Gel electrophoresis

Gel electrophoresis is used to separate DNA fragments. Electrophoresis uses an electric current to separate different-sized molecules in a sponge-like matrix. Smaller molecules move more easily through the gel pores than larger molecules.

Agarose gel can be used to separate DNA. First the gel is submersed in a tank filled with a salt solution which conducts electricity.

Using a pipette, DNA samples are loaded into slots made in the gel. The DNA is colorless but a blue dye is added, this makes it easier to track the DNA and its migration through the gel.

The phosphate groups in the DNA backbone carry negatively charges oxygen’s, giving DNA an overall negative charge. In an electric current, the negatively charged DNA moves towards the positive pole of the electrophoresis chamber.

DNA molecules then move through the gel by ‘reptation’-a reptile like snaking action through the pores of the gel. Smaller DNA fragments migrate faster and further over a given period of time than do larger fragments. This is how DNA fragments can be separated by size in the gel.

The introduction of a florescent dye, ethidium bromide, was used to stain the DNA. Ethidium bromide binds to the DNA double helix and glows in ultra-violet light. This lets researchers see where the separated DNA fragments end up.

DNA Profiling

Genes make up the blueprint for our bodies and almost every cell in the human body contains a copy of the blueprint, stored inside the nucleus. These genes are on the DNA on strands known as chromosomes. Certain portions of DNA are unique to each individual and DNA profiling is a way of establishing identity and is used in a variety of ways, such as finding out whether twins are fraternal or identical. DNA samples are usually obtained from blood.

DNA profiling is a technique used by forensic scientists to identify an individual by their respective DNA profiles. DNA profiles are encrypted sets of numbers that reflect a person's DNA makeup, which can also be used as the person's identifier.

DNA profiling involves the (partial) sequencing of genomes. Profiles tend to focus on areas of satellite of junk DNA which vary significantly between individuals. Junk DNA is used because by sequencing a number of sections a unique ‘genetic fingerprint’ can be created for an individual.

The uses for profiling DNA:

•             Paternity - to find out if the alleged father is actually the biological father of the child.

•             Twins - identical twins share the same genetic material, while fraternal (non identical) twins develop from two eggs fertilised by two sperm and are no more alike than individual siblings born at different times. It can be difficult to tell at birth whether twins are identical or fraternal.

•             Siblings - for example, adopted people may want to have DNA tests to make sure that alleged biological siblings are actually their blood brothers or sisters.

•             Immigration - some visa applications may depend on proof of relatedness.

•             Criminal justice - DNA testing can help solve crimes by comparing the DNA profiles of suspects to offender samples. Victorian law allows the collection of blood and saliva samples from convicted criminals and suspects. DNA profiles are then kept on a database.

Advantages of DNA profiling:

•             DNA tests can be applied to any human sample that contains cells with nuclei, such as saliva, semen, urine and hair.

•             DNA tests are extremely sensitive, and can be conducted using samples that would be too small for other serological tests.

•             DNA resists degeneration even after contamination with chemicals or bacteria.

•             The ability of DNA profiling to exclude a suspect means the police are able to confidently drop that line of enquiry and continue their investigation down other avenues.

Limitations of DNA profiling

•             DNA profiling can give incorrect results, due cross-contamination of samples.

•             Old DNA profiling technologies are more prone to errors, which could give false-negative or false-positive results.

•             DNA profiles can only offer statistical probability (for example, one in a million), rather than absolute certainty.

•             The more people tested, the lower the statistical probability. For example, the probability of one in a million may nosedive to one in 10,000 if enough people are profiled for a single test.

•             DNA databases stored on computer are vulnerable to exploitation via hackers.

•             Some critics point out that holding a person’s DNA profile on record is, in a sense, a violation of that person’s DNA ‘ownership’.

•             DNA evidence is easily planted at a crime scene.

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.

E.6.7 Outline two examples illustrating the adaptive value of rhythmical behavior patterns.

North American flying squirrels

They are adapted to fly at night because at night there is the most food and the least competition. Its pattern is regulated by an internal biological clock and not by external clues because even though the hours of darkness change throughout the year in North America, the flying squirrel continues to fly out at the same time no matter if it is light or dark. But external clues such as the light do come in helping the ground squirrel regulate its biological clock and adapt to its changing environment.

Fiddler Crabs

This crab only mates during a full moon or new moon as it is related to the best tidal periods.  When the moon is full or new, the gravitational pull of the moon and sun are combined. At these times, the high tides are very high and the low tides are very low. These are called Spring tides. The adaptive value of this behavior is that the mating is timed to coincide with a Spring tide so the eggs can be released during a nocturnal high spring tide so that the tiny, swimming larvae are washed far out to sea. They develop out there for several weeks and then wash into shore.