The Stories in the Bones: DNA Forensic Analysis 20 Years after 9/11

September 11, 2001 is the day that will live in infamy for my generation. On that beautiful late summer day, I was at my desk working on the Fall issue of Neural Notes magazine when a colleague learned of the first plane hitting the World Trade Center. As the morning wore on, we learned quickly that it wasn’t just one plane, and it wasn’t just the World Trade Center.

Two beams of light recognized the site of the World Trade Center attack. Today DNA forensic analysis applies new technologies to bring closure to families of victims.

Information was sparse. The world wide web was incredibly slow, and social media wasn’t much of a thing—nothing more than a few listservs for the life sciences. Someone managed to find a TV with a rabbit-eared, foil-covered antenna, and we gathered in the cafeteria of Promega headquarters—our shock growing as more footage became available. At Promega, conversation immediately turned to how we could bring our DNA forensic analysis expertise to help and support the authorities with the identification of victims and cataloguing of reference samples.

Just as the internet and social media have evolved into faster and more powerful means of communication—no longer do we rely on TVs with antennas for breaking news—the technology that is used to identify victims of a tragedy from partial remains like bone fragments and teeth has also evolved to be faster and more powerful.

Teeth and Bones: Then and Now

“Bones tell me the story of a person’s life—how old they were, what their gender was, their ancestral background.”  Kathy Reichs

Many stories, both fact and fiction, start with a discovery of bones from a burial site or other scene. Bones can be recovered from harsh environments, having been exposed to extreme heat, time, acidic soils, swamps, chemicals, animal activities, water, or fires and explosions. These exposures degrade the sample and make recovering DNA from the cells deep within the bone matrix difficult.

Bone and teeth are considered difficult sample types for DNA forensic analysis even when not exposed to harsh environments because of their unique composition. Standard preprocessing reagents and steps used for other sample types are not effective in efficiently extracting DNA from the cells within the calcium matrix of bone. Special preprocessing of bone and teeth samples is essential.

Some advances in the preprocessing that have been made since 2001 include demineralizing the bone in a buffer described by Loreille et al. in 2007. This demineralization can be complete in as little as 3 hours, shortening the time to result while improving the DNA extraction. Instead of drilling to wash off debris powder, bones are sanded with a one-time use grinding stone. The improved cleaning removes contaminants and inhibitors of DNA amplification from the surface of the bone samples.

Reading the DNA: From STR Analysis to NGS

Close to 99.9% of the DNA of any individual is the same as the DNA any other individual, which means using DNA to distinguish between two people requires significant “power of discrimination”.  In 2001, DNA forensic analysis primarily used short tandem repeat (STR) analysis for DNA fingerprinting.

STR analysis relies on sections of the human genome that do not encode proteins and show greater variability. These regions contain areas of multiple copies of repeated sequences of bases (ex. TAATTAATTAAT). The number of times one of these short sequences repeat is different in different individuals, and because of this STRs can be used to distinguish DNA from different sources. When many of these STRs are examined, the ability to distinguish individuals based on DNA increases, because it is less likely two different people will have exactly the same number of repeats for several different STR loci. In the United States there are 13 autosomal (non sex-chromosome) STR loci included in the Combined DNA Index System (CODIS) database that are used for forensic DNA analysis (Norrgard, K, 2008).

STR analysis involves several steps. The first is isolating DNA from the sample to be analyzed. Often isolating DNA is the most difficult step in the process, because samples are very small or have been degraded by the environment. The second step involves making many copies of each of the loci analyzed and then separating the amplification products using capillary electrophoresis (CE). Because a locus with two repeats would have a different molecular weight than the same locus where the repeat occurs ten times, you can separate the amplified DNA fragments based on molecular weight, and get a unique pattern or profile.

SNPs, Mitochondria and NGS: New DNA Forensics Analysis Technologies for Reading Bones

While STR analysis is extremely powerful, a newer technology, next-generation sequencing (NGS)—also called massively parallel sequencing (MPS)—has arrived on the scene. NGS can provide more data on minute variations in the genome including changes in a single nucleotide, particularly for the analysis of mitochondrial DNA (mtDNA) and single-nucleotide polymorphisms (SNPs). For degraded samples, such as charred or ancient bone, this increased information is extremely helpful.

All human cells have two sources of DNA: the nucleus, which contains the chromosomal material, and mitochondrial DNA, which is a circular piece of DNA contained within the mitochondria of cells, much smaller than the human genome within the nucleus. However, because cells contain many mitochondria (a human egg can have 60,000 of them), mtDNA is more abundant than genomic DNA—each cell only has one copy of the genome. The mitochondrial DNA encodes many of the proteins required for cellular energy production. Mitochondrial DNA is inherited through the mother, and because it only reflects the maternal lineage, analysis of mtDNA makes it easier to trace family relationships further back in time. While STR analysis looks at numbers of repeats at many places in the genome, mtDNA analysis uses sequencing to identify single-nucleotide changes.

Graphic depiction of a cut-away of an animal cell showing the nucleus with genomic DNA and the mitochondria with mtDNA now being used for DNA forensic analysis

In the 1990s, the Armed Forces DNA Identification Laboratory (AFDIL) began using mtDNA forensic analysis to identify service members recovered from Vietnam (Doyle, 2021), and in July 1998, AFDIL used mtDNA analysis to assist in the identification of Vietnam soldier First Lieutenant Michael Joseph Blassie (National Library of Medicine, Visible Proofs). In 2016, the military added NGS to their work with mtDNA and the combination of the two technological improvements have enabled the identification of hundreds of American soldiers who died in World War II and in the Korean and Vietnam wars.

By 2019, STR analysis and other forensics evidence enabled investigators to identify about 1,600 of the 2,754 World Trade Center victims. However, many remains have not been matched to reference samples because of incomplete STR profiles or the inability to get enough DNA to analyze. Recently the Office of the New York City Chief Medical Examiner received approval to use NGS on mtDNA in their forensic analyses of over 10,000 remains from the World Trade Center that have not be identified (Destefano, 2021).

Big Data: Genetic Genealogy, Familial Searching and DNA Forensic Analysis

If you read about the arrest of a suspect in the Golden State Killer case in 2018, you have probably heard about genetic genealogy. In the Golden State Killer case, detectives used a public database called GEDmatch. The investigators uploaded DNA from crime scene and looked for matches with DNA voluntarily uploaded by users of GEDmatch. Using familial DNA searching, they hunted for people potentially related to the Golden State Killer. The premise of familial searching is that close biological relatives of an unidentified DNA profile will have more STRs or alleles in common than unrelated individuals (Schweitzer, 2016).  In the Golden State case, upon finding a genetic link to the killer’s great-great-grandfather, investigators began following the entire family tree to identify the person who was arrested (Arduengo, 2018). Not only is genetic genealogy useful for unidentified DNA profiles obtained from crime scenes, but it can also be used to identify unknown victims in mass graves (Peccerelli, 2015).

Summary

The last ID of Word Trade Center victim remains was made in 2019. However, there are 10,000 remains left to be identified and many families still looking for closure about their loved ones who were in the Twin Towers on that fateful day. The technological advances made in forensics since 2001 will enable forensic scientists to close at least part of the gap between unknown remains and missing family members

Literature Cited

  1. Noreille, O.M. et al. (2007) High Efficiency DNA Extraction from Bone by Total Deminieralization. Forensic Sci Int Genet. 1(2):191–5.
  2. Norrgard, K. (2008)  Forensics, DNA Fingerprinting and CODIS. Nature Education 1(1):35
  3. Doyle, K. (in press) Technological Innovation Aids the Identification of Fallen Soldiers.
  4. National Library of Medicine. Michael Blassie Unknown No More. Visible Proofs.
  5. Destefano, A. M. (2021) Technology brings hope for closure: Advanced forensic method to be used to try to ID remains of 1,110 WTC victims. NewsDay
  6. Schweitzer, D. (2016) Familial DNA Searching for Criminal Forensics: Q &A. Promega Connections May 4.
  7. Arduengo, P. M. (2018) Questions of Genome Privacy and Protection. Promega Connections June 15.
  8. Peccerelli, F. (2015) Forensic Science in Search of the “Disappeared”. Promega Connections June 8.

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Michele earned her B.A. in biology at Wesleyan College in Macon, GA, and her PhD through the BCDB Program at Emory University in Atlanta, GA. Michele is the social media manager at Promega and managing editor of the Promega Connections blog. She enjoys getting lost in a good book, trumpet playing, knitting, and snowshoeing.

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