When was used first DNA fingerprinting?
DNA fingerprinting was first developed in 1984 by sir Jeffreys, a molecular geneticist of the University of Leicester in the UK. This technique was first used to solve a rape and murder's case in Leicestershire. At the beginning a boy, Buckland, confessed upon interrogation to be the culprit, but when the police compared his fingerprint's DNA and the DNA on the crime scene, it was found that the two samples did not match. So, Buckland was exonerated from the list of suspects by DNA evidence. Later on, the police obtained the DNA of Colin Pitchfork, which matched exactly with the one on the crime scene. In the end he was imprisoned, the sentence was mainly based on the DNA evidence.

Where can DNA be obtained from?
DNA can be obtained from biological materials, including: blood (only white blood cells, because red blood cells haven't nuclei),  semen, vaginal fluids, sweat stains, hair with roots, skin cells and dandruff, nasal secretions, urine and feces.

Who invented RFLP technique? When?
RFLP technique, which means Restriction Fragment Lenght Polymorphism, was developed by sir Alec Jeffreys in 1984. It consists in comparing DNA fragments produced after restriction enzymes digestion, according to their lenght.

What is a restriction enzyme?                   
Restriction enzymes are enzymes that cut the DNA into fragments near or at specific sequences, called  restriction sites. The sequences are palindromic, which means that the base sequence reads the same backwards and forwards, and usually they are composed by 4-6 bases

What is restriction enzyme role in nature?
The role in nature of restriction enzymes is to defend bacteria from viral infection. They have been developed inside bacteria's cytoplasm, where they can have access to the DNA of the virus and cut in in pieces.

A. Association of PCR and minisatellite-variant-repeat method MVR = 6^th ph.

B. First experiment of DNA finger-printing and possible applications in various fields =2^nd ph.

C. Other application of tandem repeats in animals =5^th ph.

D. Alec personality and attitude =8^th ph.

E. Surprising Alec's retirement and reference to identification of the dead body of Richard III = 7^th ph.

F. First application in parentage testing for maternity in a case of immigration       = 3^rd ph.

G. Serendipity in research strategies and scientific studies = 9^th ph.

H. Alec Jeffreys and his innovative method, called DNA fingerprinting = 1^st ph.

I. First case of application in forensic science: discovering the culprit of a two girls rape  = 4^th ph.

2. Why does DNA migrate toward positive electrode?
The DNA migrates towards the positive electrode because it has a negative charge, which is related to the presence of phosphate groups in the structure of the DNA.

The TBE is a buffer solution which is used to create a suitable environment for the restriction enzymes. It contains :  sodium chloride (NaCl), that provides a correct ionic strenght;  Tris-HCl, that mantains a 8.0 pH; magnesium ions (Mg ²⁺ ), enzyme co-factors  which contribute to activate the enzymatic activity. The solution is brought to 37°,  the ideal temperature for enzymes.

5. How can DNA be detected in agarose gel?
As the DNA is an invisible substance put inside a transparent gel, it's necessary to use loading dyes, including glycerol , xilen-cyanol and  bromophenol blue, that mark the boundary of the range in which can be found the DNA. In order to detect the DNA it's added an intercalant dye, that in our case is Red-gel, which makes the fragments fluorescent when exposed to the UV rays of a transilluminator.

6. Why are DNA dyes dangerous for human health?
DNA dyes are dangerous for human health because they act as mutagens:  they insert theirselves between the  strands of the DNA, deforming it. They are also  possible carcinogen substances  that can  both being  absorbed and breathed in.

Agarose gel can be made with different concentrations of agarose powder, between 0.6 and 3%, depending on the size of the fragments you want to separate. There are two main component of an agarose gel: agarose powder and buffer TAE solution, which need to be measured carefully, in order to obtain a homogeneous gel. Then, the two substances are mixed up and heated in a microwave oven, until the agarose is completely dissolved. Pay attention to use a container large and high enough to allow the solution to boil up without coming out; it's important, also, to place a loose cover (loose, because it doesn't alterate the pressure's level) on the top of the container to prevent the mixture  from splashing out. The agarose is ready when is completely clear, otherwise you will have regions of different concentration and your nucleic acid samples will not be separed correctly. Then, take it out of the microwave with protective gloves and let it cool to 60°. Add the intercalant dye in the mixture, having care to use gloves to protect the skin. Now, it's time to prepare the tray that will contain the gel: with a masking tape seal the open sides of the tray, make sure that it's placed on a horizontal surface and then insert a comb, which will create the wells in the gel. After this, pour gently the liquid gel inside the tray, eliminate quickly air bubbles with a sharp instrument. The gel will be solidified after twenty minutes, during which you must not move the tray, unless you will obtain a not uniform gel; then remove the masking tape and the comb with attention.

9. Which piece of information can be find out from a DNA pattern bands?
A band is a well-defined line, which contains a large number of DNA fragments of the same size. All the bands create a fragment pattern. The sample of DNA with the same pattern of the one found on crime scene belongs to the culprit.

36"  How long has DNA been used to identify people?
DNA technology has been used since the mid 80s, but at the moment we are using  improved and more sensitive  type of techniques.

52" The techniques used nowadays are more sensitive. How much DNA is needed to do a test?
While in the past you may have needed something like 50 nanograms or micrograms, now we are down to less than a nanogram of DNA.

1'26 "How many cells are needed to get a full profile?
It takes a vanishingly small amount of DNA to get a full profile, for example three or four cells left on a handled object by the perpetrator are enough.

1' 46" In what conditions must the exhibit be kept?
It's important to make sure that your exhibits are in bone dry conditions, unless the DNA will degrade (such as because of mould action). If it's not possible to keep them dry, you could freeze them.

1' 52" How long does DNA last if taken in good conditions?
If preserved in good conditions, DNA will last essentially forever, for this reason a cold case is not different from a fresh one

1- Voltage applied to the electrophoretic cell

2- Presence of Mg ions

3- Buffer solution pH 

4- Room temperature

5- Room atmospheric pressure

6- Gel thickness

7- Agarose gel concentration

8- DNA fragments length

9- Electrophoretic brand
10- Presence of bubbles in the gel

11- Gel tray width

12- DNA origin

Specifications:
1-The voltage applied to the electrophoretic cell is the fundamental principle of gel electrophoresis. Thanks to a high voltage, fragments of DNA run quickly through the gel;  however if the voltage is excessively high, the gel can be destroyed.
3- The buffer solution's Ph is important because, maintaining the pH at 8, the basic solution keeps the DNA's negative charge and allows it to run towards the positive pole. Chemically, in fact, the OH groups of the basic solution bond together with the phosphate group of the DNA.

7- The agarose gel concentration determinates the rate of migration of the fragments through the gel. In fact, the higher the concentration is, the slower the fragments move, because of a higher frictional force and vice versa. see(picture below)

8- The DNA fragments length: the shorter fragments move faster than longer ones. Comparing the fragments of DNA's samples with those ones of the culprit, we have been able to find out the perfect match between them, looking at their length and position.

10- The presence of bubbles in the gel, could compromise the results of the electrophoresis. In fact, if there are bubbles in the gel, it is not homogeneously distributed and the fragments can't move correctly.
 

Before the discussion, I thought that one of the most important thing was the presence of buffer solution, and it was correct; in addition I believed that magnesium ions would be in a higher position in the pyramid, but their action is more relevant during the DNA's digestion.

Room temperature (4) influences the results only if it is very high or very low, while the room atmospheric pressure (5), the gel thickness (6), the gel tray width (11) and the DNA origin (12) are almost negligible factors. In conclusion, the electrophoretic brand (9) allows to see the results from the bands pattern, but it doesn't influence them at all.