Forensic mitochondrial DNA analysis of hair shafts and naturally shed hairs is a tool to enhance the investigation of cold cases; this form of evidence historically has had severely limited utility.

Many cold cases have been re-opened in hopes that DNA profiling of evidentiary material may strengthen a case against an existing but weak suspect or identify new leads and new suspects. “Cold hits” are made when nuclear DNA (STR) profiles of semen, blood, or saliva crime scene samples are linked to convicted felon DNA profiles that are stored in the national DNA database (CODIS). A lesser-known form of DNA testing, however, also is being used for cold case investigation. In the 1990s, mitochondrial DNA (mtDNA) analysis was introduced for samples unsatisfactory for STR profiling. The earliest use of mtDNA analysis was for the identification of human skeletal remains that contained insufficient or degraded nuclear DNA, but sufficient mitochondrial DNA to aid in matching an individual to his or her maternal relatives. Since 1993, the Armed Forces DNA Identification Laboratory in Rockville, Maryland, has been using mitochondrial DNA to return the skeletal remains of military dead to their families. Identification of missing persons is also aided by this technology.

DNA can be difficult to recover from very small or environmentally challenged samples. While nuclear DNA is present in only two copies per cell, the small circular mitochondrial DNA molecule (Figure 1) is present in hundreds to thousands of copies per cell and is therefore a naturally abundant DNA molecule. During the 1990s, forensic scientists learned that while naturally shed human hair roots and hair fragments lacking any root at all do not contain sufficient nuclear DNA for routine STR typing, they contain abundant mitochondrial DNA (Figure 2). Today, the ability to perform mtDNA analysis on virtually any head or body hair is a bonus technique in the investigation of criminal cases.

The first mitochondrial DNA analysis that aided in a criminal conviction involved a suspect charged with the 1996 rape and murder of a young child in Tennessee. Since then, hundreds of cases have been examined using mitochondrial DNA analysis, and dozens have successfully been tried in the courtroom. A sizeable body of peer-reviewed scientific literature on forensic mitochondrial DNA analysis is available, and courtroom admissibility hearings have uniformly allowed its introduction worldwide.

Diagram of mitochondrial DNA molecule.

Hair shaft and naturally shed root.

Advantages and Limitations of mtDNA Analysis
A nuclear DNA match of the 13 core STR loci permits little doubt that a questioned sample has come from a known individual, except in the case of identical twins. However, because mitochondrial DNA is maternally inherited, all a woman’s offspring, her siblings, her mother, and other maternal relatives will have the same mitochondrial DNA profile (Figure 3). Mitochondrial DNA, therefore, is not a unique identifier and the test’s conclusion can be only whether or not a known individual is excluded as the donor of the questioned sample.

While non-uniqueness is a limitation, there are thousands of different mitochondrial DNA types, and the relative population frequency of almost any type is low. In most cases, at least 99% of the population will be excluded as contributors and the pool of random individuals who could have contributed the sample is less than 1% of the population. In many cases, well over 99.9% of the population may be excluded. The inability to use mtDNA in quite the same way as nuclear DNA highlights its value as a “piece of the puzzle,” meaning that mtDNA almost always supplements other information in the theory of the crime and that evidence tested by this method would rarely be the only evidence.

An advantage, however, of mitochondrial DNA use is that if a victim or a suspect is long deceased, missing, or unavailable for other reasons, a single maternal relative of that victim or suspect may provide the reference sample for comparison to crime scene samples. This feature is an added benefit for cold case investigators.

Pedigree chart showing maternal inheritance of mtDNA.

Crime Scene Hairs
A recent development in the area of post-conviction relief is the mtDNA re-examination of shed crime scene hairs previously examined using only a microscope. In a number of older cases, exonerations or new trials have been won when mtDNA analysis has proven without any doubt that a convicted offender could not have left the hair in question. In trial, a trace examiner had testified that the hair “matched” the defendant and the jury weighed this limited testimony heavily. A recent study has shown, however, that microscopic evaluations result in false positives about 11% of the time when the match was later evaluated using mtDNA analysis. Similarly, false negatives also occur: that is, hairs that might have shed light on a case were discounted by the examiner because the microscopic evaluation showed no visual “match” between an individual and a crime scene hair.

The knowledge that false negatives and positives may occur with hair microscopy provides an opportunity for the cold case investigator. By re-examining old records, laboratory and expert reports, stored evidence, and transcribed testimony from interviews, depositions, hearings, and trials, the investigator may locate previously slide-mounted hairs, or loose hairs on clothing or in envelopes or paperfolds, that were previously discounted on the basis that either “one can’t do anything with shed hairs/hairs with no root” or “the trace examiner said there was no match.” If highly probative, these sample hairs can open up new avenues for consideration of alternative or weak suspects. Similarly, crime scene hairs may help place a victim in probative relevant locations. Examples are:

  • Hair under a victim’s fingernails or in the victim’s hand
  • Hair trapped in a cracked windshield in a vehicular homicide (can place individuals in certain seats)
  • Hair superimposed on blood or other liquids at the crime scene
  • Pubic hairs in a sexual assault where the perpetrator has worn a condom
  • Hair recovered from wrappings on the body or the body bag
  • Hair recovered at autopsy
  • Hair stuck on the murder weapon, tape bindings, or ligatures
  • Hair collected from probative locations on clothing (inside underwear)
  • Hair in the mouth, throat, vagina, or rectum
  • Hair in a discarded mask or abandoned getaway vehicle
  • Hair adhering to bumper or undercarriage of a hit-and-run vehicle
  • Hair from the suspect’s vehicle (trunk or passenger seat)
  • Hair in the mechanism or tape of an explosive device

Single hairs as small as 2mm and as old as four decades have been successfully tested. It is unknown what the most extreme age for successful testing might be, but hairs over 21 years of age provide partial or full profiles in almost 80% of cases (Figure 4). Therefore, cold cases dating back to the 1960s are candidates for re-examination.

Determining the probative value of a questioned hair may be critical to a case. Unlike semen or blood, which is often present due to criminal activity such as sexual or physical assault, hairs may simply be deposited from an individual who has been innocently present before, during, or after a crime has occurred. By themselves, hairs are not indicators of any specific activity, especially since humans naturally lose 75-100 head hairs per day. Exceptions to this are forcibly removed hairs such as those yanked out during a struggle, or pubic hairs, which are not shed onto floors or furniture as often as head hairs. Since maternal relatives share the same mitochondrial DNA profile, using mtDNA analysis to investigate a within-family crime is not useful, for example, when one sibling has allegedly been murdered by another. Hairs found at the scene will be uninformative as to the correct donor based on mitochondrial DNA analysis. However, all probative hairs should be collected, for if the theory of the crime later is determined to be incorrect, and a non-family member becomes a suspect, the hairs may become useful evidence.

Collection yield of mtDNA profiles based on age of hair.

The Analytical Process
A mtDNA analysis begins when DNA is extracted from the hair. Prior to beginning an extraction, the hair is cleaned in an ultrasonic water bath, and then ground into a sterile solution (Figure 5). An enzymatic digestion and organic extraction purifies the DNA into a new sterile tube. The polymerase chain reaction (PCR) is then used to amplify two hypervariable portions of the non-coding region of the mtDNA molecule, using flanking primers. This region contains the mitochondrial DNA sequence information: the exact order of the As, Cs, Gs, and Ts that characterize that sample.

During extraction and amplification, contaminant DNA is eliminated via methods such as the use of pre-packaged sterile equipment and reagents, aerosol-resistant barrier pipette tips, gloves, masks, and lab coats, separation of pre- and post-amplification areas in the lab using dedicated reagents for each, ultraviolet irradiation of equipment, and autoclaving of tubes and reagent stocks. Questioned samples with minimal and/or degraded DNA are processed at different times and places than known reference samples.

After the appropriate amounts and regions of PCR product are amplified, sequencing reactions are performed. These chemical reactions use each PCR product as a template to create a new complementary strand of DNA in which some of the nucleotides are labeled with dye. The strands created in this stage are separated according to size by an automated sequencing machine to determine the DNA sequence. Redundancy confirms the nucleotides that characterize a particular sample.

Two forensic analysts independently assemble the sequence and agree on the final DNA sequence of the hair sample. The entire process is then repeated using blood or saliva collected from a known individual. The sequences from both samples, about 780 nucleotides long, are compared to determine if they match.

In the event of an inclusion, or match, the SWGDAM (Scientific Working Group on DNA Analysis Methods) mtDNA database, which is maintained by the FBI, is searched for the mitochondrial sequence that has been observed for the samples. The number of times that a type or profile has previously been observed is used to calculate a simple statistic which guides understanding of both the court and trier of fact about the significance of the match. It is important that a mitochondrial DNA analyst state clearly, both in a final report and in testimony, that an individual and his or her maternal relatives cannot be excluded as the donor of a questioned hair. With this statement of a “match,” the analyst may qualify the statement with additional information such as “We are 95% confident that the true frequency of this type in North American populations does not exceed 0.06%.” In other words, the analyst is saying that there is 95% confidence that at least 99.94% of North Americans will not have the type in question.

mini-glass grinder

Case #1: Terrorist Storage Locker
Armenian nationalists rented a storage locker in the Cleveland area during the 1970s for the storage of guns and explosives. Rental fees on the locker were not paid in 1996, which triggered an opening and investigation of the locker by authorities, including the Bureau of Alcohol, Tobacco, and Firearms once munitions were discovered. Several evidentiary hair fragments were collected from the locker. A 1999 mitochondrial DNA analysis of these hairs matched their profile to that of the leader of the terrorist group,Mourad Topalian. Topalian was arrested, charged, convicted, and sentenced to 37 months in prison in 2000. An “ancient” mitochondrial DNA analysis was necessary for the hairs, because their mtDNA was minimal and degraded after exposure to the heat of the storage facility. This more specialized approach allows abundant but degraded DNA, such as mtDNA in 25-year-old hairs, to be captured in smaller fragments.

Case #2: William Gregory Exoneration
William Gregory was arrested, charged, and sentenced for the attempted rape of a woman in his apartment complex after the victim identified him in a suspect line-up. There was no evidence except for six head hairs discovered in panty hose taken from the victim’s apartment for use as a mask. At the 1993 trial, a hair microscopist stated that the hairs could have come from Gregory, and this testimony was helpful to the prosecution. Post-conviction testing was performed in 2000; the six hairs shared the same mitochondrial DNA profile but had a different mtDNA profile from that of Gregory and the victim. He was released from prison shortly after testing. The case remains unsolved. This case was the first U.S. case in which mitochondrial DNA aided an exoneration.

Mitochondrial DNA analysis may supplement other tools used in cold case investigations, especially when probative hairs are discovered in old crime scene evidence collections. Until 1996, when the FBI started using mitochondrial DNA analysis, the only science routinely being applied to shed hairs was descriptive microscopy, a descriptive science prone to bias. While microscopy still has a valuable role to play in the evaluation of questioned hair evidence, it should no longer be used without confirmatory mtDNA analysis. Candidate hairs can be of any age and size and do not need any root material. In most cases when a match is obtained it is possible to eliminate well over 99% of the general population as contributors of a specific hair, with the exception of maternal relatives. With the availability of mitochondrial DNA analysis of questioned hair evidence, cold cases may be re-opened with the knowledge that a validated scientific process can be applied to valuable forensic evidence of previously limited value.

Additional Reading

  • Holland M Mand Parsons TJ (1999) Mitochondrial DNA sequence analysis: Validation and use for forensic casework. Forensic Science Reviews 11:21-50.
  • Isenberg AR and Moore JM (1999) Mitochondrial DNA analysis at the FBI Laboratory. Forensic Science Communications 1(2),
  • Melton T, Dimick G, Higgins B, Lindstrom L, and Nelson K (2005) Forensic mitochondrial DNA analysis of 691 casework hairs. Journal of Forensic Sciences 50:73-80.

(This article is taken in part from Cold Case Homicides: Practical Investigative Techniques, Richard H.Walton, editor, from the Practical Aspects of Criminal and Forensic Investigations Series, Vernon J. Geberth, BBA ,MPS, FBINA, Series Editor, Taylor and Francis Group, Boca Raton, Florida, 2006.)

Terry Melton, Ph.D., is a Forensic Examiner and the Laboratory Director of Mitotyping Technologies in State College, Pennsylvania, a private laboratory that provides DNA analysis services to law enforcement, attorneys, and other members of the criminal justice community. Terry is a Fellow of the American Academy of Forensic Sciences and a member of the editorial board of the Journal of Forensic Sciences. Terry may be reached at