A Closer Look: DNA Profiling in Forensic Investigations

“It is impossible for a criminal to act, especially considering the intensity of a crime, without leaving traces of his presence.” Doctor Edmond Locard’s exchange principle is perhaps the earliest description of trace evidence, small fragments of physical evidence transferred when objects come into contact. Trace evidence includes everything from clothing fibers, soil, and gunshot residue, to blood and semen, which can be used to link a suspect to a crime scene or with another individual. Forensic biologists focus primarily on bodily fluids, swabbing items of evidence for possible contact DNA with the goal of obtaining a DNA profile. Since the discovery of DNA fingerprinting in 1984, the evolution of instrumentation and software is creating new possibilities for forensic DNA typing. Still, maintaining evidence integrity and minimizing the risk of contamination is top priority to ensure accurate analyses.

Unique discovery becomes a powerful forensic tool

Dr. Alec Jeffreys is considered the founding father of DNA fingerprinting, but has described his discovery, like so many other scientific discoveries, as a combination of curiosity and accident. A geneticist at the University of Leicester, Jeffreys used restriction fragment length polymorphism (RFLP) to study genetic variation between individuals. The realization that single nucleotide polymorphisms (SNPs) showed little variation between individuals led him to search for short tandem repeat (STR) DNA sequences called minisatellites. Through the isolation of minisatellites, and the discovery of a short, shared sequence, the isolation of variable tandem repeat markers became possible. In 1984, in the process of testing the ability of a hybridization probe to detect multiple minisatellites in human DNA, Jeffreys unintentionally created the first DNA fingerprint. As these DNA profiles are unique to the individual, the technique was subsequently developed into a powerful forensic tool.

Genetic profiling is a variation of the DNA fingerprinting technique developed for forensic purposes. The high profile “Pitchfork Case” exemplifies its early use and significance as an investigatory tool. In the early 1980s, two young women were murdered in the village of Narborough, Leicestershire, approximately two and a half years apart. The similarities between the murders led authorities to believe they were the work of a serial killer, and a 17-year-old boy was eventually arrested. Genetic profiling exculpated the young man, and led to the identification and arrest of Colin Pitchfork.

The evolution of genetic profiling techniques

DNA fingerprinting was achieved with a combination of restriction enzyme digestion and Southern blot analysis. A major caveat of this approach is that it necessitates large, high-quality DNA samples, and forensic samples are often minute and degraded.

Some of these challenges were overcome with the emergence of polymerase chain reaction (PCR) technology. PCR uses sequence-specific primers to target and amplify STRs, followed by electrophoretic separation and detection. While this approach offers greater sensitivity compared to earlier techniques, STR targeting is still limited by small, highly degraded DNA fragments.

Single nucleotide polymorphisms (SNPs) are individual base substitutions, insertions, or deletions at single positions within the genome. They are an attractive alternative to STRs given their high abundance, low mutation rate, short amplicon length, and presence in small fragments of degraded DNA. As such, novel genetic profiling technologies are being developed to target SNPs. However, it is unlikely that SNPs will replace STRs in criminal investigations in the near future, as many large, national databases are STR-based.

The Combined DNA Index System (CODIS) is one such national database, consisting of 13 core STR loci. CODIS enables federal, state, and local forensic labs to exchange and compare DNA profiles electronically, to link serial violet crimes and known offenders. When evidence is collected from a crime scene or victim, a DNA profile of the suspect is created and can be searched against the state database of convicted offenders and arrestee profiles. If a match is confirmed, the identity of the suspected perpetrator may be obtained. Similarly, if the DNA profile from the evidence matches another crime scene DNA profile, two crimes may be linked together.

Forensics in action

India Henry is a DNA analyst at the Houston Forensic Science Center (HFSC), where she interprets DNA profiles and issues reports. She also works as a DNA technician in the laboratory where she performs extraction, quantification, amplification, and capillary electrophoresis.

“We identify bodily fluids, primarily semen and blood, and swab items of evidence for possible contact DNA in hopes of obtaining a DNA profile. We will then compare the unknown evidence sample to a known reference sample,” explains Henry.

Henry and her colleagues employ numerous different methods, largely determined by the type of case and the evidence sample. For example, when working with sexual assault kits, the swabs are not chemically tested, so Henry and her colleagues will take a portion of each swab and place it in a tube for DNA processing. “We do this to preserve as much sample as possible, since DNA analysis is very sensitive,” she explains.

Other pieces of evidence are much larger, such as clothing or bedding. Henry explains that the team first uses an alternate light source to detect possible semen and blood on the evidence, and capture photographs of fluorescence and infrared light absorbency. Possible semen stains will fluoresce under the ultraviolet light, whereas infrared light enables the detection of bloodstains. “Infrared light is especially helpful for dark items, such as a black hoodie, where it is difficult to see the bloodstains,” says Henry. “We then follow up with a chemical test, acid phosphatase (AP) for semen, and phenolphthalein (PHT) for blood. If we can visually see red-brown staining on an item, we will perform a PHT test, without the use of an alternate light source.”

The DNA processing workflow at the HFSC is entirely automated. “We use robotic instrumentation to efficiently process many samples at once,” says Henry. Following sample extraction, an automated liquid handling device performs quantification and amplification plate set-ups, and is used to set up capillary electrophoresis plates prior to separation and detection.

There are also many portable instruments and analyzers that may be used outside the lab at various crime scenes. “The benefit would be that it triages the evidence samples, lessening the amount of evidence to collect and send to the lab,” explains Henry. She adds that these instruments are useful, provided “they are validated, and the analysts are properly trained to use them and interpret the results.”

Protecting samples

As technology advances to support more sensitive forensic analyses, proper sample collection, transportation, and storage remain critical to ensure evidence integrity and minimize the risk of contamination. According to Henry, “maintaining the integrity of the evidence is the responsibility of anyone who comes in contact with the evidence. From collection to final storage, the evidence is tracked using a chain of custody.”

Sample collection is often performed by swabbing the item of evidence. Henry notes that it is imperative the swab does not come into contact with anything other than the item and area of interest, and that it is properly stored to preserve any potential DNA. “The swabs are never stored wet, which can lead to mold growth,” explains Henry. “They are dried in a swab box dryer prior to packaging in a glassine envelope. Paper storage options, rather than plastic are used to prevent moisture buildup and reduce the possibility of mold growth.” The purified DNA is also eluted into TE buffer, containing Tris and EDTA (ethylenediaminetetraacetic acid) to promote a stable environment, and frozen for preservation.

Contamination can have significant consequences for DNA analysis. The team at HFSC takes many precautions against contamination, and has several steps within their processes to check for contamination. “Our analysts wear full PPE (personal protective equipment) and decontaminate all work surfaces prior to use, in between evidence items and at the conclusion of analysis,” Henry says. “We also only handle one item of evidence at a time and maintain a staff database of DNA profiles we utilize to crosscheck our results prior to issuing a report to ensure one of our staff members did not contaminate the evidence.”

As instrumentation becomes increasingly sensitive, labs must take extra precautions to prevent contamination. “As the instrumentation becomes more sensitive, smaller amounts of DNA are detected, some of which may be extraneous. We also process our samples from low-level (contact/touch) to high-level (blood, references) to avoid cross-contamination between samples,” explains Henry.

Looking to the future

There have been significant technological advances in forensic science since the discovery of the first DNA fingerprint in the early 1980s. In addition to high-throughput instruments and automation that enable more rapid and efficient assays, more sophisticated software has the potential to enhance analyses. “Currently, we have transitioned to using a probabilistic genotyping software, to aid in the interpretation of DNA mixtures,” says Henry, “this software makes better use of the data and allows some data to be interpreted that may have previously been deemed unsuitable for analysis.” For example, the software supports analysis of complex mixtures with DNA from multiple contributors, and compares the DNA profiles against a database.

These, and numerous other developments in instrumentation and software will support forensic biologists in their efforts to create accurate DNA profiles, with significant implications for the criminal justice community.