strands
One cycle of PCR yields two identical copies of the DNA sequence
A standard reaction of 30 cycles would yield over a million copies of the DNA
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17. POLYMERASE CHAIN REACTION (PCR)
4.4.1
POLYMERASE CHAIN REACTION (PCR)
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18. PCR

19. GEL ELECTROPHORESIS Command term = STATE
4.4.2
GEL ELECTROPHORESIS Command term = STATE
In gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size.
Process:
DNA fragmented with restriction enzymes
Loaded into a gel and an electric current is applied
DNA moves towards the anode (+ end)
The fragments are separated according to their size
small fragments move faster, separate further
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20. GEL ELECTROPHORESIS Command term = STATE
4.4.2
GEL ELECTROPHORESIS Command term = STATE
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21. DNA PROFILING Command term = STATE
4.4.3
DNA PROFILING Command term = STATE
Gel electrophoresis is used in DNA profiling.
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22. DNA PROFILING Command term = STATE
4.4.3
DNA PROFILING Command term = STATE
DNA profiling is a technique by which individuals are identified on the basis of their DNA profiles.
Non-coding regions of an individual's genome contain satellite DNA - long stretches of DNA made up of repeating elements called short tandem repeats (STRs).
These repeating sequences can be excised to form fragments, by cutting with a variety of restriction enzymes.
As individuals all have a different number of repeats, they will all generate unique fragment profiles.
These different profiles can be compared using gel electrophoresis.
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23. 4.4.4
USES OF DNA PROFILING
The gel electrophoresis identifies an individual’s banding pattern
Restriction enzymes (endonucleases) target non-coding elements (STRs) that differ in length in individuals
It can be used to determine paternity and also be used in forensic investigations
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24. USES OF DNA PROFILING Is the man the child’s father?
4.4.5
USES OF DNA PROFILING
Is the man the child’s father?
Yes. The DNA sequence of the child can be mapped to show that similar bands exist in the father's sequence which are not present in the mother's sequence.
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25. THE HUMAN GENOME PROJECT
4.4.6
THE HUMAN GENOME PROJECT
The Human Genome Project was an international cooperative venture to sequence the complete human genome.
Humans have about 30,000-40,000 genes.
It was started in 1990 and was finished in 2003.
Y chromosome
X chromosome
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26. OUTCOMES OF THE HUMAN GENOME PROJECT
4.4.6
OUTCOMES OF THE HUMAN GENOME PROJECT
Mapping (locating genes)
Screening (for disease)
Treatment (gene therapy & targeted drug design)
eg. It will allow the production of new drugs based on DNA base sequences of genes or the structure of proteins coded for by these genes
Ancestry (for understanding human origins & evolution)
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27. GENETIC CODE IS UNIVERSAL Command term = STATE
4.4.7
GENETIC CODE IS UNIVERSAL Command term = STATE
Genes are able to be transferred between species because the genetic code is universal.
This means that the amino acid sequence of proteins translated from them is unchanged.
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28. GENE TRANSFER Plasmid removed from bacterial cell
4.4.8
GENE TRANSFER
Plasmid removed from bacterial cell
plasmids are self-replicating rings of DNA
Gene of interest removed from organism
Gene amplified with PCR
Gene of interest spliced into plasmid
Gene and plasmid cut with same restriction enzyme
This creates matching sticky ends like a puzzle
Spliced (glued) together with DNA ligase
Recombinant plasmid inserted into host cell
The transgenic cells should express the new trait
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29. 4.4.8
GENE TRANSFER
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30. 4.4.8
GENE TRANSFER
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31. RECOMBINATION

32. GENETICALLY MODIFIED CROPS and ANIMALS
4.4.9
GENETICALLY MODIFIED CROPS and ANIMALS
Crop plants and domestic animals have been modified over generations using selective breeding, to produce organisms with desirable characteristics.
Recombinant DNA technology can now be used to alter the genetic make-up of organisms much more quickly.
Some genetically modified organisms (GMOs) are already being produced. In 2006, 252 million acres of transgenic crops were grown globally.
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33. GENETICALLY MODIFIED CROPS and ANIMALS
4.4.9
GENETICALLY MODIFIED CROPS and ANIMALS
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34. GENETICALLY MODIFIED CROPS and ANIMALS Command term = STATE
4.4.9
GENETICALLY MODIFIED CROPS and ANIMALS Command term = STATE
You need to know two examples of the current uses of genetically modified crops or animals
Examples include:
Bt corn produces a toxin (from Bacillus thuringiensis) that kills corn borers
sheep that produce human blood clotting factor IX in their milk
synthesis of beta-carotene in Golden RiceTM (vitamin A precursor so ups nutritional value)
herbicide resistance in crops
salt tolerance in plants
delayed ripening in Flavr-Savr™ tomatoes
bacteria use to produce insulin and clotting factor VIII
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35. EFFECTS OF GENETIC MODIFICATION
4.4.10
EFFECTS OF GENETIC MODIFICATION
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36. EFFECTS OF GENETIC MODIFICATION
4.4.10
EFFECTS OF GENETIC MODIFICATION
Benefits:
Higher crop yield (lessen food shortages)
Higher yields = less land used for farming
Reduction in use of pesticides
Less expenditure, greater profits
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37. EFFECTS OF GENETIC MODIFICATION
4.4.10
EFFECTS OF GENETIC MODIFICATION
Disadvantages:
May have unknown health effects (allergy?)
Toxin may kill non-pest species
Cross-pollination to weeds
Reduced biodiversity
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38. CLONE Command term = DEFINE
4.4.11
CLONE Command term = DEFINE
Clone: a group of genetically identical organisms
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39. 4.4.12
REPRODUCTIVE CLONING
Scientists can use genetic technologies to clone whole organisms; this is known as reproductive cloning.
Farm animals or crop plants with desirable features can be cloned, ensuring a constant food supply and high productivity.
It is thought that reproductive cloning could also help to increase numbers of rare or endangered species.
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40. 4.4.10
REPRODUCTIVE CLONING
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41. REPRODUCTIVE CLONING Cells are removed from two donor organisms:
4.4.12
REPRODUCTIVE CLONING
Cells are removed from two donor organisms:
Species 1 – cell remains intact
Species 2 – nucleus is removed from cell
Cells are fused using electricity (“electro-fusion”)
Reconstructed cell is implanted into the womb of a Species 2 surrogate mother and forms an embryo
A clone of the Species 1 organism will be born.
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42. 4.4.12
REPRODUCTIVE CLONING
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43. 4.4.13
THERAPEUTIC CLONING
Scientists can use genetic technologies to produce organs, tissues and cells.
This is known as therapeutic cloning or non- reproductive cloning.
The process requires the use of stem cells and thus, is very controversial.
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44. ETHICS OF THERAPEUTIC CLONING
4.4.13
ETHICS OF THERAPEUTIC CLONING
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45. ETHICS OF THERAPEUTIC CLONING
4.4.13
ETHICS OF THERAPEUTIC CLONING
Cloning happens naturally, for example monozygotic twins.
Some may regard the in vitro production of two embryos from one to be acceptable.
Others would see this as leading to the selection of those "fit to be cloned" and visions of "eugenics and a super-race".
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46. ETHICS OF THERAPEUTIC CLONING
4.4.13
ETHICS OF THERAPEUTIC CLONING
Arguments for Therapeutic Cloning
May be used to cure serious diseases (replacing bad cells with good ones)
Stem cell research may pave the way for future discoveries
Stem cells can be taken from aborted/unviable embryos or from the umbilical cord or adult source
Cells are taken at a stage when the embryo has no nervous system and can arguably feel no pain
Arguments against Therapeutic Cloning
Involves the creation and destruction of human embryos
Stem cells are capable of continued division and may develop into tumours
More embryos are produced than are needed, so excess embryos are killed
Alternative technologies may fulfil similar roles in the future (eg. reprogramming of differentiated cell lines)
J WERBA – IB BIOLOGY 46

47. Sample questions Q1
State the name of the technique that is used to separate fragments of DNA according to their size, during DNA profiling. (1)
The diagram below represents the results of a paternity investigation. Track A is the profile of the mother of a child, track B is the profile of the child and track C is the profile of a man who might be the father.
Explain, using evidence from the diagram, whether this man is the father or not. (3)
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48. Sample questions A1 (gel) electrophoresis 1
(track C is) not (DNA from) the father; some bands on track B do not occur on A or C; these bands must be DNA inherited from the real father; band in track B that does not occur on A or C identified; Annotations to the gel may be used max
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49. Sample questions
Q2 Outline the method that can be used to amplify small quantities of DNA to obtain large enough quantities for DNA profiling. (2) Q3 Describe a technique used for gene transfer. (5) Q4 Using a named example, discuss the benefits and harmful effects of genetic modification. (9)
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50. Sample questions
A2 polymerase chain reaction/PCR; (DNA obtained from) blood/semen/hairs/other source of tissue; combined with necessary raw materials/one example of raw material; in thermal cycler / (PCR) machine; DNA replicated many times;
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51. Sample questions
A3 restriction enzymes/endonucleases cut a small fragment of DNA from an organism; same restriction enzymes used to cut DNA of plasmid / e.g. E. coli; sticky ends are the same in both cases; fragment of DNA is inserted into the plasmid; spliced together by ligase; to make recombinant DNA/plasmids; recombinants can be inserted into host cell and cloned;
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52. Sample questions A4
genetic modification is when the DNA/genotype of an organism is artificially changed; genetic modification alters some characteristic/phenotype of the organism; named example with modification (e.g. salt tolerance in tomato plants);
benefits: [5 max] allows crops to be grown where they would not grow naturally; provides more food; economic benefits; expands world’s productive farmland; reduces the need to clear rainforests to grow crops; lowers cost of production; less pesticides/fertilizers/chemicals needed so better for environment; harmful effects: [5 max] may be released into natural environment; may affect food chains / unintended effects on other organisms; may affect consumers e.g. allergies/health risks; unfair to smaller farmers who cannot compete; long-term effects are unknown; risk of cross-pollination; risk of long-term contamination of soil;
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