By P Koupparis
29 November 2002

Britain set up the world's first national DNA database at Birmingham in 1995 under the auspices of the Forensic Science Service. The original specification was based on six STR loci. After several false-match scandals the system was upgraded to ten loci in 1999.

UK legislation enables the police to collect and database DNA profiles from suspects arrested for a broad range of offences, even if charges fail to materialise or an accused is subsequently acquitted. Some people consider that to be an intolerable infringement of their rights, although, until recently, there was precious little they could do about it.

The logic underlying the DNA profiling of criminals depends on two assumptions: that criminals will continue to leave traces of their DNA at crime scenes despite knowing that a national offender database exists and that suspects will always donate genuine profiles to the database. Those assumptions may not always hold true: human intelligence is not vested exclusively in forensic scientists and police officers.

Most professional villains already know that a dilute solution of household bleach or hydrogen peroxide will destroy the DNA molecules within most types of trace evidence left at a crime scene. The criminal fraternity has now discovered how to proactively subvert DNA profiling technology to its advantage. The weakness most easily exploited relies on obtaining the PCR amplicon of a DNA profile from a discarded cigarette butt. Developing a profile from an item discarded in a public place is not illegal in the UK at the time of writing.

Whilst the science and technology surrounding forensic DNA identification is beyond the reach of most private citizens, almost anyone can acquire the simple equipment and easy-to-use PCR kits that produce amplicons. An amplicon is stable for several weeks at room temperature (when shielded from direct sunlight and UV) and will stay fresh for months in a refrigerator and years in a freezer.

Inside a PCR cocktail, billions of tiny primer molecules zip about in a frenetic jig driven by thermal kinetic energy. The primers repeatedly bump into the massive 'template' DNA molecules until some of them happen to line up and bind with their complementary base sequences (called targets). That jiggling about is called 'contention'.

Small molecules, such as primers, delineated alleles and polymerase enzymes are far more agile than template DNA molecules, which are the longest known to science. Genomic DNA is made up of a pair of anti-parallel molecular strands that couple together with a slight asymmetry that causes each strand to twist around its partner and the resulting double-strand to super-coil on itself. Each of the 3-billion base pair long template DNA molecules have to unravel and disassociate (a process known as denaturing) before contention with PCR primers can take place.

Commercial PCR reactions denature template DNA by heating to 94 °C. The primers bind to their targets when the solution is cooled to 60 °C and are extended at 72 °C. The first two thermal cycles are the most crucial since they delineate the allele molecules that are amplified by the next 20 to 30 cycles.

It is only by the end of the third cycle that fully delineated copies of the target alleles exist within the solution. With each cycle, the number of primers decreases as the number of copied allele molecules increases. Because binding and extension are driven by random contention those processes favour small, fast-moving molecules. Thus the reaction amplifies the smaller and ever more abundant copy allele molecules rather than the original template DNA targets.

After three hours a PCR reaction has run its course and is then known as an amplicon: a highly concentrated solution of target allele molecules.

The amplicon is the most potent source of cross-contamination in the forensic laboratory. A single, microscopic droplet contains billions of PCR-favoured allele molecules. It is such a powerful contaminant that modern laboratories are designed to progress jobs through one-way departments that isolate DNA specimen extraction and preparation areas from those where amplicons are created and handled.

The amplicon is the key to subverting DNA profiling technology. The tiniest trace of amplicon will contaminate a fresh PCR cocktail and swamp any template DNA with its own ready-to-go alleles.

Police use three principal DNA specimen collection methods: the cheek swab (also known as a buccal swab), hair extraction with the root-ball attached and blood drawn from a vein.

Those three collection methods can be compromised with relative ease when the specimens are destined for profiling with a multiplex PCR kit, as most are these days.

For some time now, the DNA profiles of countless innocent people may have been archived on the national DNA database by crafty villains. Not only by salting crime scenes with other people's discarded cigarette butts, but also by submitting false DNA profiles to the database.

The simplest way to beat the cheek swab is to use the amplicon of another person's DNA profile as a mouthwash shortly before swabbing. However, that may not always be practical. Under arrest conditions, a cheek swab may be compromised with a home-made confection incorporating someone else's amplicon. Spiking a hot, saturated sugar solution with an amplicon produces a mixture that can be set into the shape of a boiled sweet or lozenge. The sweet can be protected with a thin plastic coating that allows it to stay in the mouth indefinitely until it is crunched, whereupon it releases billions of PCR-subverting molecules.

The rogue allele molecules will contaminate the cheek swab and pass through to the PCR stage of the offender profiling process, where, because of their small size and huge numbers, they are preferentially amplified by the polymerase chain reaction.

Applying a spiked oil-based dressing to the hair and scalp prior to sampling can compromise the hair root specimens collected by some police forces. An amplicon emulsified with 250 grams of a hair dressing like Brylcreem can remain active for several weeks when stored in a cool, dark place.

A person's blood can be compromised for at least several hours, perhaps longer, by the intravenous injection of a diluted, filtered amplicon before blood is drawn for analysis. The effect is temporary and believed to be harmless.

In each case, a PCR-based analysis of the compromised specimen will yield the DNA profile of another person instead of the suspect's - all else being equal. The substitute profile should be from an individual of the same sex and preferably the same ethnic group as the suspect. The laboratory protocols currently in use will not detect the deception.

The technique works most effectively when the PCR kits used to produce the subverting and reporting amplicons are of the same type. In the UK, criminal profiling is most likely to be carried out with the multiplex SGM Plus kit manufactured by PE Applied BioSystems. In the US, the FBI's thirteen-STR profiles can be developed by more than one type of kit, in which case amplicons of a profile developed with the most likely kits can be combined to cover the possibilities.

When arrested in the UK, a professional villain can now assume a false identity and receive the benefits of a first-time offender, often including bail, which can be skipped with impunity while the Home Office continues to deny that DNA profiling is vulnerable to corruption. Conventional fingerprints are not automatically cross-referenced with DNA profiles, for reasons that are now obvious.

For those who can harvest allele variations from public ashtrays, it is possible to create a customised masking profile in which the substitute alleles are one repeat-length longer than the subject's profile. The PCR's stutter bands will mask some level of template DNA amplification, allowing such an amplicon to maintain deception at very low concentrations of specimen contamination.

If the integrity of a national criminal DNA database can be undermined with something as simple as a spiked sweet, how many potential injustices lie hidden within that database?

© Copyright P Koupparis 2002. All Rights Reserved.

A comprehensive web resource for PCR/STR technology is at: http://www.cstl.nist.gov/biotech/strbase/

STR reference book: Forensic DNA Typing

DNA Evidence: is it safe to convict?

DNA Evidence: science or smoke and mirrors?