Forensics

DNA profiling uses non-coding, repetitive DNA — specifically short tandem repeats (STRs) or variable number tandem repeats (VNTRs).

It is suitable because the number of times the sequence is repeated varies greatly between individuals, so the resulting pattern of bands is (almost) unique to each person.

During PCR the mixture is repeatedly heated to about 95 °C to denature (separate) the DNA strands.

A normal DNA polymerase enzyme would be denatured at this high temperature — its active site would change shape — and would have to be replaced every cycle.

Taq DNA polymerase is obtained from a bacterium that lives in hot springs, so it is heat-stable (thermostable) and is not denatured at 95 °C. This means the same enzyme can be used throughout all the cycles, allowing the process to be automated.

DNA contains many phosphate groups, which give the molecule an overall negative charge.

When an electric field is applied across the gel, the negatively charged DNA fragments are attracted towards the positive electrode (anode) and move through the gel.

The gel acts as a molecular sieve / mesh: smaller fragments move through it more easily and travel further, while larger fragments are held back nearer the wells. The fragments are therefore separated according to size.

PCR is an in vitro method of making many copies of a chosen region of DNA. It is carried out in repeated cycles, each with three stages at different temperatures.

In denaturation (~95 °C), the DNA is heated so that the hydrogen bonds between the complementary bases break and the double helix separates into two single strands.

In annealing (~50–60 °C), the mixture is cooled so that short single-stranded primers bind (anneal) to the start of each target sequence by complementary base pairing. Primers are needed for the polymerase to begin building.

In extension (~72 °C), the heat-stable enzyme Taq DNA polymerase adds free DNA nucleotides to each primer, building a new complementary strand along each template strand.

Because each strand becomes the template for a new double-stranded molecule, the amount of DNA doubles every cycle ($N = N_0 \times 2^n$). After many cycles (e.g. 30) the chosen region has been amplified millions of times.

(a) Using $N = N_0 \times 2^n$ with $N_0 = 80$ and $n = 12$:

$N = 80 \times 2^{12} = 80 \times 4096 = 327\,680 \text{ copies}$

So there are 327 680 copies of the target sequence after 12 cycles.

(b) The number of copies doubles every cycle, so the amount of DNA increases exponentially rather than by a fixed amount. Because each cycle multiplies the total by two, even a very small starting number is multiplied by $2^n$, which becomes very large after only a modest number of cycles. This is why a trace sample recovered from a crime scene can be amplified to enough DNA to produce a clear profile.

First, DNA is extracted from the crime-scene sample (for example from blood, hair roots or skin cells). Because the sample is often tiny, the chosen non-coding repetitive regions (STRs/VNTRs) are then amplified by PCR so that there is enough DNA to analyse.

The amplified DNA is then separated by gel electrophoresis. The fragments are loaded into wells in a gel and an electric field is applied; because DNA is negatively charged it moves towards the positive electrode (anode), and smaller fragments travel further through the gel, so the fragments are separated according to size. This produces a pattern of bands — the DNA profile.

The same procedure is carried out on a sample of DNA from the suspect, run in an adjacent lane of the same gel so the two can be compared directly.

The two profiles are then compared band by band. If all the bands in the suspect's profile line up at the same positions as those in the crime-scene profile, the profiles match, which is strong evidence that the crime-scene DNA came from that suspect. If any bands differ, the suspect is excluded.

DNA is negatively charged because of its phosphate groups, so it is attracted to the positive electrode (anode) and moves towards it.

If the wells are placed at the positive end, the DNA will try to move towards the positive electrode but is already there — so the fragments will move off the edge of the gel (towards the positive electrode, out of the gel) rather than separating across the gel.

As a result the student will see no separated bands within the gel — the DNA runs off the end. To work correctly, the samples must be loaded at the negative (cathode) end so the fragments migrate across the gel towards the positive electrode, separating by size as they go.

Several lines of evidence are combined. Soon after death, body cooling (algor mortis) can be used: metabolism stops so the body cools towards the surrounding temperature at a roughly predictable rate, and the core temperature gives an early estimate. Rigor mortis is also useful in the first day or so: after death respiration stops, so no ATP is made; without ATP the actin and myosin filaments stay bound, so the muscles stiffen, and later they relax again as the proteins decompose — the stage of rigor narrows the time.

For longer intervals, the stage of decomposition (caused by the body's own enzymes and by microorganisms) gives a broad estimate, and forensic entomology is used: different insect species colonise the body in a predictable succession (blowflies and their maggots first, then other flies and beetles), so identifying the species present and the developmental stage of the larvae dates the colonisation, often to days or weeks.

Several methods are used together because each one is only reliable over a particular time window and is affected by external factors. Cooling and rigor only help in roughly the first day and are influenced by ambient temperature, clothing and body size; decomposition and insect succession take over for longer periods but their rates also depend on temperature. Because no single method is precise, combining them gives a narrower and more reliable estimate of the post-mortem interval and reduces the effect of the limitations of any one method.

(a) Using $N = N_0 \times 2^n$ with $N_0 = 1$ and $n = 25$:

$N = 1 \times 2^{25} = 33\,554\,432 \text{ copies}$

To 3 significant figures in standard form this is $\mathbf{3.36 \times 10^{7}}$ copies.

(b) During each cycle the double-stranded DNA is denatured into two single strands, and each single strand acts as a template for a new complementary strand built by Taq polymerase during extension. Because every strand becomes a new double-stranded molecule, the total number of double-stranded molecules doubles each cycle — one molecule becomes two, two become four, and so on, giving the $2^n$ relationship.

(c) In practice the yield is slightly lower because, as the cycles continue, reagents become limiting — for example the primers or free nucleotides run low, or the enzyme activity falls — so not every template is copied in every cycle. (Any one valid reason, e.g. reannealing of product strands, is acceptable.)

(a) The crime-scene bands are at 18, 26, 41 and 55 mm. Comparing each suspect:

• S1 (18, 31, 41, 60) matches at 18 and 41 but differs at 26→31 and 55→60, so S1 does not match.
• S2 (18, 26, 41, 55) matches at all four distances, so S2 matches the crime-scene sample.
• S3 (22, 26, 48, 55) matches only at 26 and 55, so S3 does not match.

Therefore Suspect 2 (S2) matches the crime-scene profile, because every band is at the same distance, meaning every fragment is the same size.

(b) During electrophoresis the negatively charged DNA moves towards the positive electrode through the gel mesh. Smaller fragments move through the mesh more easily and faster, so in a given time they travel a greater distance. The bands that have travelled the greatest distance (here 55 mm) are therefore the smallest fragments, while those nearest the wells are the largest.

(c) A profile uses only a limited number of repeat regions, so although the chance is extremely small, it is not impossible for two unrelated people (or close relatives) to share the same pattern of bands by chance. A match therefore shows the suspect cannot be excluded and is very likely to be the source, but it does not prove it with absolute certainty; matching more regions lowers the probability of a chance match further.