Genetic engineering and biotechnology

1. Genetic engineering and biotechnology Daniel Njuguna - DAIS 2012

2. Assessment statements 4.4.1 Outline the use of polymerase chain reaction (PCR) to copy and amplify minute quantities of DNA. 4.4.2 State that, in gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size. 4.4.3 State that gel electrophoresis of DNA is used in DNA profiling. 4.4.4 Describe the application of DNA profiling to determine paternity and also in forensic investigations. 4.4.5 Analyse DNA profiles to draw conclusions about paternity or forensic investigations. 4.4.6 Outline three outcomes of the sequencing of the complete human genome. 4.4.7 State that, when genes are transferred between species, the amino acid sequence of polypeptides translated from them is unchanged because the genetic code is universal. 4.4.8 Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast or other cell), restriction enzymes (endonucleases) and DNA ligase. 4.4.9 State two examples of the current uses of genetically modified crops or animals. 4.4.10 Discuss the potential benefits and possible harmful effects of one example of genetic modification. 4.4.11 Define clone. 4.4.12 Outline a technique for cloning using differentiated animal cells. 4.4.13 Discuss the ethical issues of therapeutic cloning in humans.

3. Polymerase chain reaction (PCR) • Laboratory technique which takes a very small quantity of DNA and copies all the nucleic acids in it to make millions of copies of the DNA • Way to ensure that enough DNA for analysis can be generated

4. Gel electrophoresis • Laboratory technique used to separate fragments of DNA in an effort to identify its origin • Enzymes used to chop up DNA strands into fragments • Fragments are placed into small wells in the gel • Gel is exposed to an electric current • Heaviest, largest and least charged particles do not move easily through the gel • Smallest, least massive and most charged particles pass through the gel to the other side easily • Intermediate particles are distributed in between • In the end, the fragments leave a banded pattern of DNA to be used in DNA profiling

6. DNA profiling • Process of matching an unknown sample of DNA with a known sample to see if they correspond • Also referred to as DNA fingerprinting • If, after separation by gel electrophoresis, the pattern of bands formed by two samples of DNA fragments are identical, it means that both came from the same individual • If the patterns are similar, it means that the two individuals are most probably related

7. Applications of DNA profiling • Parentage testing • e.g. resolving paternity disputes • Forensic investigations • e.g. match suspects samples & those found at the scene of crime • Ecosystems forensic • e.g. birds migrating patterns & nesting habits • Evolution credibility • e.g. matching fossil samples with those of organisms living today

8. Paternity Investigation • Determine who is the biological father of the child, male 1 or male2 • The DNA fragments in the child comes from the mother and father • A band present in the child must come either from the mother or from the father • Compare male 1 with the child & the mother then male 2 with the child & the mother. • The bands on the child's fragments are either found on the mother or the male1. • Therefore Male 1 is this father of this child.

9. Forensic Investigation – Case 1 • A specimen of DNA is taken from the victim or the crime scene • DNA samples are taken from the 3 suspects. • The bands are compared to associate the suspects but to eliminate the victims DNA from the specimens • Of the 3 suspects, who is likely to have been at the scene of crime? • the bands on the specimen are matched by the bands on the Suspect 1, therefore Suspect 1 was present at the crime scene • Is this evidence sufficient to convict the suspect? • The law will still require to prove a crime was committed and then that Suspect 1 really committed the crime

10. Forensic Investigation – Case 2 • Sexual assault has been committed and two suspects are under investigation • DNA profile was carried out and the result are shown in the opposite diagram • From the DNA profile, who of the two suspects committed the assault? • The two bands visible on the evidence (sperm DNA) matches the DNA sample from suspect 1

11. TOK Aspects • How would you feel if you were to find out from DNA profiling that your father with whom you’ve lived with for 18 years was not your biological father? • What effect would such a result have on the relationships between siblings or between spouses? • What kind of emotions might someone feel after spending 18 years in prison, and then being freed thanks to a DNA test?

12. The Human Genome Project • Commitment by world’s scientific community to determine the location & structure of all genes in human chromosomes • Started in 1990 & by 2003, 99.9 % of the work of sequencing human DNA was completed • It involved sequencing 3 x 109 base pairs in human DNA then mapping the genes i.e. listing & finding the locus of each human gene

13. Outcomes of the sequencing of the complete human genome • improved understanding of genetic diseases because more disease causing genes are now known • production of medicine based on DNA sequences to cure diseases & or remove disease causing genes through genetic engineering • to determine fully which genetic diseases any individual is prone to & genetic screening can lead to preventive medicine • it provides more information about evolutionary paths by comparing similarities & differences in genes between species • research into a particular disease now can focus on only the gene(s) that are relevant to the disease

14. TOK Aspects • The human genome project maps the DNA sequence of human and could be seen as a recipe of what it is to be human. • This is a “reductionist view” which does not account for complexity of life stemming from interactions between our genes & the environment • What does the sentence, “We are all the same; we are all different,” mean? • Can one genetic group be considered genetically superior to another?

15. Gene transfer • Technique of taking a gene out of one organism (donor) and placing it in another organism (host) • When genes are transferred between species, the amino acid sequence of polypeptides translated from them is unchanged because the genetic code is universal. • e.g. cold resistant gene from arctic fish is transferred to tomatoes to make them more resistant to cold and frost • Proteins used by fish to resist icy temperature of arctic waters are now produced by the modified tomato

16. General technique for gene transfer

18. Basic technique used for gene transfer • Preparing a vector for the transferred gene • Plasmids (small circular DNA molecules) from bacteria are used as vector to carry the desired gene into a host cell. • Plasmids are removed from a bacterium & cut using restriction enzyme (endonuclease) leaving complementary 'sticky ends' of unattached hydrogen bonds in the plasmid • Obtaining the gene for transfer • DNA containing gene of interest is isolated from the donor organism • Restriction enzymes are used to cut out the useful gene that is to be transferred leaving the 'sticky ends' of unattached hydrogen bonds • Preparing a recombinant DNA • recombinant plasmid is used as a vector to carry the desired gene into a host cell • Gene of interest is placed inside the open plasmid using DNA ligase (i.e. gene splicing) to produce a recombinant plasmid • Recombinant plasmid is re-inserted into the bacterium • Isolation of transformed cells • Bacterium allowed to grow and proliferate, it expresses the desired gene by synthesizing whatever protein the gene codes for • the bacterium is used to insert the DNA carrying the foreign desired gene into the chromosome of a host cell • Host cells are grown in a culture medium • An organism is generated from a cell clone & all of its cells carry the desired gene & may express it as a new trait

19. Genetically modified organisms (GMOs) • Genetically modified (GM) crops & animals are called genetically modified organisms (GMOs) or transgenic organisms. • Their genetic material has been changed to include specific genes, usually from another species • Examples of transgenic organisms in use today includes: tomatoes, corn, rice, sheep, cow & mice

20. Uses of transgenic organisms (genetically modified crops & animals). Transgenic organism Current use of transgenic organism Produce toxins to particular insects hence reducing use of insecticides & increasing yield Pest Resistant Corn Transgenic tomato plants carry the gene for salt tolerance, frost tolerance or to ripen without becoming soft hence keep for long Tomatoes transgenic rice contains the gene for the manufacture of beta-carotene used by the body to make retinol essential for normal vision Golden Rice Transgenic sheep produce human clotting factor (factor IX) in milk which is isolated & used by haemophiliacs Sheep Cow genetically modified cows contain human genes for making medically important proteins e.g. insulin & growth hormone)

21. Potential benefits of genetic modification • GMO results in increased yield thus providing more food in regions where there is food shortage • Yields of crops with specific dietary requirement such as vitamins and minerals • Crops that do not spoil so easily during storage, thus economic benefits • GM animals produce proteins required for medical purposes & higher meat yields • less pesticides & fertilizers needed so reduces environmental impact • expands world’s productive farmland & reduces the need to clear rainforests to grow crops

22. Possible harmful effects of genetic modification • Modified genes may be released into natural environment where they may affect food chains resulting in reduction in biodiversity • GM food may affect consumers causing health risks such as allergies • Producing GM foods is unfair to smaller farmers who cannot compete with large scale farmers • long-term effects of GM are unknown • risk of cross-pollination producing “supper plants” which out-compete naturally occurring varieties • There are risk s of long-term contamination of soil

23. Potential benefit and possible harm of one named example of gene transfer between species • Gene transfer details • Btgene transferred from bacterium (Bacillus thuringiensus) to maize • specific benefit • corn borer (insect pest) killed by Bt toxin produced by maize plant thus increasing crop production • less pesticides (chemicals) are needed so better for environmentdue to less negative impacts • specific harmful effect • non-target insects may be killed by Bt toxins as well • risk of cross-pollination may introduce Bt gene to unintended species thus producing super weeds

24. Clones and cloning • A clone is a group of genetically identical organisms or a group of cells derived from a single parent cell. • Cloning is producing identical copies of genes, cells or organisms • Producing copies of an organism is called reproductive cloning while producing cells, tissues or organs for treating patients is called therapeutic cloning.

25. Technique for cloning using differentiated animal cells

26. Technique for cloning using differentiated animal cells • Somatic cell from donor sheep udder was collected and cultured • Unfertilized egg cell collected from another sheep & nucleus removed • Using a zap of electrical current, the egg cell and the nucleus from the cultured somatic cell were fused together • New cell developed in vitro and started to form an embryo • Embryo placed in the womb of a surrogate mother sheep • Embryo developed normally • Dolly was born and presented as a clone of the original donor sheep This is Known as reproductive cloning

27. Therapeutic cloning in humans • Therapeutic cloning involves creation of an embryo to supply embryonic stem cells for medical use & stem cell research • The technique used is same as the one used for Dolly • Human embryos are produced & allowed to grow into a blastocyst (hollow ball of cells) which is broken open & the stem cells are cultured • Therapeutic cloning aims at cell therapy where diseased cells are replaced with healthy ones • Cell therapy is used for patients suffering from; leukemia, Parkinson’s disease, skin burn & growing new corneas for people with visual impairment.

28. Ethical issues of therapeutic cloning in humans • Is it ethically acceptable to generate a new human embryo for the sole purpose of medical research? • Religious groups belief that every embryo contains a ‘soul’. Should we destroy a ‘soul’ to safe an individual’s life whose destiny is death? • Many scientists doubt the existence of a ‘soul’ since a ‘soul’ can not be observed or measured and hence there is no data - based evidence of its existence. Is it ethical to deny a cancer or diabetic patient treatment on the basis of saving a ‘soul’ who’s existence is uncertain?

29. Ethical issues of therapeutic cloning in humans Arguments for: Arguments against: • Cell therapy is used to treat patients with; leukemia, Parkinson’s disease, sever skin burns etc. thus saving lives & reducing pain; • Embryos left over from IVF treatment or those that have stopped developing could be used for treatment instead of being destroyed; • Stem cells are taken before the embryos develop nerve cells so they can’t feel pain; • Risk of tissue rejection during tissue transplant reduced. • There are fears it may lead to reproductive cloning & if embryos are not destroyed they will develop into a clone of the adult; • Use of embryonic stem cells involves creation & destruction of human embryos; • Embryonic stem cells are capable of repeated cell division & may turn into tumours; • Every human embryo is a potential human being & should be given a chance of developing.

31. Revision Questions • (a) Outline the use of polymerase chain reaction (PCR) to copy and amplify minute quantities of DNA. [2] • (b) Outline some of the outcomes of the sequencing of the human genome. [3] • (c) Outline a basic technique for gene transfer. [6] • (d) Explain the methods and aims of DNA profiling. [8] • (e) Using a named example, discuss the benefits and harmful effects of genetic modification. [9] • (f) Genetic modification involves the transfer of DNA from one species to another. Discuss the potential benefits and possible harmful effects of one example of genetic modification in a named organism. [9]