DNA Testing Diversifies

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How and why the forensic value of mitochondrial DNA is growing

As millions of CSI fans can testify, DNA is an increasingly important factor in linking crime scene evidence to individual suspects. And, like the television series, this technique has branched out. The core technology that began entering courtrooms in the mid-1980s is "nuclear DNA testing", or nDNA. More recently, two complementary regimes have been joining the forensic mainstream — mitochondrial DNA (mtDNA) testing and Y-STR, which checks for short tandem repeats on Y-chromosome.

Although mtDNA has been used in at least 100 cases in the US since 1996, it made its way in September into a Canadian court for just the second time since first being admitted in 1999. This very limited adoption could reflect the many issues related to mtDNA that have surfaced in the US, and which have also been raised in Canada. At the forefront of these issues is the possibility that mtDNA testing is more prone to contamination than nDNA testing. In addition, mtDNA testing is often performed on types of evidentiary samples that further increase this risk of contamination by other DNA, either at the crime scene or in the laboratory.

This problem stems from the fact that even a single contaminating cell can introduce hundreds of copies of mtDNA molecules into the process, while that same cell would only be able to insert two copies of nDNA. Such cells could come from sample collectors, scientists analyzing the samples, cross contamination from other samples, or exogenous DNA in the air.

Besides contamination, another key issue surrounding mtDNA is the phenomenon of heteroplasmy. Heteroplasmy is defined as the presence of more than one mtDNA type within an individual, another issue that has been addressed in the literature.

In comparison with nDNA testing, mtDNA is less discriminatory, and should only be used when the former either fails or is unobtainable. It is worth noting that when nuclear DNA yields are low, complete mtDNA profiles can be obtained because of the presence of higher number of mtDNA copies per cell as compared to nDNA. For this reason, mtDNA can be a useful forensic tool for specialized sample types such as shed hairs.

A necessary alternative

Shed hairs or hair shafts (without roots/follicles) are often discovered at crime scenes. Although nDNA profiles are unique to individuals except identical twins, it cannot be recovered from all types of evidentiary samples. For example, hair shafts do not contain nuclear DNA in significant amounts, whereas mtDNA is often preserved within the hair shafts.

Similarly, skeletal remains from missing persons contain nuclear DNA but reliable nuclear DNA profiles cannot always be obtained from these remains, rendering mtDNA profiling as the only alternative. In general, mtDNA testing is recommended in cases where nDNA testing is not possible due to low quantity, high level of degradation or absence of nuclear DNA.

There are three possible outcomes in a mtDNA test: "exclusion", "cannot exclude" and "inconclusive". Since mtDNA profiles from all maternal relatives are expected to be same, it cannot be used as a unique identifier like nDNA profiles and the term "cannot exclude" is recommended instead of "match" or "inclusion". For example, if mtDNA profiles from suspect and crime scene samples are identical, then it is reported as "mtDNA profile from crime scene sample cannot be excluded as originating from the suspect or his/her maternal relatives", or a similar statement.

At least two differences between samples are reported as "exclusion". One difference is considered as "inconclusive". If a mtDNA profile from an evidentiary sample cannot be excluded as originating from the suspect or his/her maternal relatives, the probability that a randomly selected individual unrelated to suspect would coincidentally share the observed mitochondrial DNA profile is estimated and reported.

Calculating the odds

Established databases comprising many thousands of mitochondrial DNA profiles from unrelated persons are used to determine the rarity (probative value/statistical significance) of a given mtDNA profile. The mtDNA population database compiled by the FBI’s Scientific Working Group on DNA Analysis Methods is used in forensic casework by North American laboratories.

Since a mtDNA profile is considered as "one DNA marker", the product rule used for nDNA markers cannot be employed to calculate random match probability of mtDNA profile. Probability numbers are therefore higher in mtDNA matches than nDNA, since the currently accepted practice is to use the 'counting method', where a count of the total number of times a particular haplotype is present in the population database is used along with conservative approach called confidence intervals.

In nDNA evidence, the statistical probability of someone other than the accused having the same DNA is extremely small (usually less than one in the population of the world). Whereas, mtDNA probability values are large (the lowest probability is the total size of the current database, i.e. one in 4,839 individuals).

But mtDNA evidence is still considered as reliable genetic evidence because approximately 99 per cent of the general population can be excluded as potential donors (with 95 per cent certainty) in most cases. mtDNA evidence is equally reliable for exclusionary cases as nuclear DNA testing.

DNA regions containing the most variability among individuals are utilized in forensic DNA testing because the more variable the DNA region being tested is, the less likely the DNA profile will match by chance alone. The majority of the variation between two maternally unrelated individuals is found in the two Hypervariable Regions — HV1 (nucleotide positions 16024 to 16365) and HV2 (nucleotide positions 73 to 340) of Control Region of mtDNA — both of which are tested in forensic casework.

Conducting the analysis

Although the initial steps of DNA isolation and amplification of DNA are similar for all DNA testing technologies, mtDNA testing differs entirely at the analysis stage. While nDNA analysis calls for the separation of amplified DNA segments according to their size, mtDNA analysis compares the sequence of segments from evidentiary and known samples is obtained and compared. A further comparison takes place using the Revised Cambridge Reference Sequence, with resultant differences being noted for position and any type of change.

Guidelines published by international forensic groups, including the FBI, are used to ensure consistency among forensic labs in terms of technology and contents of the final reports.

To become accredited, a laboratory must have appropriate contamination control measures in place. For example, Tyvek cover-alls are worn during evidentiary DNA processing, DNA profiles from the laboratory staff are on file for comparison, positive pressure "clean lab" and air flow hoods prevent exogenous DNA (if any) from entering sample tubes, and reagents and equipment are sterilized by autoclave and/or UV irradiation where applicable. Appropriate reagent blanks and negative amplification controls (no DNA added) are processed along with samples to monitor the occurrence of DNA contamination.

Molecular World Inc. handled the mtDNA work on this fall's court case, the first use of this technology in a Canadian criminal trial. The firm, based in Thunder Bay, is the only private Canadian company accredited to offer mtDNA services, and has completed many of these tests for law enforcement and defense during last year. The results of this work can therefore be expected to serve as genetic evidence in future Canadian cases.

Amarjit Chahalaa, Arlene Lahtiaa, Curtis Hildebrandtaa, Jennifer Aguirreaa and El Moltoa,b
a Molecular World Inc., 1 South Cumberland Street, Thunder Bay, ON, Canada, P7B 2T1, Phone: 877-665-9753
b Department of Anthropology, Social Science Centre, University of Western Ontario, London, ON, Canada, N6A 5C2