Within non coding regions between genes there are short sequences of bases called core sequences that repeat themselves over and over again, sometimes up to 100 times. These repeated regions of DNA are called mini-satellites or variable number tandem repeats. Different people have different numbers of repeated core sequences; therefore they have different sized mini-satellites. The greater the number of repeats the longer the mini-satellite.
DNA fingerprinting is based on two observations. Firstly, the number of repeats of a core sequence tends to vary considerably from person to person. Secondly, each individual has 50-100 different types of mini –satellite made form different core sequences. There is virtually no chance of two people having matching mini-satellites that are all the same length, unless they are identical twins. Some mini-satellites can be similar in length, therefore containing the same number of repeats. The trick in DNA profiling is to choose mini-satellites that show the most variation between people.
To make a DNA fingerprint, a very small sample tissue containing cells with a nucleus, such as, blood (0.5cm3), one hair root or semen (0.005cm3), is taken into the laboratory where the DNA is extracted. This is done by shaking the sample in a mixture of water-saturated phenol and chloroform. The proteins precipitate out leaving pure DNA dissolved in the water layer.
Certain restriction enzymes are added to the DNA to cut it. These enzymes recognise specific base sequences and so cut at specific points close to but not within the mini-satellite region, leaving the mini-satellites intact. A number of DNA fragments are produced of different lengths, some of which contain the mini-satellite.
The DNA fragments are separated according to size by electrophoresis. This involves placing the DNA fragments in wells at one end of a block of agarose gel. An electric current is then passed through the gel. The pieces of DNA carry negative charges and so move towards the positively charged electrode. The smaller fragments move through the gel more quickly than the larger fragments and consequently travel further through the gel. The fragments are therefore separated into bands. The pattern of bands is invisible at this stage. To show the bands containing the mini-satellite, a probe is added. This is a single strand of complementary DNA which cannot attach to the existing double-strand of DNA. The block of gel is immersed in an alkaline solution to separate the double-stranded DNA fragments into single strands.
Southern blotting is used to transfer the single-stranded DNA fragments onto a nylon membrane. This involves putting a thin sheet of nylon over the gel and covering it with absorbent paper towels. This draws the DNA fragments up into the membrane by capillary action, with their relative positions remaining unchanged. The fragments are then fixed to the membrane by exposure to UV light.
A radioactive DNA probe is used to bind onto and reveal the location of a certain type of mini-satellite. This involves immersing the nylon membrane in a solution containing the radioactive DNA probe. DNA probes consist of a single strand o a length of DNA made up of sequences of bases complementary to the core sequence. The probes used in forensic work are commonly of a type that will bind only at one specific site or locus and are known as single-locus probes. Excess robes are washed away. The process can be repeated with different radioactive probes, which bind to different core sequences and thus identify different mini-satellites.
The mini-satellite regions that have been picked up by radioactive probes have to be made visible by putting an X-ray film over the nylon membrane. The places where radioactive probes have bound to DNA fragments will emit radiation which fogs the film. This creates a pattern of bands known as a DNA fingerprint. It looks like the barcode found on supermarket products.