Effects of Aging on DNA for Forensics

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Effects of Aging on DNA for Forensics
By Alyson Saadi
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What is Forensics?

• application of a broad spectrum of sciences to
  answer questions of interest to the legal system
• Or
• a great subject for TV shows

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Forensic samples used to extract DNA

• Blood
• Semen
• Saliva
• Sweat

• Urine
• Bone
• Hair
• Epithelial Cells

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What can DNA give us?

• Gender (STR)
• Ancestry
• Maternal Linkage (Mito)
• Paternal Linkage (Y-STR)
• So what about AGE?

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Benefits of Age in Forensics

• Determination or estimation of age could help to
  build a profile of an unknown suspect
• (Serial Killers and Rapists)
• Could also help in identification of unknown
  persons (Jane Does, Mass Disasters)

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Downfalls of Age in Forensics

• Any scientific test has room for error
• With incorrect age estimation you could
  potentially bias people to rule out the wrong
  suspects

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Current Methods of Forensic Aging

• Dental Characteristic
• Anthropological Characteristics (FACES)
• Potential Eye Witness

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Estimating age of humans based on telomere
shortening
Akiko Tsujia, Atsushi Ishikoa,b, Tomoya
Takasakia,1, Noriaki Ikedaa,
Purpose
To estimate age using DNA based on telomere
shortening, we determined the terminal
restriction fragment (TRF) length, as telomere
length, using Southern blot analysis of
peripheral human blood and blood stains. All
blood stains had been stored at room temperature
for 5 months.
Akiko Tsujia et. Al Estimating age of humans
based on telomere shortening, Forensic Science
International 126 (2002) 197199.
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Methods

• DNA was extracted from the peripheral blood of 60
  Japanese
• individuals between 0 and 85 years of age, using
  the
• phenol/chloroform method.
• As telomeres have no restriction enzyme cleavage
  sites, these
• sites located to a greater degree inside of the
  subtelomeric
• domain and inside of the telomere structure were
  cut.
• Terminal restriction fragment (TRF) length from
  such sites to the
• end of chromosome was identified as telomere
  length by
• Southern blot analysis.

Akiko Tsujia et. Al Forensic Science
International 126 (2002) 197199.
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Methods

• One microgram of DNA was digested with the
  restriction
• Enzyme HinfI and RsaI.
• Following DNA digestion, the DNA fragments
• were separated by agarose gel electrophoresis and
• transferred to a nylon membrane by Southern
  blotting.
• The blotted DNA fragments were hybridized to a
• probe specific for telomeric repeats and
  incubated
• with an antibody

Akiko Tsujia et. Al Forensic Science
International 126 (2002) 197199.
11
Methods

• Finally, the immobilized telomere probe was
  visualized by a
• highly sensitive chemiluminescense substrate.
• The average TRF length was determined by
  comparing
• signals relative to a nuclear weight standard on
  X-ray film.
• Correlation between age and the average TRF
  length of
• each blood sample was calculated as it was for
  blood
• stains, all samples being from the same blood and
  stored
• at a room temperature of 22 C for 5 months.

Akiko Tsujia et. Al Forensic Science
International 126 (2002) 197199.
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The average TRF length clearly showed a tendency
to shortening with aging
Results
Akiko Tsujia et. Al Forensic Science
International 126 (2002) 197199.
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Results
A straight line was obtained by regression
analysis between the average TRF length detected
and the age
Akiko Tsujia et. Al Forensic Science
International 126 (2002) 197199.
14
Results
The detected average TRF length of each sample
can be included in the following formula to
roughly calculate age of the subject (blinded
tests) age 0.0095y 148.9 7.037 (y
average TRF length bp, 7.037 standard
error) This calculation can be made regardless
of age of the subject
Akiko Tsujia et. Al Forensic Science
International 126 (2002) 197199.
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Important to note
The estimated age calculated from TRF length
depends on both environmental and genetic
factors. Age of the subject could be determined
even from 5-month-old stains. If the DNA is cut
to a small size and the TRF length is shorter
than that used in our study, 500 bp, our method
is not so applicable.
Akiko Tsujia et. Al Forensic Science
International 126 (2002) 197199.
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An Improved Method for Determining Telomere
Length and Its Use in Assessing Age in Blood and
Saliva
by Peter Lahnert
Purpose
It is disputed whether telomeres shorten with
age. The proposition of this study was to answer
this question.
Peter Lahnert Gerontology 200551352356
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Methods
Peripheral blood or saliva was collected from
healthy Caucasians DNA was isolated and
electrophoresis performed The gel was blotted on
nylon membrane Prehybridization and
Hybridization Immunological Detection and Color
Reaction The mean lengths of the telomeres were
estimated at the peak position of hybridization
signal
Peter Lahnert Gerontology 200551352356
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Results
With the aid of an amazingly simple
method consisting of color reaction combined
with long- term electrophoresis and weak voltage
it has been proved that the telomeres do, in
fact, shorten during in vivo ageing.
Peter Lahnert Gerontology 200551352356
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Results for Blood

Figure 1 shows that even with the naked eye it is
now possible to assess the age of an individual
from a drop of blood
Peter Lahnert Gerontology 200551352356
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Figure 2a shows that the data obtained with an
image analysis by a scanner are of a
substantially higher accuracy
Peter Lahnert Gerontology 200551352356
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Results for Saliva

• The results are similar for saliva, but there
• are three persons who would be wrongly
• allocated if the specimens were pieces of
• evidence in a real criminal investigation.

Peter Lahnert Gerontology 200551352356
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Results
Telomere length lt 8 kb the blood belongs to a
person older than 55 years of age
Telomere length gt 8.5 lt 10 kb the person is
likely to be younger than 50
years Telomeres length gt 10kb not possible to
determine whether the person is old or
young
Peter Lahnert Gerontology 200551352356
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Discussion
There is a publication in which the authors
claim that they can estimate the age of a person
by the telomere length in blood. If one follows
with an open-mind my criticism mentioned below,
nobody can take this publication seriously.
Peter Lahnert Gerontology 200551352356
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Discussion

• First of all, the discernible points in figure 2
  are at most 48 in number.
• Second, the distance between the 21,226-bp band
  and the 8,576-bp band is about 14 mm. The
  distance between the 8,576-bp band and the
  7,427-bp band is less than 1 mm and the distance
  between the 7,427-bp band and the 4,973-bp band
  is about 2 mm. How can this be correct? How can
  it be possible that the distances between the
  21,226-bp band and the 8,576-bp band are so huge
  and the distances between the 8,576-bp band, the
  7,427-bp band and the 4,973-bp band are so tiny?

Peter Lahnert Gerontology 200551352356
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Discussion
Third, it would be better if the lanes with
telomeres from old subjects and the lanes with
telomeres from young subjects are arbitrarily
mixed, for example in lanes 1, 2, 5, 7 and 9 old
subjects, in lanes 3, 4, 6 and 8 young subjects
and so on. This procedure would counterbalance
any source of measurement error, Fourth, if one
wants to know whether a physiological property
changes with age, one should compare young and
old persons. In figure 2 of Tsuji et al., most of
the persons are middle-aged. Fifth, in my method
for telomere length measurement there are some
persons with very long telomeres. In figure 2 of
Tsuji et al., there are no subjects with very
long telomeres.
Peter Lahnert Gerontology 200551352356
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Conclusions
To improve the benefit for the forensic medicine
the telomere length in blood and saliva in
middle-aged persons should also be determined. I
have omitted this because the main aim of this
paper was to test whether the telomeres shorten
at all. It goes without saying that the new
method described in this work is not only
suitable for the determination of age in blood or
saliva. It can be used for all questions where
the determination of the exact length of
telomeres is required, e.g. the telomere length
of cancer cells, the telomere length of different
plants or animals, testing whether or not
inhibitors of telomerase have an effect on the
telomere length. Although the method I have
developed works very well, it is certainly not
perfect.
Peter Lahnert Gerontology 200551352356
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Response to Peter Lahnerts criticism
regarding Estimating age of humans based on
telomere shortening
First, some points looked like one because of an
overlap. Secondly, about the size marker, we
did not notice the gap of the size position when
we set up the figure 1 . The positions of 21226,
8576 and 7427 bp are correct and that of 4973 bp
is much below. The correct positions of these
bands were mentioned in our next paper . Third,
figure 1 was arranged in order of age and was the
picture of re-electrophoresis for the
contribution to the paper. The first
electrophoresis was of course done
randomly. Fourth, we used the same numbers for
each age group as an ideal method however, very
young or very old healthy persons who gave their
informed consent were few, consequently we used
middle aged subjects. Fifth, of course we
occasionally detected the long telomere however,
we did not detect the very long telomeres.
A. Tsujia, et. Al Gerontology 200551416.
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Repsonse to Peter Lahnerts criticism regarding
Estimating age of humans based on telomere
shortening
Mr. Lahnerts method is also fundamentally a
Southern blot method, and is not novel. Each lane
is very long in his method, and it is considered
that deciding where the start and end points in
the smear are is difficult because of its length,
although the smear was stained by color. We
recommend another method such as the
hybridization protection assay in place of the
Southern blot method. Generally, the cell cannot
divide when its telomere length becomes 5 kb.
Therefore, scattering of the telomere length
appears narrow in old persons. In our data, the
tendency towards narrowing in old compared with
middle aged persons also had been shown.
Consequently, we conclude that middle age was a
rough estimation compared with the very young and
old. Finally, we believe that the rough telomere
shortening based on aging occurs due to various
factors during aging, and we think that age
estimation using our method is useful for
forensic medicine.
A. Tsujia, et. Al Gerontology 200551416.
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We have covered the potential of age estimation
using blood and saliva
What about the other most tested biological
sample? Semen
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Increased telomere size in sperm cells of mammals
with long terminal (TTAGGG)n arrays
Kozik A, Bradbury EM, Zalensky A Mol Reprod Dev.
1998 Sep51(1)98-104.
Using several independent methods we demonstrate
that the telomeres in the sperm of human, porcine
and bovine are elongated by 69, 24, and 14,
respectively, in comparison with somatic tissues.
Therefore, increased sperm telomere length is a
feature preserved throughout mammalian evolution.

• Alexander Kozik, et. Al Molecular Reproduction
  and Development 5198104 (1998).
• .

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Results

• Alexander Kozik, et. Al Molecular Reproduction
  and Development 5198104 (1998).
• .

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Results

• Alexander Kozik, et. Al Molecular Reproduction
  and Development 5198104 (1998).
• .

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Future explorations
How may increased telomere length in sperm effect
age estimations for forensics?
Could you estimate the same age range regardless
of sample type?
Could gene expression contribute to age
estimations
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References

• Akiko Tsujia, Atsushi Ishikoa, Tomoya Takasakia,
  Noriaki Ikeda Estimating age of humans based on
  telomere shortening, Forensic Science
  International 126 (2002) 197199.
• A. Tsujia, A. Ishikoa, N. Ikeda Telomere
  Shortening and Age Estimation in Forensic
  Medicine Gerontology 200551416.
• Alexander Kozik, E. Morton Bradbury, and Andrei
  Zalensdy Increased Telomere Size in Sperm Cells
  of Mammals With Long Terminal (TTAGGG)n Arrays,
  Molecular Reproduction and Development 5198104
  (1998).
• Peter Lahnert An Improved Method for Determining
  Telomere Length and Its Use in Assessing Age in
  Blood and Saliva Gerontology 200551352356.