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.
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