Crime scene investigation is about to get more affordable and efficient, as researchers from the National Center for Forensic Science in Orlando, Florida, have found a cheaper and faster method to identify a wide range of body fluids.
The most prevalent methods to analyse biological evidence at a crime scene have been unable to identify body fluids including vaginal secretions, menstrual blood or skin. “There are also limited, and largely only presumptive tests available for the identification of saliva as well,” explained Dr. Erin Hanson who conducted the study.
Dr. Hanson and Dr. Jack Ballantyne are co-authors of the study, published in F1000Research, which introduces a new method of body fluid identification that relies not on proteins, but on another unique biomarker: messenger RNA. “All body fluids and tissues can be easily identified using the new mRNA profiling and the developed HRM assays,” said the researchers.
Chasing the Clues from mRNA
Identifying the nature of biological material present on evidence from a crime scene can shed light on the circumstances. Biological materials in the form of bodily fluids such as blood, semen, and saliva, or tissues such as skin, each contain unique proteins that can identify a particular sample. Unfortunately, protein detection techniques are variable and labor intensive. They’re also imperfect, as they’re not able to conclusively detect all bodily fluids and tissues.
Besides proteins, body fluids and tissues also contain messenger Ribo Nucleic Acid (mRNA). This genetic material, a sort of “carbon copy” of DNA and the precursor to a protein, contains biomarkers that – like proteins – are specific for the type of tissues or body fluids present. The biomarkers hidden in the mRNA can determine which fluids are present in a mixture and may help investigators reconstruct the crime scene events.
To correctly identify the sometimes complex mixtures of body fluids and tissues found on an item or a person, forensic analysis relies on very specific and sensitive assays. Often there is only a limited amount of sample available, and it may not always be pristine, which makes analysis difficult. This is another problem that the mRNA detection method seems to overcome, as mRNA biomarkers can be successfully detected even when the sample is partially degraded or has been affected by environmental factors.
RNA profiling techniques
Using mRNA detection to identify body fluids on evidentiary items is not a new technology, but the techniques that are currently available in this area are both expensive and time consuming. The most commonly used techniques typically involve capillary electrophoresis (CE); a separation technique based on the differences in migration speed of analyzed substances within an applied electric field, or quantitative real-time polymerase chain reaction (qRT-PCR). PCR is also commonly used in forensic analysis of DNA samples.
To measure mRNA with this method, the mRNA is first reverse-transcribed into DNA using a reverse transcriptase enzyme. Next, the small amounts of DNA are multiplied by using a DNA template consisting of at least one pair of specific primers (DNA fragments that bind to the piece of DNA one is looking to identify), deoxyribonucleotides (DNA building blocks), a buffer solution and a DNA polymerase (enzyme capable of building DNA). A fluorescent probe that binds the target sequence of DNA is added to measure the generation rate for the specific DNA product.
Both CE and qRT-PCR require the use of expensive fluorescently labelled primers or probes for detection and only three or four markers at the time can be detected with qRT-PCR assays, as each marker requires a different fluorescent dye. This means that to consider a larger amount of markers in a sample, laboratory analysts will need to perform multiple PCR reactions, driving up both the costs of the investigation, as well as precious time and labour.
High Resolution Melt RNA profiling
To simplify mRNA profiling assays, and to reduce the time and costs of analysis, the two researchers investigated possible alternative assays for the identification of common forensically relevant biological fluids and tissues. They were successful, and developed a High Resolution Melt (HRM) assay, which requires only unlabeled PCR primers and a single fluorescent dye.
“As its name suggests, HRM is a technique that permits the identification of specific PCR products (i.e. amplicons) by their melting temperature (Tm),” said Dr. Ballantyne. “An amplicon’s precise melting temperature is dependent upon its sequence, length and the ionic strength of its environment.”
Since HRM assays only require the use of unlabeled PCR primers and a single fluorescent dye, the technique is cheap compared to existing methods for mRNA analysis.
The development of multiplex assays enables multiple types of tissues to be detected per reaction, saving both time and money. After the fluorescent dye is added to saturation, the DNA is amplified. When the amplified product is then melted slowly, the DNA strands separate and release the bound fluorescent dye into solution. By measuring the fluorescence with respect to the temperature, a distinct and characteristic melting curve is obtained making it possible to analyse several amplicons in the same tube.
Developing such multiplex assays however, is quite a challenging task, as the biomarkers for different body fluids or tissues may have overlapping melting curves. Even amplicons of different size may have similar resulting melting temperatures, depending on the amplicon sequences. To solve this problem, Hanson and Ballantyne tested several different biomarkers to develop an assay that could identify a mixture of 6 tissue and body fluids at one time. With their method they have now been able to successfully identify biomarkers for skin, blood, menstrual blood, saliva, vaginal secretions and semen by the presence of their distinct melt peak, even when these tissues and body fluids are present as a mixture.
Another difficulty in identifying body fluids and tissues in forensic investigations is the often limited availability of the sample. Therefore, the HRM assay needs to be very sensitive to be able to identify even the smallest trace of a body fluid. In their study, Hanson and Ballantyne show that their assay could correctly identify the presence of skin cells on a swab of a computer mouse, showing that correct identifications can be made on objects that were touched or handled at a crime scene.
The development of compatible multiplex RNA biomarkers makes it possible for laboratories to create a customized HRM assay to suit their specific needs. Each HRM assay takes about two hours to perform. This is similar to the time it takes to perform a qRT-PCR, but because more samples can be tested per reaction, the HRM assay saves time overall.
At the moment, Hanson and Ballantyne have only tested their new method for body fluid identification in a laboratory setting, but after further optimization and validation the HRM assay may soon be used for forensic casework and reduce both the costs and time of crime scene investigation.
“The developed HRM assays incorporate the specificity and sensitivity of RNA profiling for body fluid identification, but provide a rapid, and relatively inexpensive, means by which to identify all forensically relevant fluids and tissues,” Dr. Hanson concluded. “Definitive body fluid identification helps provide additional context to a DNA profile, indicating a behavioral activity that led up to its deposition at the crime scene.”