4.4.1 Outline the use of polymerase chain reaction (PCR) to copy and amplify minute quantities of DNA

Why might we want to amplify minute quantities of DNA?

Polymerase chain reaction is a laboratory technique that is used to copy DNA (basically used to amplify small quantities of DNA). The machine used to make copies of the DNA is called the thermocycler (a device used to amplify segments of DNA via the PCR)

Image of a thermocycler:

Image source: http://upload.wikimedia.org/wikipedia/commons/thumb/d/da/Pcr_machine.jpg/220px-Pcr_machine.jpg

This process is useful when only a small amount of DNA is available but a large quantity is needed so that tests can be done on it. 

A real life example of when only a small amount of DNA is available but a large quantity is needed is in forensics. Where perhaps only a small amount of blood or tissue is found at a crime scene and a lot of tests needs to be done on them (or some of the DNA needs to be given to the defense so that they can run their own tests for verification). 

It is in this situation where it would be useful to amplify small quantities of DNA. 

INTERESTING NOTE: PCR requires high temperatures and a DNA polymerase enzyme that we can get from Thermus aquaticus (which is a bacteria that lives in hot springs) 

Citation: http://www.ibguides.com/biology/notes/genetic-engineering-and-biotechnology

Answers to the past paper questions

c) Discuss the ethical arguments for and against the cloning of humans: [4]

Arguments FOR cloning:

- Clones may provide tissues or organs necessary for transplantation to save people's lives. Clones are genetically identical, therefore if a clone is made for someone who is sick and needs a transplant then that clone may be used to provide that organ (e.g a liver). Not only would the clone be beneficial as organ donors but because they are genetically identical to that person. The chance of rejection is significantly lower. (for example cloning could be used to lower the chance of rejection when: growing skin to repair a serious burn or  growing a new heart muscle to repair a damaged heart)
-Some people argue that cloning is 'playing god', however, it can be argued that identical twins are made by cloning and therefore in fact cloning is actually a natural process.
-clones can be beneficial because they can be used in research such a transplant research, if clones are used instead of animals the results and the tests will be a much more representative. Therefore using clones for research could lead to medical breakthroughs such as in cancer or regeneration research.

Arguments AGAINST cloning:

-cloned individuals are more likely to have health problems (such premature aging)
-Moreover, clones will be actual humans but the fact that they are identical to someone, perhaps may lead to physiological problems if they feel that they have loss value because they are not unique.
- cloning is an expensive process and some may argue that this is a misallocation of resources and this money would be better of being used on other things forms of health care where there is less of a risk for unknown consequences.

c) Outline a basic technique for gene transfer involving plasmids [5] 

Plasmids are basically circular chromosomes that are most typically found in bacteria. Therefore gene transfer involving plasmids normally refers to gene transfers involving bacterias such as E. Coli (gene transfer used for insulin production).

How this one is that first we need to isolate and cut out the gene that we want to transfer using an enzyme called a restriction enzyme. Then the plasmid in the bacteria is cut out using the same restriction enzyme. This way the sticky ends of the gene of interest and the plasmid match up. The gene of interest is then inserted into the plasmid and they are 'glued' together using an enzyme called DNA ligase. The newly interested gene of interest along with the plasmid collectively is called a recombinant plasmid.

Now the bacteria has a new gene in it's genome and it will express it. And when replicating that new gene will also be replicated.

b) Outline a technique for transferring genes between species [5] 

First of all we need to isolate and cut out the gene that we want to transfer with an enzyme called a restriction enzyme. Then the same restriction enzyme is used to cut a plasmid or chromosome in the organism that we would like to transfer the gene of interest to. Both the gene of interest and the plasmid will have sticky ends (the ends left exposed)- which because it was cut with the same restriction enzyme will match up. The gene is then inserted into the chromosome or plasmid and 'glued' together using another enzyme called DNA ligase. If a plasmid was used the collective plasmid along with the new gene is called a 'recombinant plasmid'. Now the organism that the gene of interest was placed in will express that gene. As the cell divides the recombinant plasmids are clones and many more copies of it will be produced.

TASK: Discuss the potential benefits and possible harmful effects of one example of genetic modification

Genetically modified organism chosen as an example: A species of tomato that was genetically modified to be more tolerant to higher levels of salt in the soil. 

BENEFITS: 

-This would help farmers in improving food production globally (if the tomatoes are more resistant to higher levels of salt then this would make it easier to grow in more regions-now including regions of higher salinity). 

-Farmers can be more in control of what crops they produce because normally without genetic modification, farmers rely on the randomness in breeding. Therefore genetic modification makes the process less of a gamble and farmers are more likely to get a generation of tomato plants that are just as desirable (in this case resistant to high soil salinity). 

-can help people in developing nations who are starving because their soil is of high salinity therefore normally without genetic modification has how fertility. This genetically modified tomato will enable them to grow at least tomatoes which would reduce hunger to a degree. 

HARMFUL EFFECTS: 

-long term effects of these tomatoes in the wild. seeds from the genetically modified tomato may be eaten by animals and carried out of the area to neighboring fields. The genes from the genetically modified tomato could be integrated into wild species. These tomatoes may gain an unnatural advantage over other tomato species and have an advantage in taking over the habitat which would lead to the extinction of other tomato species in that area which could potentially affect the ecosystem if other animals feed on that particular species of tomato. 

-Also the genes could cross species. It has been proven possible that it is possible to cross genes in the laboratory and so there is a possibility in nature as well that the genes may cross species and potentially this could be harmful. (NOTE: gene leakage can occur across species in plants in general, so the risk is not only on tomato varieties but to other plants too as they can acquire the modified genes!)

-Risks for allergies: Someone could not be allergic to natural tomatoes but are allergic to genetically modified tomatoes. There is no convenient way to tell the difference (in other words cannot determine which tomato is genetically modified and which is not be simply looking at it) and this would be highly problematic for those who are allergic. 

-Large portions of the human food supply will be in the hands of a small number of organizations-  and this would be problematic if the human food supply is dependent upon a very small number of organizations and failure to effectively produce could have a global effect. As a large food supply being controlled by a very small number of organizations there is a greater chance that there would be a mistake that could lead to a humanitarian catastrophe. 

-Decrease in biodiversity- critics argue that the proliferation (rapid reproduction) of genetically modified tomatoes may lead to a decrease in biodiversity either because people stop growing natural tomatoes or the seeds of these tomatoes become integrated into the wild and have an unfair advantage over natural tomatoes (or a combination of both) leading to the possible extinction of natural tomatoes= reduction of biodiversity. Which could be problematic because if a disease to genetically modified tomatoes is introduced then potentially all the genetically modified tomatoes are vulnerable (as they have a similar genetic make up with a few variations due to random mutation) whereas if there was more diversity it is less likely that all tomatoes will be affected and in the event of a disease that kills a particular species we still have other species of tomato to turn to as another source of food. 

Citation: http://www.csa.com/discoveryguides/gmfood/overview.php

TASK: Research the bio-engineering technique

Gene transfer- taking a gene out of on organism and putting it into another organism is called gene transfer.

Simple summary steps as to how this is done:

First we need to CUT the section of the DNA to get the gene that we need and then PASTE the gene into the organism that we are transferring the gene to: (The cutting and pasting process is known as GENE SPLICING)

The ‘scissor’s used for cutting these base sequences are in fact enzymes. To be exact they are called restriction enzymes (molecular scissors) called endonucleases. 

They find and recognize the specific sequence of base pairs on the DNA molecule. Then they cut the DNA at the specific points (normally about four to six base pairs long). The gene is cut and then it is released and then is removed from the donor organism. 

After cutting the PASTING is done by another enzyme called DNA ligase. It recognizes the parts of the base sequences that have to be stuck together (sticky ends) and attached them to the plasmid (circular DNA found in bacteria) or chromosome of the new organism. 

The DNA of the plasmid or chromosome is usually cut with the same restriction enzyme used to cut the DNA from the first organism in order to make room for the gene that is going to be transferred. 

(Also the fact that both DNA where cut from the same restriction enzyme implies that the sticky ends will have the same DNA code as the end of the going to be pasted gene: thus allows the stranded to be more easily matched up) 

Then the enzyme LIGASE is used to join the join between the transferred gene and the plasmid or chromosome.

NOTE: restriction enzymes will work on DNA from other organisms because DNA is chemically identical as the bases are always the same (it is just the order of bases that varies).

Now the next step is copying DNA (or DNA cloning): now a HOST cell is needed. Cells such as yeast cells can be used but the most popular is E. coli (a type of bacteria). In which case is Escherichia coli is used, gene splicing involves plasmid. 

Once the gene has been transferred to the plasmid the plasmid with the new gene is called a recombinant plasmid.  This recombinant plasmid is then used as a vector to introduce this new gene into an organism’s genome.

The recombinant plasmid is then placed inside the host bacterium and to encourage growth and reproduction it is put in ideal conditions. (Such as putting in a bioreactor where the bacterium will be in a liquid that is full of nutrients and kept at the right warm temperature).

Then the host cell make copies of the gene (new gene is copied as well). But not only that since the gene is part of it’s genetic make up, the host and it’s newly reproduced bacterium begins to synthesize whatever the protein the gene codes for.

A real life example is getting E. Coli to make human insulin (a protein to treat diabetes) by producing a recombinant plasmid that involves the human gene for making insulin. 

Citation: http://www.biotechnologyonline.gov.au/biotec/cutpaste.html