Forensic mitochondrial DNA analysis: A different crime-solving tool.

On a December evening in 1994, a woman and her 4-month-old son were abducted and left to die in a wooded area in Pennsylvania. Although the woman's husband was an early suspect, detectives soon realized that he had not been involved in the crime. Before long, his jealous ex-girlfriend became the prime suspect in the murder investigation. Due to the careful collection of trace evidence from the victim's vehicle, investigators located a hair, stained with the victim's blood, on the back of the driver's seat. Laboratory tests performed on this hair and a sample from the suspect demonstrated that the evidentiary hair had the same mitochondrial DNA sequence as the one from the suspect and possibly could have come from her. Later, the suspect was tried and convicted of killing both the woman and her baby.

The FBI Laboratory began analyzing mitochondrial DNA (mtDNA) in casework in June 1996. Since that time, the DNA Analysis Unit II, using mtDNA sequencing techniques, has processed approximately 500 cases and recently has created the National Missing Persons DNA Database to assist the law enforcement community with missing person cases. Mitochondrial DNA analysis, while similar to the forensic nuclear DNA analysis (1) found in the news so often in the past few years, has several differences that impact its analysis. Because current cases, as well as many cold cases housed in the archives of law enforcement agencies, potentially could benefit from mtDNA analysis, it becomes important for investigators to understand the capability of this technique as a crime-solving tool. (2)

SCIENCE OF MITOCHONDRIAL DNA

Found in almost every cell in the human body, DNA, an abbreviation for deoxyribonucleic acid, contains the information that enables the body to function and gives everyone a unique appearance. DNA is composed of four building blocks, called bases, represented by the letters A, C, 0, and T. These bases form a structure known as a double helix because it is composed of two strands of DNA and looks similar to a twisted ladder or a circular staircase. In this structure, two bases comprise each rung of the ladder or step in the staircase. In mtDNA analysis, the order of the bases provides the forensic scientist with a basis for distinguishing between unrelated individuals. A phone number analogy can illustrate the importance of the order of the bases in DNA. The phone number 555-1234 would reach one particular individual when dialed, whereas a phone number containing the same digits in another order, such as 555-4321, would contact an entirely different individual. In a similar manner, forensic scientists can use the order of bases in mtDNA to distinguish between unrelated individuals.

DNA can be found in two separate locations within most cells in the body. As an analogy, the yolk and the white make up the two major components of an egg. Likewise, nuclear DNA is found in the nucleus of the cell, which is similar to an egg yolk. Two copies of nuclear DNA are found in each cell: one copy from the father and one copy from the mother. Because nuclear DNA is inherited from both parents, it remains unique to individuals, with the exception of identical twins. Over the past few years, nuclear DNA analysis has played a pivotal role in the adjudication of several important cases, thereby garnering much attention from the media. However, mtDNA analysis can offer the law enforcement community some equally noteworthy assistance in solving crimes.

Mitochondrial DNA differs from nuclear DNA in its location, its quantity in the cell, its mode of inheritance, and its sequence. Mitochondrial DNA is located in structures, called mitochondria, found in the outer layer of the cell, much like the egg white. While the nucleus of the cell contains two copies of nuclear DNA, cells may contain hundreds of mitochondria, each of which may contain several copies of mtDNA. Thus, mtDNA has a greater copy number than nuclear DNA. This characteristic of mtDNA proves useful in situations where the amount of sample is very limited. Typical sources of evidence suitable for mtDNA analysis include hairs without tissue, bones, and teeth.

In humans, individuals inherit mitochondrial DNA strictly from their mothers. (3) Thus, the mtDNA sequences obtained from maternally related individuals, such as a brother and a sister or a mother and a daughter, will exactly match each other in the absence of a mutation. This characteristic of mtDNA is advantageous in missing person cases as any maternal relative of the missing individual can supply reference samples. However, mtDNA analysis is limited when compared with nuclear DNA analysis in that it cannot distinguish between individuals of the same maternal lineage or individuals who have the same mtDNA sequence by chance.

Given the many different circumstances that can surround a case, sometimes advantages exist in analyzing mtDNA over nuclear DNA for forensic purposes. First, the location and structure of mtDNA protect it from degradation when exposed to the environment. Mitochondrial DNA is buried deep within the cell and has a circular structure, which protects it from deterioration. Also, DNA is bound and protected by a substance, called hydroxyapatite, found in teeth and bones. Second, the high copy number of mtDNA gives the forensic scientist a better chance of locating and amplifying a piece of undergraded DNA in a sample. Finally, the maternal inheritance of mtDNA can prove advantageous in cases involving missing persons, even though this fact also makes it less discriminating than nuclear DNA because any person who is a member of the same maternal lineage will have the same mtDNA sequence. Mitochondrial DNA also is highly variable between unrelated individuals. In fact, the scientific community has not yet seen all o f the variation that exists between human mtDNA sequences.

STEPS IN mtDNA ANALYSIS

Currently, the forensic analysis of mtDNA remains labor-intensive. Several biological techniques are combined to obtain an mtDNA sequence from a sample. The steps of the mtDNA analysis process include primary visual analysis; sample preparation; DNA processing, including extraction, amplification, quantification, and sequencing; and data analysis.

Primary Visual Analysis

Primary visual analysis of a sample set to undergo mtDNA analysis proves important because the techniques used to obtain an mtDNA sequence consume that evidence. Trace evidence examiners document the visual analysis through photography and written notations for future reference. The first step in the analysis of a hair involves a microscopic comparison of an evidentiary hair and a sample population of reference hairs. If the hair from a questioned source exhibits similar microscopic characteristics as hair from a known source, examiners perform mtDNA analysis to determine, on a molecular level, if the hair is consistent with reference standards from a particular individual. At times, hairs, such as body hairs or hair fragments, are not microscopically compared prior to mtDNA analysis because they are not suitable for such examination.

In instances in which bone or tooth material is collected, forensic anthropologists or odontologists inspect the tissue first. If the tissue is of human origin, mtDNA analysis can be used in conjunction with medical, anthropological, and odontological examinations to assist in the identification process. If available, X rays or other medical records often are considered preferable to mtDNA analysis for identification purposes.

Sample Preparation

DNA analysts clean evidentiary samples prior to the mtDNA sequencing process to remove contaminating materials surrounding or adhering to the sample. This step ensures that the sequence of the DNA obtained from the sample originates from the sample and not from exogenous human DNA.

The cleaning process for hair samples uses a detergent treatment in an ultrasonic water bath, which removes possible contaminating residues from the hair. The hair sample is then placed in an extraction solution and ground using a small mortar and pestle, resulting in a mixture that contains both the cellular material and the released DNA.

Bone and tooth samples also undergo a cleaning process. To clean a bone or tooth, an analyst sands the exterior to remove any extraneous material that may adhere to the surface. Then, the analyst removes a small sample, grinds it into a fine powder, and places the powdered bone and teeth in a solution to release the DNA from the cells.

DNA Processing

To extract DNA, analysts expose the cellular mixture from the sample preparation step to a mixture of organic chemicals that separate the DNA from other biological material, such as proteins. Analysts then purify the DNA sample to prepare it for the amplification process, which makes many copies of the target DNA. The DNA of interest must be amplified because initially it is not present at a concentration high enough to be detected by laboratory instrumentation used in the analysis. After the DNA is copied, it is purified and quantified prior to determining the order of the bases in the DNA fragment. The sample undergoes a series of chemical reactions and is then placed in an instrument that "sequences" the bases in the DNA sample.

Data Analysis

The FBI Laboratory has established guidelines for the interpretation of differences and similarities between sequences. Basically, samples cannot be excluded as originating from the same source if sequence concordance (the presence of the same base or a common base at every position analyzed) exists between them. In cases of sequence concordance, at least two examiners independently analyze each sequence obtained. (4)

The human body contains trillions of cells, each of which can contain hundreds to thousands of copies of mtDNA. A complete homoplasmy (the same mtDNA sequence) for each of these copies is unlikely because of the immense amounts of mtDNA present in the body. Thus, heteroplasmy (the occurrence of more than one mtDNA type at a particular position or region in a DNA sequence) is expected to be present at some level in all individuals, though not always detectable with current instrumentation. (5)

The level of heteroplasmy may not always be the same in various tissues. In cases where heteroplasmy is thought to occur, examiners can sequence additional samples to determine if the heteroplasmy is visible in other tissues. Obviously, further testing cannot always be performed on a crime-scene sample of limited quantity, but it can prove helpful for interpretation of known samples. In most instances, the presence of heteroplasmy makes data interpretation more complex, but does not render the data nonfunctional.

The FBI Laboratory has collaborated with other laboratories to compile an mtDNA population database containing the sequences from major racial or ethnic groups: Caucasians, Africans, Asians, Native Americans, and Hispanics. The database currently contains 4,142 mtDNA sequences from unrelated individuals in a forensic index and another 6,686 sequences gathered from published literature. (6) However, the laboratory updates the database regularly and, thus, constantly increases its size.

When a sequence from a sample of questioned origin is the same as a sample of known origin, laboratory personnel search the mtDNA population database for this sequence. The FBI Laboratory lists the number of observations of a sequence in each racial subgroup of the forensic database in a report of an mtDNA examination. For example, a sequence might be seen five times in the database samples of Caucasian descent and one time in the database samples of Hispanic descent, yet not appear in the remaining database subgroups.

Currently, the FBI Laboratory does not provide frequency estimates of mtDNA types in laboratory reports because of restrictions involving the mtDNA database size. The FBI states only the number of occurrences of an mtDNA sequence in the current database. The number of mtDNA sequences present in the general population is unknown because scientists have not observed all of them yet. However, statistical methods exist for calculating an upper-bound estimate of the frequency of mtDNA types with zero, or very few, occurrences in a database of limited size. This upper-bound estimate describes the highest frequency expected for a particular mtDNA sequence using the database. As the database grows in size, the frequency estimates for individual mtDNA profiles will become more and more refined.

QUALITY ASSURANCE IN mtDNA TESTING

Forensic mtDNA analysis uses some extremely sensitive techniques. Because the laboratory analysis may begin with very few copies of the mtDNA of interest (if a sample is degraded), the presence of any foreign DNA in the sample can harm the analysis. Any extraneous DNA that is amplified with the evidence has the potential to interfere with the interpretation of the sequence data. Thus, the FBI Laboratory performs many precautionary measures to prevent contamination of evidence and to ensure the quality of results. (7)

For example, the FBI Laboratory follows the quality assurance standards established by the DNA Advisory Board (now called the FBI Standards for Forensic DNA Testing Laboratories) and the American Society of Crime Laboratory Directors-Laboratory Accreditation Board (ASCLDLAB). The laboratory also has two external proficiency tests conducted annually for all unit personnel working cases and retains a record of these tests. In addition, the laboratory follows general and specific precautions designed to minimize contamination, including the physical separation of pre-and postamplification areas; the separation of evidence from questioned and known sources in time and, often, space; the proper cleansing of work spaces and instruments; and the use of control samples. The FBI Laboratory uses positive and negative controls in mtDNA processing to monitor amplification and sequencing and does not proceed with the mtDNA analysis if these controls fail to meet established criteria.

CONCLUSION

Forensic mitochondrial DNA analysis has become a valuable crime-solving tool for law enforcement investigators. Work is ongoing in a new program, the National Missing Persons DNA Database, at the FBI Laboratory. This program has a mission to create a database of the mtDNA sequence of the remains of missing individuals and their maternal relatives. In addition, several state and local crime laboratories are validating mtDNA analysis for implementation into their casework capabilities.

The future of all DNA analyses looks promising as techniques for smaller-scale and higher-throughput testing become available. While mtDNA analyses do not provide the discrimination potential of some nuclear DNA tests, mtDNA data often are the only information that examiners can gather from degraded evidence, which is either old or has been exposed to the environment for a significant period of time. The development of forensic mtDNA sequencing over the past decade has been helpful to many past cases and will continue to provide useful information to the law enforcement community in the future.

Endnotes

(1.) For information on collecting DNA evidence, see "The Microscopic Slide: A Potential DNA Reservoir," FBI Law Enforcement Bulletin, November 2000, 18-22.

(2.) The author adapted this article from a research paper previously published in the July 1999 issue of Forensic Science Communications, at http://www.fbi.gov. For additional information, contact the DNA Analysis Unit II, FBI Laboratory, 202-324-4354.

(3.) For additional information, see M.M. Holland and T.J. Parsons, "Mitochondrial DNA Sequence Analysis--Validation and Use for Forensic Casework," Forensic Science Review 11 (1999): 21-50.

(4.) The FBI Laboratory DNA Unit II Mitochondrial DNA Analysis Protocol (Rev. 7/01) contains a more detailed description of the FBI Laboratory's mtDNA interpretation guidelines.

(5.) Supra note 3.

(6.) K.W.P. Miller and B. Budowle, "A Compendium of Human Mitochondrial DNA Control Region: Development of an International Standard Forensic Database," Croatian Medical Journal (2001).

(7.) Supra note 4.

RELATED ARTICLE: CASES SOLVED USING MITOCHONDRIAL DNA ANALYSIS

Murder Investigation

In September 1994, a mother and her 4-year-old daughter were at a friend's house. The mother was going out on a date and arranged to have her friend watch her daughter. During the course of the evening, the mother's friend decided to go out and left the victim and her own children with her sister's boyfriend. Later, the boyfriend put the children to bed, with the 4-year-old girl sleeping in one of the twin beds in the room she shared with her mother. At about 2 a.m., a family friend, who had been at the house repairing a screen door earlier in the day, returned after drinking heavily. He went into the same bedroom where the girl was sleeping to lie down. The sister's boyfriend, who assumed that the friend would pass out in the other twin bed in the room, continued watching a movie. He heard two noises coming from the bedroom, but thought they were car doors slamming.

The girl's mother arrived home about an hour after the friend and talked with the sister's boyfriend for approximately 20 minutes. Then, she went to check on her daughter. Finding the bedroom door locked, she yelled for the friend to open it. When he did not, she obtained a knife from the kitchen to unlock the door. Upon entering the room, the mother found both beds empty and the girl and the family friend, both without clothing, on the floor of an adjacent utility room. The girl was unconscious and cold to the touch; the friend was unresponsive. The mother immediately took her daughter to the hospital, but all attempts to resuscitate her failed, and she was pronounced dead at 3:30 a.m. The medical examiner found several hairs clinging to her body and evidence of sexual abuse. The FBI Laboratory performed mtDNA testing on the hairs found at the crime scene and on the victim's body. Analysts compared the results to the mtDNA profile for the family friend. All sequences were the same, and the sequence was not present in the FBI's database of 742 individuals at the time. The male was sentenced to life without parole for felony murder, plus two concurrent 25-year sentences for the rape convictions.

Missing Person

In the fall of 1979, a 61-year-old patient wandered away from a U.S. Department of Veterans Affairs medical facility. Despite an extensive search, authorities never located the missing man. Over 10 years later, a dog discovered a human skull in a wooded area near the facility. The DNA Analysis Unit II of the FBI Laboratory received the case in the winter of 1999. The laboratory determined that the mitochondrial DNA profile from the missing patient's brother matched the mitochondrial DNA profile from the recovered skull and provided the information to the local medical examiner. Subsequently, the remains were declared to be those of the missing patient and returned to the family for burial.