Extraction
A sample of tissue containing cells with a nucleus (e.g. blood, a hair root or semen containing a few sperm cells) is taken to the laboratory where DNA is extracted by shaking the sample in a mixture of water-saturated phenol and chloroform.
Proteins precipitate out, leaving pure DNA dissolved in the water layer.
Some facts:
Required amount of tissue:
- 0.5 cm³ of blood
- 0.005 cm³ of semen
- one hair root
Digestion
Certain restriction enzymes are added to the DNA to cut it. The enzymes recognise specific base sequences and so cut only at specific points, close but not within the required minisatellite region. This process produces a no. of DNA fragments of different lengths, some of which contain the minisatellite.
Separation
1. The DNA fragments are separated according to their size by electrophoresis:
- DNA fragments are placed in wells at one end of a block of agarose gel.
- An electric current is then passed through the gel.
- The pieces of DNA are carrying negative charges and so will move towards the positively charged electrode.
- Smaller fragments move trough the gel more quickly than the larger fragments and consequently travel further through the gel. Therefore the fragments are separated into bands.
2. The pattern of bands on the gel is invisible at this stage. A probe is added, containing a single strand of complementary DNA, which cannot attach to the existing double-strand of DNA. So the bands become visible.
3. Next step is separating the double-stranded DNA fragments into single strands by immersing the block of gel in an alkaline solution.
4. To transfer the single-stranded DNA fragments onto a nylon membrane a technique called Southern blotting is used.
This involves putting a thin sheet of nylon over the gel and covering it with absorbent paper towels. The absorbent paper draws the DNA fragments up into the membrane by capillary action, with their relative positions remaining unchanged. Later these fragments are fixed to the membrane by exposure to UV-light.
Hybridisation
A radioactive DNA probe is used to bind onto and reveal the location of a certain type of minisatellite. The nylon membrane is immersed in a solution containing the radioactive DNA probe. See picture
DNA probes consist of a single strand of a length of DNA (made up of sequences of bases complementary to the core sequence). The commonly used probes in forensic work bind only at one specific site or locus → known as single-locus probes. Excess probes are washed away. The process can be repeated with different radioactive probes, which bind to different core sequences and thus identify the different minisatellites.
Final stage in DNA fingerprinting
- Making visible the minisatellite regions that have been picked up by the radioactive probes by putting X-ray film over the nylon membrane.
- Places where radioactive probes have bound to DNA fragments will emit radiation, which will fog the film → a pattern of bands is created known as a DNA fingerprint (not unlike the bar code found on items in supermarkets).
Humans have two copies of most chromosomes (one from father, one from mother side). Each individual should therefore have two of each type of minisatellite (not necessarily with same no. of repeats/lengths).
Forensic laboratories commonly use 3 or 4 single-locus probes on a single sampling, giving a DNA print with up to 6 or 8 bands. As only one of each chromosome is inherited from each parent, half of the bands should match those of the mother and half those of the father.
DNA fingerprinting can be used to identify suspects in a crime. EXAMPLE:
Analysing the results
- First visual inspection is used whether two DNA fingerprints match.
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If there is a match, an automated scanning system is used to calculate the length of the DNA fragments denoted by the bands (how far have the fragments travelled through the gel compared with the distance travelled by DNA of known length, called markers.) → DNA profile then expressed as a set of 6 to 8 numbers.
- Calculating the odds of somebody else in the population having the same DNA fingerprint as the suspect.
- This typically works out to be 1 in 30 for a single band.
- However, by multiplying the odds together from 8 bonds, analysts can predict the chance of an identical DNA fingerprint being produced from 2 different individuals as hundreds of millions to one.
Problems with contamination
There are three main problems:
- Forensic samples are rarely pure: a blood sample may be contaminated with DNA from bacteria and fungi.
- A delay in collecting the sample could decompose the DNA (some sites normally cut by the restriction enzymes could have been lost) → longer or shorter fragments are produced, forming a different banding pattern.
- Ions in the contaminants could affect the charge on the DNA fragments, thus causing them to travel through the gel at a faster or slower rate.
