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Forensics Research: Blood, Science, and Social Justice

Forensics Research: Blood, Science, and Social Justice

“Crime is common. Logic is rare. Therefore it is upon the logic rather than upon the crime that you should dwell.” 
-- Sherlock Holmes (The Adventure of the Copper Beeches, by Arthur Conan Doyle)

Forensics is a glorified area of science. This in large part owes to TV and movies. For everyday folks, the word “forensics” conjures up images of murder scenes, flashing police lights, fingerprints, and telltale bloody weapons. The realities are a bit less glamorous, but this field has been highly rewarding for me because of the many challenges and discoveries it presents.

I got into forensics because I grew up watching detective movies and was fascinated by Sherlock Holmes’ intellect. I knew movies weren’t reality and so I pursued the science behind it. I studied biochemistry and explored different types of research within that vast field.

Forensic sciences was more appealing to me because it seemed to offer a quick turnaround between academic research for a new method and application of such a method in real forensic cases.

It may very well excite you, too. So in this article, I’ll go into the nitty-gritty of the academic and scientific background you’ll need to get in and get ahead. Perhaps I can pique your interest, like a murder mystery.

What Actually is Forensics?

Forensics incorporates a number of scientific disciplines and applies these to serve justice. Most disciplines of forensics require at least a bachelor’s degree in a relevant field; including accounting, digital and multimedia sciences, photography, and radiology.

Crime scene investigators typically have a high school diploma and specific training in such investigation, either as a part of a university or vocational degree.

Edanz - Forensics ResearchForensic fields that require advanced knowledge commonly acquired in a master’s or PhD program include anthropology, odontology (study of the structure and diseases of teeth), engineering, pathology (both medical doctors and nurses), psychiatry, jurisprudence, toxicology, and criminalistics. Biologists, chemists, and those with a degree in similar disciplines will typically work in toxicology or criminalistics laboratories.

Toxicology focuses on drug detection through use of instruments capable of detecting either illicit drugs or their metabolites. This detection is usually done via bodily fluids (saliva, blood, or urine). Advanced degrees in chemistry, toxicology, and/or pharmacology are recommended and involve laboratory research. Research in toxicology typically focuses on improving methods to detect drugs that are abused. Improving these methods relies on obtaining faster or more sensitive results.

New methods are also researched for detecting newly available synthetic drugs, which are increasingly an element of criminal activity and accidental death.

Criminalistics typically includes those with advanced degrees in chemistry, molecular biology, genetics, biology, and similar. Those with chemistry-related degrees can perform tasks regarding the analysis of trace gunshot residue, explosives, hairs, and fibers.

One of the main goals of research in forensic sciences is to improve current methods for sensitivity, speed, or cost. For example, recently, paper chips able to detect explosive residues with high sensitivity has been described, such as in this TEDx Talk. These chips are highly portable and can be used with minimal training, making them ideal for troops deployed to conflict zones.

Those with degrees more related to biology will typically analyze biological fluids and DNA extracted from such evidence. I’ll touch specifically on DNA a bit later on.

More information regarding different fields of forensics and degrees required can be found through the American Academy of Forensic Sciences.

With that background, let’s take a deeper dive into the specific sciences within forensics, set alongside a real-life application of them.

The Modern-day Forensic Scientist

When DNA sequences were initially studied with the purpose of identifying individuals, it brought a colossal revolution in forensic sciences. By comparing lengths of specific loci, it became possible to determine if two DNA samples originated from the same individual.

Nowadays, forensic scientists continue seeking ways to put DNA to work for justice; either by pursuing faster, newer, and more sensitive methods or by studying if DNA can allow the determination of physical features. The high demand reduces the time between establishing a new methodology and using it to solve crimes, which is a strong motivator for doing research with forensic applications every day.

Imagine this. You’re watching a new crime drama. In the show a violent murder has occurred, the police arrive at the scene and tape it off. Only detectives and crime scene unit can get inside this area. DNA samples are collected and sent to a lab.

In this case, the police have run out of other investigative leads, there are no eyewitnesses, and the only hope is that DNA from the drop of blood near the kitchen knives matches someone in the database.

The DNA laboratory finally sends the results … no match between the evidence DNA and the database. It’s a dead end for the investigation. As time goes by, other cases get higher priority with the police and the case goes cold. The end.

That TV show would probably be canceled after one episode, right?

Real life, however, often differs from what we see on TV, but this outcome was the reality for several violent crimes investigated in the United States. In fact, only about 59% of violent murder crimes in the US were solved in 2016.

How did the forensic laboratory analyze the DNA from the blood?

The suspect must have cut himself when he grabbed the knife, and didn’t notice. The drops of blood collected at the scene arrived at the forensic laboratory for analysis. A DNA analyst extracted the genomic DNA from the blood and quantified it.

Generally speaking, a small drop of blood is sufficient to obtain a complete DNA profile, yielding more than 1 ng/μL (ng: nanogram; one billionth [10−9] of a gram) of extracted DNA. If the DNA is not fragmented and is from a single source (not a mixture of people) 1 ng of DNA can be sufficient to get a complete profile. The quality and presence of mixtures can only be determined by using some of the DNA extracted, and sometimes there isn’t enough material to repeat any of the procedures.Edanz - Forensics Research

Check here for a visual.

This time, they’re lucky. There is DNA leftover in case they need to repeat or perform other tests.

In the US from 1998 to 2016, a profile was considered to be complete if it showed results for 13 specific loci. In 2017, the FBI increased the number to 20. Over the years, forensic researchers chose the core loci for human identification from non-coding regions of DNA, called microsatellite regions. Those loci are also called short tandem repeats (STRs) and consist of a sequence of 2–7 nucleotides repeated over and over.

The analyst continues the procedure by amplifying the extracted DNA using polymerase chain reaction (PCR) with 20 primer pairs that flank the STRs (multiplex PCR).

These multiplex PCR reactions are considered more efficient because they avoid wasting DNA. Because STRs are located in non-coding regions of DNA, changes in the number of repeats are common in the human population without effect on gene products.

The sequence of genomic DNA is unique for each individual (except for monozygotic twins), and results from a combination of DNA from the progenitors. As a result, the same person may have different sizes of the same STR in each chromosome – one inherited from the mother and one from the father. The range of possible numbers of repeats per STR (alleles) is known and the size varies from 100 to 500 base pairs. A size ladder also called a “size standard” is used in the analysis to convert base pair size to repeat number.

The multiplex PCR is complete and the analyst can now separate the amplified fragments by size.

The separation of amplified products is done by capillary electrophoresis (CE), which works similarly to a polyacrylamide gel. After loading the PCR product in a small tube, the analyst turns the instrument on and voltage is applied to the capillary tube filled with a resolving polymer. As a result of voltage, the DNA migrates inside the capillary and its size is detected and registered in a computer connected to the CE.

Each tube has the amplified fragments of 20 STRs and some alleles of one STR can have the same size, overlapping with other alleles of other STRs. To separate overlapped alleles, primers used in multiplex PCR are fluorescently tagged. STRs that have alleles with the same size will have primers with different fluorescent tags and can therefore be separated by color.

The analyst starts to see peaks in the computer screen, which correspond to each allele. When the analysis ends, with the click of a button, the analyst anxiously instructs the software to include peaks of the same color in separate charts and to use the size standard to attribute repeat number to each peaks according to their base-pair length.

The final profile is ready! The analyst searches the DNA database to determine if there is a match between the blood found at the crime scene and any profile previously deposited in the database. Unfortunately, no luck! The profile from the blood evidence collected at the crime scene does not match any of the profiles in the database.

The forensic analyst writes the report describing his findings. In his desk is a stack of articles describing how in research laboratories scientists are using DNA on other ways beyond STR sizes to help solve crimes. He realizes that one day he may be using some of that research to help solve situations like this one, where no suspect has been found not even in the databases. For now, he can only write his report and hope that one day this profile now entered in the database may help solve other crime.

Maybe at that point, other evidence will point to a suspect whose profile matches this and both crimes can be solved.

The analyst sends the report to the detectives. Reading the report was, alas, a big disappointment. No match with the database and no witnesses, cameras or other leads, it seems like it’s the end of this police investigation.

However, one of the detectives remembers something he saw in a conference just last month. Some group somewhere presented their research showing how they can produce a computer-generated sketch based on DNA. He searches his notes and found the contact information. It has something to do with SNPs, he read. He shows this to his superior and they decide to try it.

Single Nucleotide Polymorphisms (SNPs)

SNPs are variations on the human genome that consist in single nucleotide differences. For example, one allele of the same gene can have an adenine at a specific location, whereas the other allele has a cytosine. It has been established that certain SNPs are more common in people with similar ancestry. In the United States, some companies have commercialized that knowledge-providing services for the general public that allow them to know more about their ancestors and origins.

In forensic sciences, SNPs have been widely explored in two main ways, one as a replacement for STRs, and second as a genetic marker for specific physical traits. The idea behind using SNPs instead of STRs relies in the fact that SNPs are abundant in the genome and smaller in size, making them ideal loci to obtain profiles from samples with little or degraded DNA. However, a large number of SNPs would have to be analyzed to obtain a statistical power comparable with STRs.

As a genetic marker for physical traits, specific SNPs can code for eye color, skin complexion, and hair color. Forensic research has studied these differences to determine phenotypes from DNA. If DNA is left at a crime scene and no suspect has been found, the DNA sample can be analyzed to determine which SNP variations are present.

Facial features are more complex than eye or hair color, therefore many years of research were necessary to determine them from SNPs. In 2014, it was demonstrated how computer models can predict facial shapes from SNP analysis.

Gathering all the scientific data found to date allowed the development of a software that provides a computer-generated sketch based on SNPs. Thanks to this tool, several cases have already been solved including the violent deaths of a couple in North Carolina.

Check here for a visual.

The same technology can be used in other types of police investigations, such as missing persons. Occasionally, bones or partial human remains are found several years after the disappearance of a loved one. If enough DNA is available from the remains, the computer-generated reconstruction can point investigators toward the identity of the victim, thus providing the family with some form of closure.

However, physical features coded in SNPs are not age-dependent; therefore, SNPs alone cannot provide information related to a person’s age. For that reason, the computer-generated sketch may be of a 25-year-old, whereas the suspect may be 50 years old and looks quite different. Epigenetics may provide a solution to that gap by offering information on age prediction based on DNA methylation.

DNA Methylation Can Predict Chronological Age

DNA methylation is a form of epigenetic variation and has been researched for several years in clinical studies, mostly related to aging, cancer, and other pathologies. Recently, forensic scientists have looked at DNA methylation to determine chronological age from DNA samples. The sequence of DNA provides the code that allows protein synthesis, but it does not dictate in which cells those proteins are formed. Gene expression is not a product of DNA sequence; otherwise, two monozygotic twins would look alike even at older ages, and that is not always the case.

Forensics - Indian Twins
photo credit: Perumalnadar https://commons.wikimedia.org/wiki/File:Indian_Twins.jpg

DNA methylation refers to the presence of methyl groups in the 5′ carbon of specific cytosines in the genome. As the majority of those cytosines are followed by a guanine, they’re called CpGs. Changes in DNA methylation such as the loss or gain of a methyl group in a CpGs can lead to changes in gene expression. The methyl groups are covalently attached to the cytosines, therefore any changes in methylation occur in biochemical reactions as a result of environmental factors including pollution and exposure to toxics or simply as a result of lifestyle choices, such as exercise and diet.

DNA methylation can be determined from the same DNA extracted from the evidence collected at the crime scene. Before PCR, the DNA is bisulfite-modified which converts any unmethylated cytosine into uracil, while maintaining methylated cytosines. PCR follows by amplifying the CpGs of interest and copying uracils as thymines and methylated cytosines as cytosines. Sequencing the PCR product allows determining the percentage of methylation for each CpG.

Researchers have identified specific sets of individual CpGs that quantified together allow the determination of biological age. DNA methylation age calculators have been published online for the use of research groups. Some age calculators have been developed to predict age from the results of mass parallel sequencing technologies that provide quantification of DNA methylation for over 27,000 or 450,000 CpGs, and others were developed to be used with only a few CpGs.

To date, the biggest challenge in using this technology for forensic cases relies in the amount of genomic DNA necessary to quantify DNA methylation. Since the bisulfite reaction fragments the DNA, most technologies require hundreds of nanograms of genomic DNA as start material, which is not compatible with forensic applications. Researching teams in forensic laboratories have been focusing in optimizing the existing technology to quantify DNA methylation from low amounts of DNA.

DNA typing technologies such as STR analysis have revolutionized forensic sciences and allowed the conviction of many guilty suspects and exoneration of many innocent ones. However, unveiling the complexity of DNA mechanisms does not stop with knowing the length of STRs. Using SNPs, police is already able to get a sketch of suspects, increasing the number of convictions and identifying missing persons from skeletal remains. In the near future, the use of DNA methylation may further help to determine the age of a suspect based on the DNA he inadvertently left behind.

This means research must continue.

Present Roles and Needs in Forensics Research

Despite the technological advances, methods to identify body fluids are still based on protein quantification and are not confirmatory. Most of the identification methods lack specificity and can show positive results for several body fluids. Such presumptive methods can therefore be disputed in court. Researchers have focused on quantifying messenger RNA or micro RNA transcripts to identify body fluids, however DNA may be a better candidate for the task. Specific gene expression occurs as a result of DNA methylation patterns in specific CpGs in the genome. Determining which CpGs are methylated or unmethylated for forensically relevant body fluids can constitute a confirmatory method accepted in court.

The CSI effect caused jurors to expect DNA samples and conclusive results from such samples. As a result, crime scene investigators and police officers greatly increased the number of evidence items collected from a crime scene. The result is that more samples are submitted to a DNA laboratory for analysis. Similarly, mass disasters yield large numbers of samples to analyze, and little time to do so. As a result for the high demand in increased throughput, forensic researchers have been focused on developing a rapid and direct method where DNA extraction is avoided and the PCR amplification as well as analysis takes hours rather than days.

Also related to the increased number of evidence items to be processed in little time and/or at low cost, is the backlog of rape kits still waiting to be analyzed in the United States. Evidence collected in rape cases isn’t always tested, either because the suspect confessed, the victim recanted, or a decision was made not to prosecute. As a result, millions of rape kits have been stored and never analyzed. The increase of DNA profiles in databases suggested rapists typically would repeat the crime. For that reason, an effort was made to analyze all the rape kits stored, regardless of the resolution of the case. By doing so, profiles of rapists are available for comparison, even if at first they do not correspond with a known suspect. However, when a suspect is found, and if considered guilty, that may mean the end of several rape cases, some of which may be added to the prosecution plea. Despite this, the laboratories don’t always have the personnel, equipment, or funds to deal with the reduction of the rape kit backlog. Additionally, rape samples are typically mixed samples with a high ratio of female to male DNA. Research laboratories have been focusing on developing improved methods able to separate the male from the female cells prior to DNA extraction. Such improvement results in lower costs or reduced time of analysis per sample.

Generally speaking, forensic research searches for faster, cheaper, and more sensitive methods. Technological development relies on the cooperation of multiple disciplines to answer a common question and this is how, even in forensics, a multidisciplinary teamwork is necessary for the evolution of the field and the search for justice.

Even though the daily reality of a forensic analyst or researcher may not be as glamorous as seen on TV shows, the work is crucial for establishing the circumstances of a crime. Without the unbiased eye of a scientist, or the technologies developed by science, numerous criminals would stay free and have the opportunity to make more victims. Forensic sciences certainly comprise stimulant research looking at emerging technologies and applying them to solve crimes; or use already known methods, improving them, to better identify or detect evidence of a crime. Overall, forensic sciences welcome the contribution of expert scientists from around the world and who have an interest in improving the justice system, which ultimately helps in providing closure for families and for victims of crimes.

Joana Antunes graduated with a masters in biochemistry in her native country of Portugal. She then decided to pursue a PhD with a research focus in forensic sciences, specifically DNA. In 2011 she joined the University in Florida, as she feels the United States is a country highly focused in her field of choice. Dr. Antunes is now dedicated to furthering her knowledge in the field and disseminating research in forensic sciences.

Joana Antunes