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

PCR is the cloning or amplification of DNA, copies need to be made to rapidly increase the amount of DNA, this is useful if the source of DNA is small.

Firstly in a PCR reaction, DNA samples are heated to 94-96⁰C for several minutes to denature (separate into single strands) the target DNA.

The temperature is then lowered to 50-65⁰C for several minutes to let left and right primers to base pair to their complementary bases. The primers are designed to bracket the DNA region to be amplified.

Temperature is then raised again to 72⁰C for several minutes to allow Taq polymerase to attach at each priming site and synthesize a new DNA strand.

Temperature is raised to 94-96⁰C again to denature the DNA like in the beginning thus starting the process again

Animation: http://www.dnalc.org/resources/animations/pcr.html

Why might you want to amplify minute quantities of DNA?

We need to clone DNA for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. 

Past Paper Questions on Bio engineering

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

The definition of clone is a group of genetically identical organisms derived from a single parent. These clones are made using laboratory techniques and starts with the production of human embryos. Therefore there are many issues with cloning; some arguments against it are that generating a new human embryo just for research is unnatural and wrong and is opposed by some religions. Cloning humans could also reduce the value of the human and clones may have health problems and they are more likely to die from complications. Cloning is also an expensive process and not fully successful so there could be better allocation of resources

On the other hand, the arguments for cloning are that it may help provide tissues and skin to repair burns, organs for transplantation and can lead to future medical breakthroughs.

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

Gene transfer involves plasmids which is a small piece of circular DNA. The plasmid is first removed from a cell and cut with restriction enzymes. The genes from another organism is also cut with the same restriction enzyme and spliced together with the plasmid using DNA Ligase. This plasmid is called a recombinant DNA which is then put into the genome of a host cell and then a fermenter to be cloned.

Outline a technique for transferring genes between species [5]

Gene transfer between species is when a gene from one organism is taken and placed in another organism. Firstly the gene for transfer has to be obtained; this is done by using restriction enzymes to cut out the useful gene. A plasmid, small circular DNA molecules found in bacteria, are cut with the same restriction enzyme and the gene cut out is spliced together with the plasmid using DNA Ligase. This plasmid is called a recombinant DNA which is then put into the genome of a host cell and then a fermenter to be cloned. The bacteria with the Recombinant DNA are then grown by asexual reproduction.

4.4.10 Discuss the potential benefits and possible harmful effects of one example of genetic modification.

A genetically modified organism (GMO) is an organism containing a transplanted gene.

An example is in the genetically modified salt tolerance in tomatoes. Plants like tomatoes couldn’t grow in salty conditions since this hypertonic soil water results the death of the plant however once the gene for salt tolerance was introduced in the tomato it now has the opportunity to be grown in saltier areas.

Benefits of GMO:

§  Increased yields particularly in regions of food shortage.

§  Yields of crops with specific dietary requirement such as vitamins and minerals.

§  Crops that won’t spoil so easily during storage.

§  Higher meat yields in genetically modified animals thus reducing hunger.

§  GM breeding is faster more specific and certain.

§  Plants are genetically modified to resist pests and extreme weather conditions which extends its survival.

Disadvantages and concerns of GMO:

§  Genetically modified animals and plants are considered un-natural and unsafe for human consumption.

§  There is a risk of the escape of 'genes' into the environment where they can be passed to other organisms with unknown effects. The genes could give an unnatural advantage for certain species over others.

§  There is a risk for allergies from the GM plants and animals.

§  It is worrisome that most of the food in the world could be controlled by a small number of corporations.

§  Decrease in biodiversity.

§  Gene transfers can be potentially harmful to animals

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.

Gene transfer is when a gene from one organism is taken and placed in another organism.

Stage 1 is obtaining the gene for transfer. This is done by using restriction enzymes to cut out the useful gene that is to be transferred; this leaves sticky ends on each side of the gene.

Stage 2 is preparing a vector for the transferred gene. This is done by using plasmids which are small circular DNA molecules found in bacteria. These are cut with the same restriction enzyme and this leaves the same complementary 'sticky ends' in the plasmid like the ones on the gene.

Stage 3 is the when gene has to be copied and this is when the gene is combined with the DNA. The gene is glued into the plasmid using DNA ligase. This plasmid is called a recombinant DNA and it can be used as a vector.

Stage 4 is the isolation of the transformed cells. The vector is then put into the genome of a host cell (bacterium, yeast or other cell) as the bacteria gives the plasmid the ideal conditions to grow and this is done by putting it into a bioreactor. Many of the cells remain untransformed but some of the cells are transformed to contain the recombinant DNA and these transformed cells will be separated from untransformed. 

Stage 5 is obtaining the final product. This is done when the transformed cells are isolated and put into a fermenter the right to be cloned. The bacteria with the Recombinant DNA are then grown by asexual reproduction and the final step is to isolate and purify the product called downstream processing.