4.4.1 Polymerase chain reaction
4.4.2 Gel electrophoresis
4.4.3 DNA profiling
4.4.4 DNA profiling and applications in paternity and forensic investigations.
4.4.5 Interpretation of paternity and forensic investigations.
4.4.6 Human genome project.
4.4.7 Gene transfer
4.4.8 Gene transfer techniques.
4.4.9 Genetically modified crops and animals
4.4.10 Hazards and benefits of genetic modification.
4.4.11 Definition of clone.
Outline means to give a brief account or summary
PCR is the cloning of DNA (amplification).
Copies are made and the amount of DNA can be rapidly increased. Useful if the source of DNA is small.
Temperature is used instead of enzymes like helicases (95oC ).
DNA polymerase is thermostable to protect it against the reaction temperatures.
This is an automated process and can produce sufficient DNA in 20 cycles.
State means to give a specific name, value or other brief answer without explanation or calculation.
Sample of fragmented DNA is placed in one of the wells on the gel.
An electrical current is passed across the gel.
Fragment separation is based on charge and size.
Large fragments move slowly.
Negative fragments are moved to the right.
Gel after staining:
This diagram shows the separation of 6 separate mixtures of DNA.
The dark bands to the left are those with a large molecular mass or a positive charge
(a) contains 5 fragments of DNA. Each bands corresponds to a group of DNA molecules of the same size and charge.
(b) and (c) have the same bands. They are identical
State means to give a specific name, value or other brief answer without explanation or calculation.
Gel electrophoresis is used in DNA profiling.
Satellite (Tandem repeating) DNA are highly repetitive sequences of DNA from the non coding region of DNA.
Different individuals have a unique length to their satellite regions.
These can be used to differentiate between one individual and another.
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:
Forensic crime investigations
Parentage Issues
Animal breeding pedigrees
Disease detection
Describe means to give a detailed account.
Paternity Investigation:
Trying to determine who are the biological parents of a child.
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
Comparing male 1 with the child then male 2 with the child.
Interpretation:
The bands on the child's fragments are either found on the mother or the male1.
Male 1 therefore is this father of this child.
None of the Male 2 bands appear in the child
Forensic Investigation:
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
Interpretation:
Note that the bands on the specimen are matched by the bands on the Suspect 1.
This means that Suspect 1 was present at the crime scene.
The law will still require to prove a crime was committed and then that Suspect 1 committed the crime
Analyse means to interpret data to reach conclusions.
In the above example the description has been extended to an interpretation.
Outline means to give a brief account or summmary.
Begun formally in 1990 the international projects aims where:
identify all the approximate 30,000 genes in human DNA.
determine the sequences of the 3 billion chemical base pairs that make up human DNA.
store this information in database.
improve tools for data analysis.
transfer related technologies to the private sector.
address the ethical, legal, and social issues (ELSI) that may arise from the project.
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:
State means to give a specific name, value or other brief answer without explanation or calculation.
The genetic code is universal
All known organisms use the same genetic code.
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.
Outline means to give a brief account of summary.
Stage 1: obtaining the gene for transfer:
Restriction enzymes are used to cut out the useful gene that is to be transferred.
Note the 'sticky ends' of unattached hydrogen bonds.
Stage 2. Preparing a vector for the transferred gene:
Plasmids are small circular DNA molecules found in bacteria.
These can be cut with the same restriction enzyme as above.
This leaves the same complementary 'sticky ends' in the plasmid
The plasmid can be cut at particular sites. These are called restriction sites and some are named in the diagram.
Stage3. Recombinant DNA
(a) plasmid that will be the vector
(b) plasmid cut at restriction site Pstl
(c) Source DNA cut with same restriction enzyme as plasmid to (d)
(e) 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
Recombinant DNA is introduced into the host cells
Many cells remain untransformed
Some cells are transformed to contain the recombinant DNA.
These transformed cells must be separated from untransformed
Stage 5. Product manufacture.
The transformed bacterial cells are isolated.
They are introduced into a Fermenter to be cloned.
The bacterial population grows by asexual reproduction.
The Recombinant DNA is copied along with the rest of the bacterial genome.
In a fermenter the conditions for growth and reproduction are controlled.
Once the bacteria express the transformed gene the product is produced.
The next (long ) step is to isolate and purify the product. This is called downstream processing.
State means to give a specific name, value or other brief answer without explanation or calculation.
Genetically modified organism (GMO) is an organism containing a transplanted gene.
The organism will express the gene and synthesis the protein.
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.
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.
Rice does not contain retinol or beta-carotene (used by the body to make retinol).
Rice does contain a molecule that is normally used to make beta-carotene.
The gene and enzymes to manufacture are missing from rice.
Genetically modified rice contains the gene for the manufacture of beta-carotene.
Source of the gene is either Erwinia bacterium or the common daffodil.
The transgenic rice is usually yellow in colour because of the accumulation of beta-carotene.
This transgenic rice is then crossed with local strains of rice.
Herbicides: Roundup
Weeds growing amongst a crop use up soil nutrients that would otherwise be used by the crop plant.
This competition of resources reduces the productivity of the crop plant and therefore the efficiency of farming.
Herbicides can be used prior to crop planting to kills weeds.
The herbicide cannot be used after crops have been sown as they will also kill the crop.
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.
However, Cotton, Corn and Soybeans have been genetically modified to contain an enzyme that breaks down glyphosate.
This makes these crops resistant to the herbicide.
Herbicide can then be use after the crop has grown to prevent the reoccurrence of weed competition.
Discuss means to give an account including, where possible, a range of arguments for and against the relative importance of various factors, or comparisons of alternative hypotheses.
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:
Increased yields particularly in regions of food shortage.
Yields of crops with specific dietary requirement such as vitamins and minerals.
Crops that do not spoil so easily during storage.
GM animals produce similar effect including higher meat yields.
The disadvantages or concerns about GMO usually can be found:
The foods (animal and plant) are considered un-natural and unsafe for human consumption.
There is a risk of the escape of 'genes' into the environment where they may be passed to other organisms with unknown effects.
define means to give the precise meaning of a word, phrase or physical quantity.
Syllabus statement: ' Clone: a group of genetically identical organisms or a group of cells derived from a single parent'.
Outline means to give a brief account or sumarry.
Somatic cells are differentiated that is specialised to a particular function. In the case of Dolly (1996) the cell was taken from the udder of the original sheep (1) that was to be cloned.
Sheep (2) provides the egg cell after being stimulated to superovulated by the use of the hormone FSH.
The nucleus is removed from the egg cell. This removes the genetic information of sheep (2).
The cells are fused combining the nucleus of sheep (1) with the egg cell from sheep(2). The egg cell retains its ability to replicate chromosomes and divide by mitosis.
The cell is grown 'in-vitro' until it reaches the16 cell stage this will then be implanted into a surrogate mother sheep.
Sheep (3) is the surrogate mother sheep and is not related to any of the other sheep. There is a normal gestation period before the 'Clone lamb' is born.
Lamb (4) is dolly the clone of sheep (1). They are genetically identical. However they will experience a different set of environmental conditions.It should be noted that this technique was tried many times before it was successful.
Discuss means to give an account including, where possible, a range of arguments for and against the
relative importance of various factors, or comparisons of alternative hypotheses.
The discussion is about the creation of an embryo to supply stem cells for medical use.
Research what is meant by therapeutic cloning.
Decide what the ethical issues are in therapeutic cloning.
What is an embryo?
Where would they be obtained from? Alternatives?
Try to make yourself aware of the stance of interest groups on the issues.
Some useful links:
Primer on Ethics and Human Cloning