4.4.1 Polymerase chain reaction
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4.4.2 Gel electrophoresis
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Sample of fragmented DNA is placed in one of the wells on the gel.
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An electrical current is passed across the gel.
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Fragment separation is based on charge and size.
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Large fragments move slowly.
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Negative fragments are moved to the right.
Gel after staining:
This diagram shows the separation of 6 separate mixtures of DNA.
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The dark bands to the left are those with a large molecular mass or a positive charge
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(a) contains 5 fragments of DNA. Each bands corresponds to a group of DNA molecules of the same size and charge.
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(b) and (c) have the same bands. They are identical
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4.4.3 DNA profiling
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Gel electrophoresis is used in DNA profiling.
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Satellite (Tandem repeating) DNA are highly repetitive sequences of DNA from the non coding region of DNA.
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Different individuals have a unique length to their satellite regions.
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These can be used to differentiate between one individual and another.
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There are different types of 'DNA fingerprinting' for different circumstance
(a) The mothers chromosome provides a DNA STR cutting the chromosome with particular restriction enzymes.
(b)The fathers chromosome provides the same fragment using the same restriction enzymes.
(c) The mother DNA fragment placed in the well of the gel.
(d) The mother DNA fragment placed in the well of the gel.
(e) Mothers fragment produces 5 STR and moves a short distance along the electrophoresis gel.
(f) fathers fragment produces 2 STR and moves a longer distance along the electrophoresis gel.
(g) The child is heterozygous for the fragment having on homologous chromosome form the father and one form the mother.
Both 5 STR and 2 STR are shown in the electrophoresis.
The technique can be used in:
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4.4.4 DNA profiling and applications in paternity and forensic investigations.
Paternity Investigation:
Interpretation:
Forensic Investigation:
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A specimen of DNA is taken from the victim or the crime scene.
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DNA samples are taken from the 3 suspects.
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The bands are compared to associate the suspects but to eliminate the victims DNA from the specimens
Interpretation:
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4.4.5 Interpretation of paternity and forensic investigations.
see above interpretations.
4.4.6 Human genome project.
Begun formally in 1990 the international projects aims where:
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To help achieve these goals, researchers also are studying the genetic makeup of several nonhuman organisms. These include the common human gut bacterium Escherichia coli, the fruit fly, and the laboratory mouse.
External Links:
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4.4.7 Gene transfer
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The genetic code is universal
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All known organisms use the same genetic code.
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Therefore in principle if we transfer a gene from one species to another it should still be transcribed and translated into the same protein.
As with all of biology its the 'exceptions that prove the rule' and so it is with the 'Universal genetic code. There are in fact some prokaryotes which have one or two different code specifications. However they do not adopt a radically different system. Once more this demonstrates the emergent properties of complex biological systems.
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4.4.8 Gene transfer techniques.
Stage 1: obtaining the gene for transfer:
Stage 2. Preparing a vector for the transferred gene:
Satge3. Recombinant DNA
Expression vectors: usually if a eukaryotic gene is inserted into the genome of a prokaryote it make very little of the desired gene product. Therefore additional factors are included in the vector plasmid 'package' which includes types of RNA. The final plasmid as outlined above containing these additional factors is called an' Expression vector'.
Stage4. Isolation of transformed cells
Stage 5. Product manufacture.
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4.4.9 Genetically modified crops and animals
Factor IX : A human clotting factor is produces by genetically modified sheep. The protein (factor IX) is expressed in milk from which it must be isolated before use by haemophiliacs.
A ewe is treated with fertility drugs to create super-ovulation.
Eggs are inseminated.
Each fertilised egg has the transgene injected.
A surrogate ewe has the egg implanted for gestation.
Lambs are born which are transgenic, GMO for this factor IX gene.
Each Lamb when mature can produce milk.
The factor IX protein is in the milk and so must be isolated and purified before use in human.
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Tomato salt tolerance:
Plants find it hard to grow in salty conditions since this hypertonic soil water results in desiccation, wilting and death of the plant.
Tomato plants have now been genetically modified to carry the gene for salt tolerance.
The origin of the gene was a weed called Arabidopsis thaliana.
The transgenic tomato plant can tolerate plants.
This now provides the opportunity for a crop to be grown in an otherwise sterile soil.
Links:
Rice with retinol:
Retinol deficiency:
Retinol (Vitamin A1) is essential for the development of an effective immune system, normal vision and growth. Where a child lacks Retinol they have stunted growth and in extreme cases blindness. A serious complication arises from the combined of malnutrition, disease and retinol deficiency. The coincidence of these three conditions together is seen in some third word nations. In Zambia more than 54% of the population of children have retinol deficient diets. Even if children have sufficient food (calories) the problem is that is not a balanced diet. These children experience retarded growth for their age group and vision problems are common. Vision problems usually begin with a loss of night vision and then ultimately complete blindness. As might be expected these communities find it very difficult to support children with these multiple problems. To make matters worse diseases such as malaria and measles (known killer diseases) disable or kill large numbers of children as they have ineffective immune systems, another consequence of retinol deficiency.
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Rice does not contain retinol or beta-carotene (used by the body to make retinol).
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Rice does contain a molecule that is normally used to make beta-carotene.
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The gene and enzymes to manufacture are missing from rice.
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Genetically modified rice contains the gene for the manufacture of beta-carotene.
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Source of the gene is either Erwinia bacterium or the common daffodil.
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The transgenic rice is usually yellow in colour because of the accumulation of beta-carotene.
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This transgenic rice is then crossed with local strains of rice.
Herbicides: Roundup
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Weeds growing amongst a crop use up soil nutrients that would otherwise be used by the crop plant.
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This competition of resources reduces the productivity of the crop plant and therefore the efficiency of farming.
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Herbicides can be used prior to crop planting to kills weeds.
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The herbicide cannot be used after crops have been sown as they will also kill the crop.
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The major herbicide in use is called 'Glyphosate', anyone who has gardened will know this as 'Roundup' which is the market name for the product.
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However, Cotton, Corn and Soybeans have been genetically modified to contain an enzyme that breaks down glyphosate.
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This makes these crops resistant to the herbicide.
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Herbicide can then be use after the crop has grown to prevent the reoccurrence of weed competition.
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4.4.10 Hazards and benefits of genetic modification.
The advantages and disadvantages of GMO is a controversial topic with wide political, environmental, health and social effects. The following issues can be applied specifically to the above examples of GMO.
The benefits of GMO include:
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Increased yields particularly in regions of food shortage.
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Yields of crops with specific dietary requirement such as vitamins and minerals.
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Crops that do not spoil so easily during storage.
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GM animals produce similar effect including higher meat yields.
The disadvantages or concerns about GMO usually can be found:
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The foods (animal and plant) are considered un-natural and unsafe for human consumption.
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There is a risk of the escape of 'genes' into the environment where they may be passed to other organisms with unknown effects.
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4.4.11 Definition of clone.
Syllabus statement: ' Clone: a group of genetically identical organisms or a group of cells derived from a single parent'.
4.4.12 Cloning in differentiated animal cells.
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4.4.13 Ethical issues of therapeutic cloning
The syllabus demands that the student discuss the 'Ethical issues of therapeutic cloning in humans'.
The discussion is about the creation of an embryo to supply stem cells for medical use.
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Research what is meant by therapeutic cloning.
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Decide what the ethical issues are in therapeutic cloning.
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What is an embryo?
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Where would they be obtained from? Alternatives?
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Try to make yourself aware of the stance of interest groups on the issues.
Some useful links:
Primer on Ethics and Human Cloning
New Scientist Journal
Webliography/ Cloning
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