Review article on current practices in mitochondrial DNA analysis
Current practices for performing forensic mitochondrial DNA (mtDNA) sequence analysis, as employed in public and private laboratories across the United States, have changed remarkably little over the past twenty years. Alternative approaches have been developed and proposed, and new technologies have emerged, but the core methods have remained relatively unchanged. Once DNA has been recovered from biological material (for example, from older skeletal remains and hair shafts), segments of the mtDNA control region are amplified using a variety of approaches, dictated by the quality of the sample being tested. The amplified mtDNA products are subjected to Sanger-based sequencing and data interpretation is performed using one of many available software packages. These relatively simply methods, at least in retrospect, have remained robust, and have stood the test of time. However, alternative methods for mtDNA analysis remain viable options (for example, linear array assays and dHPLC), and should be revisited as the desire to streamline the testing process, interpret heteroplasmy and deconvolute mixed mtDNA profiles intensifies. Therefore, it is important to periodically reassess the alternative methods available to the mtDNA practitioner and to evaluate newer technologies being put forth by the scientific community, such as next generation sequencing.
Review article on mitochondrial DNA heteroplasmy.
Heteroplasmy, the presence of more than one type of mitochondrial DNA in an individual, holds implications for forensic analysis of specimens such as blood, hair, and skeletal material. That is, what can we conclude about the likelihood that heteroplasmic specimens could or could not be from known individuals? Originally believed to be quite rare in healthy individuals, we now know that heteroplasmy exists at some level in all tissues on a predominantly homoplasmic background. A substantial body of general literature covers the biological origins of heteroplasmy, especially its transmission to new offspring and during life, the methodology for its detection, and its distribution in different tissues. In addition, the forensic community has contributed many observations on the characteristic appearance of heteroplasmy in relevant regions of the mtDNA control region and its appropriate treatment in forensic science. As a result of this growing understanding of a relatively simple biological phenomenon, we conclude that heteroplasmy can be expected to play a role in forensic interpretation on a regular basis, and that knowledge of its biological underpinnings contribute to just, conservative, and scientifically appropriate interpretational guidelines. For the PDF file of this manuscript, published in Forensic Science Review, January 2004, click here.
Forensic mitochondrial DNA analysis of 691 casework hairs.
A five year retrospective review of mitochondrial DNA (mtDNA) analysis on 691 casework hairs was carried out by abstracting case folder data on the frequency of obtaining full, partial and no mtDNA profiles with respect to age, condition and appearance of each hair, and type of submission whether loose or slide mounted. Also recorded were both the frequency of mixtures and the incidence and location of sequence heteroplasmy. Overall, a full or partial profile was obtained for > 92% of hairs. With increasing age of the hair, the likelihood of obtaining a full profile decreased, although "mini-primer sets" were extremely useful in many cases in recovering abundant, but degraded, mtDNA to capture a partial profile. With increasing color and diameter of the hair, characteristics we assess as "robustness", the likelihood of obtaining a profile increased even though the fragment used for analysis was, on average, smaller for more robust hairs. Full or partial profiles were obtained on more than 80% of the 114 hairs + 1.0 cm. Mixtures were observed in 8.7% of hairs tested; the frequency of this result increased with the age of the hair and was presumed to be due to exterior surface contamination that could not be sufficiently cleaned prior to extraction, since the overall level of laboratory contamination was low. The frequency of sequence heteroplasmy in hair was 11.4%, and both hot-spot and novel sites were observed. In about one-third of the heteroplasmy observations, another sample in the case such as hair, blood, or buccal cells showed either the same heteroplasmic site or a nucleotide substitution at that site.
Forensic mitochondrial DNA analysis of 116 casework skeletal samples. Between February 1999 and May 2005, 116 DNA extractions were completed on skeletal remains from routine casework. Overall, at least a partial mitochondrial DNA (mtDNA) profile was obtained on 83.6% of samples. Skeletal remains fell into two general categories: (1) samples for body identifications submitted by law enforcement and (2) samples submitted to answer historical or family identity questions. Body identification cases were more likely to yield full mtDNA profiles whereas historical cases were more likely to result in partial profiles. Overall, the ability to obtain a full or partial profile primarily reflects the difference in the average age and condition of the samples in these two categories and thus, difference in the quantity and quality of the DNA. Cremated remains were uniformly unsuccessful, whereas infant/fetal remains were uniformly successful. Heteroplasmy in skeletal remains was observed at a rate similar to that in hair (~10%). For body identification cases, skeletal remains had the same mtDNA profile as the accompanying reference sample in 50% of cases.
Routine Forensic Use of the Mitochondrial 12S Ribosomal RNA Gene for Species Identification. Since July 2004, Mitotyping Technologies has been amplifying and sequencing a ~150 base pair fragment of mitochondrial DNA (mtDNA) that codes for 12S ribosomal RNA, to identify the species origin of non-human casework samples. The ~100 base pair sequence product is searched at http://blast.ncbi.nlm.nih.gov and the species match is reported. The use of this assay has halved the number of samples for which no mtDNA results are obtained and is especially useful on hairs and degraded samples. The availability of species determination may aid forensic investigators in opening or closing off lines of inquiry where a highly probative but challenging sample has been collected.