DNA Profiling

[blank_start]Short Tandem Repeats[blank_end] (STRs) are short DNA sequences repeated many times. They're found within [blank_start]introns[blank_end] (intragenic regions) that are non-coding. They're inherited in the same way as genes in the coding regions - [blank_start]exons[blank_end] (expressed regions). STRs occur at the same [blank_start]locus[blank_end] on both chromosomes of a [blank_start]homologous[blank_end] pair. The number of repeats on each of the homologous chromosomes can be different. The number of repeats at a locus varies between individuals. There's a large amount of variation in the number of repeats at each locus. Therefore 2 individuals are highly unlikely to have the same combination of STRs.

STRs are inherited in the same way as alleles of a gene, with offspring receiving one repeated sequence randomly from each parent.

What can DNA profiling be used for?

DNA profiling is widely used in [blank_start]legal proceedings[blank_end]. It is generally thought to produce a result that is almost [blank_start]unique[blank_end] to the individual and a near [blank_start]certain[blank_end] indication of guilt in criminal trials. This is true even for [blank_start]twins[blank_end] due to the accumulation of mutations during a person's life. But because the DNA profile only analyses a few [blank_start]repeated sequences[blank_end], it is less likely to be completely unique. This is a particular problem if individuals being tested are closely [blank_start]related[blank_end]. In court, a forensic scientist estimates the chance of seeing the same DNA profile in the general population; this is usually in excess of [blank_start]1 billion[blank_end].

DNA samples can come from almost all [blank_start]biological tissue[blank_end] - animal or plant.

Examples of sources of DNA samples:

The tissue sample is broken down in a [blank_start]buffer[blank_end] solution (includes salt and detergent) to disrupt the cell membranes. The small suspended particles, including the DNA, are separated from the rest of the cell debris by filtering or [blank_start]centrifuging[blank_end]. [blank_start]Protease[blank_end] enzymes are incubated with the suspension to remove proteins. Then, cold [blank_start]ethanol[blank_end] is added to precipitate out the DNA. Several stages of washing the DNA in buffer solution follow.

[blank_start]Restriction enzymes[blank_end] cut a DNA sample into fragments only where their [blank_start]specific restriction sequence[blank_end] occurs. The names of the restriction enzymes reflect the [blank_start]bacteria[blank_end] from where they originate. If restriction sites are either side of an [blank_start]STR[blank_end] sequence, that fragment of DNA will remain intact, but it will cut away the rest of the [blank_start]genome[blank_end]. The repeated sequences remain [blank_start]intact[blank_end]. They only cut at specific base sequences, usually 4 or 6 base pairs long. Restriction enzymes are found naturally in bacteria where they cut up invading [blank_start]viral DNA[blank_end]. [blank_start]Endonucleases[blank_end] means they cut the DNA at sites within a strand of DNA.

Polymerase Chain Reaction: The sample is placed in a reaction tube with DNA [blank_start]polymerase[blank_end], DNA primers and [blank_start]nucleotides[blank_end]. The reaction tube goes into the PCR thermal cycler. It's heated to [blank_start]95[blank_end] degrees C which separates the double stranded DNA. It's then cooled to [blank_start]55[blank_end] degrees C which optimises the binding of the [blank_start]primer[blank_end] to the target DNA sequence in the sample. It's then heated to [blank_start]70[blank_end] degrees C which is the [blank_start]optimum[blank_end] temperature for the heat stable DNA polymerase. The polymerases attach and [blank_start]nucleotides[blank_end] are added, extending the DNA from the primer. After 2 cycles, copies that are just the [blank_start]STR[blank_end] sequence fragment are produced. As the cycle continues, huge numbers of the targeted DNA fragments are produced.

What are DNA primers?

In gel electrophoresis, DNA fragments are separated according to their colour

Gel Electrophoresis: DNA placed on [blank_start]agarose[blank_end] or polyacrymalide, which provide a stable medium through which the fragments can move (porous). The gel is submerged in a [blank_start]buffer[blank_end] solution, and connected to [blank_start]electrodes[blank_end] that produce a potential difference (voltage across the gel). The [blank_start]negatively[blank_end] charged DNA fragments migrate through the gel according to their overall size and [blank_start]charge[blank_end]. In a given time, smaller fragments end up closer to the [blank_start]positive[blank_end] electrode. A reference sample with fragments of known length may be added to the gel. This is known as a ladder or marker. The fragments are measured in base pairs.

The gel is quite [blank_start]fragile[blank_end] and the DNA fragments are [blank_start]double stranded[blank_end] after electrophoresis if restriction enzymes have been used.

What is DNA probe?

Southern blotting is used to transfer the fragments to a more resilient [blank_start]nylon[blank_end] or nitrocellulose membrane. The membrane is placed directly onto the gel and a wad of dry absorbent [blank_start]paper[blank_end] is placed on top. This acts as a wick to draw [blank_start]buffer solution[blank_end] up through the gel, carrying [blank_start]DNA fragments[blank_end] onto the membrane. During this process, the fragments maintain their positions relative to each other and are denatured into [blank_start]single[blank_end] strands, exposing the base sequences. The membrane is then incubated with an excess of DNA [blank_start]probe[blank_end]. After allowing time for the probe to bind to any complementary sequences (hybridise), any unbound probe is washed away.

Probes may be radioactive, labelled with [blank_start]radioactive phosphorous[blank_end], or labelled with a [blank_start]fluorescent marker[blank_end].

With a radioactive probe...

With a fluorescent probe...

A [blank_start]single[blank_end] band occurs on the profile where a person's maternal and paternal chromosomes have the [blank_start]same[blank_end] number of repeats at a particular locus. [blank_start]2[blank_end] bands occur on the profile if the 2 chromosomes have a [blank_start]different[blank_end] number of repeats at the same locus. There must be a [blank_start]reference profile[blank_end] for comparison. The reference profile may come from a suspect in a murder investigation, a relative in the case of identifying a corpse, or from the parents when establishing paternity.

The DNA [blank_start]primers[blank_end] have a fluorescent tag attached to them which allows the system to be automated. As the DNA fragments with their fluorescent tags move through the gel, they pass a [blank_start]laser[blank_end], the [blank_start]dye[blank_end] in the tag fluoresces and the coloured light is detected. This effectively gives a time that it has taken for the fragments to pass through the gel. Passing a separate set of fragments of known length through the gel allows the length of time for passage through the gel to be calibrated with fragment size. Several STR loci can be analysed at once by using tags that fluoresce at different [blank_start]wavelengths[blank_end] giving different colours for each of the STR loci. A computer processes the information from the detector displaying the results of the gel electrophoresis as a [blank_start]graph[blank_end].