Introduction to DNA Profiling

Here we are, the technique considered by many to be a holy grail of forensics. But what is DNA profiling and how does it work? Well first things first, the term DNA profiling refers to the sequencing of an individuals DNA. 

When DNA profiling there are 3 possible outcomes:

1. Exclusion - the sample DNA does not match the suspects, thus ruling them out. This can be used to prove innocence or rule out suspects.

2. Inconclusive - due to damage through contamination or DNA degradation, further information must be sought, and the test must be carried out again.

3 Inclusion - The DNA is a match. However as DNA is not unique the significance of the match is calculated by quantifying the random match probability (Pm)'. The (Pm)' is a statistical test that estimates the probability of two unrelated people sharing the same DNA profile. Where markers are not linked (so inherited independently), the (Pm)' can be estimated by multiplying individual allele frequencies within a sample population. Therefore the more loci that are included in the analysis and the greater their heterozygosity the smaller the value of (Pm)' will be. A smaller (Pm)' means that the DNA being profiled is more likely to have come from the suspect. 

Despite DNA profiling's high levels of specificity there are a few problems with the technique. The first is that DNA will degrade over time, meaning it is harder to produce conclusive results with the test. The second major problem is that DNA from related people is extremely similar so has a much higher (Pm)' value. This means in cases between family members DNA is much less useful.

We'll end the introduction with a quick case study:

   1983, a girl in Narborough, Leicestershire is found dead with sigs of rape. A semen sample recovered from her body was tested and showed that the murderer was blood group A and had an enzyme profile shared by about 10% of the male population. However this was the only evidence recovered, and the investigation made no progress.

   1986, a second girl in the same area, was also found raped and murdered. The semen sample recovered from the body showed the same characteristics as the one from the case 3 years ago. The police established this connection between the 2 cases. The police believed that they were the work of the same man and that he lived in the vicinity. Their investigation led them to Richard Buckland, who was arrested. When interrogated he confessed to the second girls murder but denied having any involvement in the first. Buckland was DNA profiled, as were the semen samples. The girls had in fact been killed by the same man, but this man was not Buckland. 

   Having lost their primary suspect the police tried a different approach. They conducted the first ever mass DNA screening. All men in the vicinity (roughly 5000) had a blood sample or saliva swab taken. From these those exhibiting a matching enzyme profile and blood group where then DNA profiled. However despite all this they had nothing. There were no matches. Once again the investigation hit a wall. 

    Six months later however a woman reported that she had overheard a man claiming to have provided DNA in place of his friend, Colin Pitchfork. Colin Pitchfork was subsequently dragged in, and his DNA profile, unsurprisingly, was a match, inclusive. By 1988 he had been sentenced to life in prison. 

Colin Pitchfork on his arrest

   This case study helps show how DNA evidence alone is rarely enough to find or convict a suspect, but is very effective at excluding suspects. Thanks for reading! I'll be back soon, and we're going to be getting into some harder science! Over and Out!

Testing for Blood

You've probably all seen it in TV dramas; the crime scene looks clean as a whistle, not a trace left. That is until the CSI unit rolls in, sprays a mysterious liquid all over the place, off go the lights, out come the UV lamps and suddenly the room is covered in ethereal blue stains. Well you may be happy to learn that that's one of the few things that forensics shows largely get right.

Presumptive tests for blood reveal blood where it may not have been found with the naked eye. This could be due to it blending well with the background, seeping into cracks, or possibly just occuring in trace amounts. Luckily presumptive tests can help a forensic scientist find and identify blood at a crime scene.

Presumptive tests for blood are usually based on haemoglobin's ability to catalyse the oxidation of certain reagents. The most common reagent is Hydrogen Peroxide Solution (H202(aq)). These tests also commonly use reagents that change colour on oxidation. Phenolphthalein which goes from colourless to pink when oxidised.

To test a stain, a small circular piece of absorbant card (diameter=25mm) is folded twice to form a point. A small amount of the stain being tested is then scraped onto this point. a drop of phenophlthalein is added by a drop of hydrogen peroxide, with the bright pink colour showing a positive result. It's important to note that this test does not differentiate between human blood and other kinds of blood. 

However in some situations the colour change tests are not appropriate. Maybe the crime scene has been scrubbed clean, or you need the blood 'intact' for DNA profiling. This is were luminol comes into play. In the luminol test an alkaline solution containing both luminol and an oxidising agent (for example Hydrogen Peroxide), is sprayed onto the area being tested. Where blood is present luminol is catalytically oxidised and produces a distinct glow. 

Luminol before and after

I hope this has helped to illuminate the basics of blood identification. Until next time! Over and out!

Immunoassays in the Analysis of Drugs and Poisons

I'm back, and this time we're going to be looking at one of the most powerful techniques in the toxicologists arsenal, the immunoassay. No matter the sample size or the specificity or broadness of molecules your trying to ferret out, the immunoassay is both practical, accurate and relatively cheap.

In forensics the immunoassays are used mainly for identifying the presence of drugs in or poisons in both ante and post mortem samples. Immunoassays can be highly specific, picking out only a single compound (e.g. mephamphetamine), or a group of compounds. Immunoassays are useful because not only are they highly sensitive, they are also able to analyse large samples relatively quickly and require no preliminary extraction phase.

Immunoassay relies on the antibody-antigen reaction, which occurs in the mammalian immune system. When an antigen enters the body, an antibody specific to it i produced. The antibody binds with the antigen forming an antigen-antibody complex. In immunoassays specific antibodies are used to detect and measure the level of the analyte of interest.

In immunoassays, fixed quantities of the specific antibody to the analyte and a labelled form of that sample are added to the sample being tested. The labelled molecules and the analyte (if it's present) will competitively attempt to bind with the antibodies, in inversely proportional numbers. So you can measure the reduction in concentration of the labelled poison to work out the concentration of a drug in the sample. 

However in some types of immunoassay, it isn't possible to distinguish between the labelled poison bound to the antibody and that remaining free in solution, so physical separation of the two phases is required before measurement. These are known as heterogenous immunoassays (for example radioimmunoassay). However immunoassays where this is not necessary are called homogenous immunoassays (for example fluorescence polarisation immunoassay). These two examples are actually both very good examples of immunoassays. 

Radioimmunoassays: In radioimmunoassays, the poisonous molecules are labelled with an appropriate radioisotope (for example iodine-125) and the radioactivity is measured. The concentration of unlabelled poison molecules in the test mixture is determined by reference to a standard curve. This graph is produced by adding increasing concentrations of poison to a fixed quantity of labelled poison and antibodies. 

Fluorescence Polarised Immunoassays: A suitable fluorescent substance is used to label the analyte. It's possible to distinguish between labelled analytes bound to the antibody and analytes free in solution. This is because the former emits polarised fluorescence, but the latter generates unpolarised fluorescence.

Thanks for reading, over and out!