mtDNA Basics
Mitochondrial DNA (mtDNA) provides a valuable
locus for forensic DNA typing in certain circumstances.
The high number of nucleotide polymorphisms
or sequence variants in the two hypervariable
portions of the non-coding control region can
allow discrimination among individuals and/or
biological samples.
The likelihood of recovering mtDNA in small
or degraded biological samples is greater than
for nuclear DNA because mtDNA molecules are
present in hundreds to thousands of copies per
cell compared to the nuclear complement of two
copies per cell. Therefore, muscle, bone, hair,
skin, blood and other body fluids, even if degraded
by environmental insult or time, may provide
enough material for typing the mtDNA locus.
In addition, mtDNA is inherited from the mother
only, so that in situations where an individual
is not available for a direct comparison with
a biological sample, any maternally related
individual may provide a reference sample.
The Analytical Process
A mtDNA analysis begins when total genomic DNA
is extracted from biological material, such
as a tooth, blood sample, or hair. The polymerase
chain reaction (PCR) is then used to amplify,
or create many copies of, the two hypervariable
portions of the non-coding region of the mtDNA
molecule, using flanking primers. Primers are
small bits of DNA that identify and hybridize
to or adhere to the ends of the region one wishes
to PCR amplify, therefore targeting a region
for amplification and subsequent analysis.
Care is taken to eliminate the introduction
of exogenous DNA during both the extraction
and amplification steps 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. In casework, questioned
samples are processed at different times than
known samples and they are usually processed
in different laboratory rooms.
When adequate amounts of PCR product are amplified
to provide all the necessary information about
the two hypervariable regions, 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 As, Ts, Cs, and Gs (nucleotide bases) that
make up the DNA sequence are labeled with dye.
The strands created in this stage are then separated
according to size by an automated sequencing
machine that uses a laser to "read"
the sequence, or order, of the nucleotide bases.
Where possible, the sequences of both hypervariable
regions are determined on both strands of the
double-stranded DNA molecule, with sufficient
redundancy to confirm the nucleotide substitutions
that characterize that
particular sample.
At least two forensic analysts independently
assemble the sequence and then compare it to
a standard, commonly used, reference sequence.
The entire process is then repeated with a known
sample, usually a buccal swab, saliva, or blood
collected from a known individual. The sequences
from both samples, about 780 bases long each,
are compared to determine if they match. The
analysts assess the results of the analysis
and determine if any portions of it need to
be repeated.
Finally, in the event of an inclusion or match,
the EMPOP mtDNA database is searched for the
mitochondrial sequence that has been observed
for the samples. The analysts can then report
the
number of observations of this type based on
the nucleotide positions that have been read.
A written report is provided to the submitting
agency.
Non-Forensic Uses
While mtDNA is useful for forensic examinations,
it has also been used extensively in two other
major scientific realms.
First, there are a number of serious human
diseases caused by deleterious mutations in
gene-coding regions of the mtDNA molecule, which
have been studied by the medical profession
to understand their mode of inheritance.
In addition, molecular anthropologists have
been using mtDNA for three decades to examine
both the extent of genetic variation in humans
and the relatedness of populations all over
the world. Because of its unique mode of maternal
inheritance it can reveal ancient population
histories, which might include migration patterns,
expansion dates, and geographic homelands. Recently
mtDNA was extracted and sequenced from a Neanderthal
skeleton. These results allowed anthropologists
to say with some conviction that modern humans
do not share a close relationship with Neanderthals
in the human evolutionary tree.
While all the applications of mtDNA, including
forensic, are relatively recent, the general
methods for performing a mtDNA analysis are
identical to those used in molecular biology
laboratories all over the world for studying
DNA from any living organism. There are several
thousand published articles
regarding mtDNA.
Advantages and Disadvantages
MtDNA has advantages and disadvantages as a
forensic typing locus, especially compared to
the more traditional nuclear DNA markers that
are typically used. As mentioned above, mtDNA
is maternally inherited, so that any maternally
related individuals would be expected to share
the same mtDNA sequence. This fact is useful
in cases where a long deceased or missing individual
is not available to provide a reference sample
but any living maternal relative might do so.
Because of meiotic recombination and the diploid
(bi-parental) inheritance of nuclear DNA, the
reconstruction of a nuclear profile from even
first degree relatives of a missing individual
is rarely this straightforward. However, the
maternal inheritance pattern of mtDNA might
also be considered problematic. Because all
individuals in a maternal lineage share the
same mtDNA sequence, mtDNA cannot be considered
a unique identifier. In fact, apparently unrelated
individuals might share an unknown maternal
relative at some distant point in the past.
Interpretation of Results
At the present time the available forensic database
of human mitochondrial DNA sequences has 29,143
sequences available for a search of a casework
sequence, of which 11,392 sequences originate
from Northern America. The current convention
in the event of an inclusion (a match between
questioned and reference sample sequences) is
for the analyst to report the number of times
the observed sequence is present in the database
to provide some idea of its relative frequency
in the database. To determine the frequency
within a population group, a frequency statistic
may also be used, and a 95% or 99% confidence
interval is placed around the calculated frequency
to account for the inherent uncertainty in the
frequency calculation.
While most types appear to be rare or at least
infrequent in each of the ethnic databases (African
or African-origin, Asian or Asian-origin, Caucasian
or European-origin, and Hispanic), there is
one type which is seen in around 7% of Caucasians.
However, almost two thirds of newly-typed samples
have novel sequences, so we have not yet uncovered
all the variation present in the general human
population. For novel types, a 95% or 99% upper
bound frequency calculation may be performed.
In general, the pattern observed in most populations
around the world, with the exception of a few
populations of anthropological interest, is
that the vast majority of sequences are uncommon,
and relatively few types present at frequencies
greater than 1% in the databases. Because of
this fact, it will be possible to exclude greater
than 99% of the population as potential contributors
of a sample in most cases, except where one
is dealing with a more "common" type.
In contrast, a multilocus nuclear DNA typing
profile provides vastly superior discriminatory
power, such that we can now approach the possibility
that a typed individual has a unique profile
with respect to any other person in the world.
Therefore, mtDNA can never provide the resolution
of individuality that
nuclear typing can. For this primary reason,
it should be reserved for cases or samples for
which nuclear typing is simply not possible.
Samples Typically Chosen for mtDNA
Typing
Candidates for mtDNA typing analyses would most
likely be: 1) shed hairs with no follicle, tissue,
or root bulb attached, 2) hair shaft fragments,
3) bones or teeth which have been subjected
to long periods of high acidity, high temperature,
or high humidity, 4) tissue (skin, muscle, organ)
that has been previously unsuccessfully typed
for nuclear markers, and 5) stain or swabbing
of materials that has been previously unsuccessfully
typed for nuclear markers.
Hair roots, when available, should be removed
from the shaft and processed separately for
nuclear DNA markers prior to attempting mtDNA
analysis on the hair shaft. Hair shafts or fragments
are only suitable for mtDNA analysis as they
can contain fewer than 100 copies of the mtDNA
molecule and virtually no nuclear DNA. The same
is generally true for older skeletal remains.
While mtDNA typing of stains is possible, it
is more likely that mixtures will be obtained,
due to the extreme sensitivity of this form
of typing in samples that unlike hairs and bones
are difficult to clean before DNA extraction.
Duration and Throughput of mtDNA Analyses
Finally, it must be noted that mtDNA analyses
are the most rigorous and time-consuming of
DNA forensic analyses. Based on informal statistics
available from all laboratories performing these
typings, the rate of throughput is approximately
3-4 cases/analyst/month.
The reasons for this include: 1) small/degraded
samples requiring numerous PCR reactions to
obtain sufficient DNA template for sequencing,
2) exhaustive procedures to control for contamination,
and 3) sequencing analyses of both strands of
DNA in both hypervariable regions.
In addition, for some types of samples, especially
hairs, mtDNA analysis is more likely to consume
the whole sample than nuclear DNA typing. For
example, a single mtDNA analysis could be performed
on a 0.2-2 cm hair fragment. A 4 cm fragment
could have duplicate testing for confirmation
of the sequence. In both cases the fragment
would be totally consumed. However, a root ball,
follicle, or skin tissue attached to a hair
would also be consumed in a nuclear typing effort.
For both mtDNA and nuclear DNA testing there
is a possibility that sufficient extracted DNA
might remain for duplicate testing in another
lab. Swatch, swab, stain, bone, and tooth analyses
are less likely to consume all material, as
these samples can often be divided, although
the difficulties of obtaining enough DNA for
analysis could result in consumption of these
materials as well. For the reasons above, pre-analysis
documentation (microscopy, photography) is desirable.
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