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ORIGINAL RESEARCH PAPER
COMPARATIVE ANALYSIS OF PRESERVATIVES USED FOR
EFFICIENT FORENSIC DNA EXTRACTION FROM FETAL TISSUES
Deepali Jain1, Biswamitra Parua2, Naresh Kumar3& J.R. Gaur4
ABSTRACT
DNA extraction and its profiling for forensic analysis is a
normal practice in the 21st century. Sample preservation
isa significant step for obtaining adequate data from the
samples. This study has been done for comparative
analysis of the preservatives used to preserve fetal tissue.
The phenol-chloroform extraction methods used for the
extraction and various kits by Thermo fisher were used
for the controlled results. PCR amplification kit has been
utilized for subsequent development. The amplification of
the samples such as fetus in formalin, fetus in normal saline,
fetus limb in normal saline, fetus toe in normal saline and
fetus toe in formalin. DNA profiles of all the samples were
generated after RT-PCR and conventional PCR. Based on
those DNA profiles data, Standard deviation and mean
were derived. The probability plot of samples data showed
the normal distribution, so the normal distribution of the
graph has been analyzed for all the samples, based on
this histogram plotted and the mean pie chart. Results
concluded that fetus toe in normal saline is more effective
for DNA extraction than fetus limb in normal saline. Even
saline solution acted as a better preservative rather than
formalin
Keywords: Fetus, DNA, Normal Saline, Formalin, RT-
PCR, DNA profile
INTRODUCTION
DNA extraction plays a significant role to create a DNA
profile. This fur ther helps to make data points for
analysis.Preservation of DNA is also a critical step to
gettingaccurate results.India holds 140th rank in gender
parity, 148th in gender gap data published by the world
economic forum14.
Such a massive population with improper economic
distribution leads to a lot of conflict within the community,
which leads to logarithmic growth of the crime rate in India.
Relatively, crime against women is increasing daily,
including hugely heinous crimes like sexual assault, rape,
and others.
We all know the case of Delhi Gang Rape (2012), Kathua
rape (2018), Priyadarshani Mattao(1996) and many more.
Most of the cases were not even reported to the police.
Some of them were dealt with by local panchayat or any
person with positional power with zero knowledge of the
law.
As a torchbearer of action against injustice, forensic
data is being utilized for more accurate results. While in
any such case of the sexual offence occurs, a gynecologist
must examine and preserve all the crucial evidence for the
Justice of the accused.
This preservation method indulges fetus and placental
tissue with the help of formalin; here, fetal tissue was
submerged into formalin solution. The formaldehyde
component partially breaks DNA in due course of time,
leading to improper or inadequate DNA profiles5.
DNA technology is a robust and powerful tool in the
prevailing technology standards for criminal justice
systems. The quantity and quality of extracted DNA impact
the success of its analyses and the whole quality of the
final DNA profiling. There is also a PCR inhibitor, which
inhibits and creates hindrance in the reaction12. To avoid
such a scenario, we use the AMPFlSTR® Identifiler Plus®
TM PCR amplification kit.
1. M.Sc., PhD, Assistant Professor, School of Forensics,
Risk Management & National Security, Rashtriya
Raksha University, Lavad, Gandhinagar, Gujarat-
382305 (INDIA)
2. M. S c., Fo rensi c Sci e nce, Depart ment of
Biotechnology, Babasaheb Bhim Rao Ambedkar
University, Lucknow- 226025 (INDIA)
3. M.Sc., PhD, Senior Scientific Officer, Biology Division,
Fo r ensi c Sc ience La b orato r y, Ro hini, De lhi-
110085(INDIA)
4. M.Sc., PhD, Director, School of Forensics, Risk
Management & National Security, Rashtriya Raksha
University, Lavad, Gandhinagar,Gujarat- 382305
(INDIA)
Corresponding Author
Dr. Deepali Jain, M.Sc., PhD
Assistant Professor,School of Forensics, Risk
Management &National Security, Rashtriya Raksha
University, Lavad, Gandhinagar,Gujarat- 382305 (INDIA)
Email: deepali.jain@rru.ac.in
DOI No: 10.5958/0974-4568.2021.00005.3
30
METHOD AND MATERIALS
SAMPLE COLLECTION
Ten Fetal tissue samples were collected from the biology
division, State Forensic Science Laboratory, New Delhi. All
the samples were in good condition for further analysis.
SAMPLE PREPARATION
The well-preserved fetal tissue was transferred to the
laboratory for examination. The phenol-chlor oform
extraction method is used for DNA extraction. The tissue
sample is washed multiple times to avoid contamination. It
took days to wash & dilute formalin from tissues. In labs,
samples were kept in a deep fridge at around -20 C to prevent
denaturation or degradation.
To get most of the purified DNA sample, it is transferred
to the small 1.5ml tube along with 25ìl dithiothreitol, 500 ìl
forensic buffers. Later on, 40l was added and 25l Pk and
left for incubation on the ‘Thermo mixer.Sodium acetate,
along with isopropanol liquid utilized to form a DNA pallet.
At last, the DNA pallet got a wash in 2 repetitive cycles
with 70% and 100% alcohol6.
This DNA extraction process from saline submerged
fetal tissue is not up to the mark to provide sufficient DNA.
Then, we run DNA Sample RT-PCR (Real-time PCR) machine
within proper procedure and protocols. RT-PCR results help
identify the gender of the sample along with the quantity
of DNA it may avoid the deletion of Y allele on amelogenin2.
All research done until now points to the significant
fact that amelogenin has a distinct difference in size and
nucleotide sequence in the either gender. This difference
be t wee n the two pheno t ypes is a sensi tive sex
determination for very minute DNA samples produced from
unknown fetal tissues.
Amelogenin gene present on X-chromosome has 106
base pairs in length, whereas this gene present on Y-
chromosome has 112 base pairs. This information can
provide a vital distinction between male and female sex
determination and highlights those females have two
identical amelogenin genes present on X-chromosome2.
In contrast, males have two different genes present on
both the sex chromosomes. This difference in male and
female genotypes can be an indispensable tool with
excellent specificity sensitivity and is financially suitable
for modem forensic science.
The Quantifiler® duo kit of applied Biosystem is best
to exclude the misrepresentation of females in place of males
due to deletion of Y allele in amelogenin11,2. Sometimes
amelogenin provides negative feeds on gender detection
to avoid using a quantifier duo kit. This kit contains unique
components like Taq Man®, RNA component H1 (RPPH1)
gene), (SRY) gene, Internal Positive Control (IPC) which
binds to the protein of the sample to exclude any false
result11.
DNA’S AMPLIFICATION
We used phenol-chloroform solution, which is also a PCR
inhibitor, which inhibits and creates hindrance in the
reaction. IdentifilerTM PCR amplification kit was used for
ampilification4.
Here, amplicons are loaded into data collection
application ABI Applied Biosystems 3500xL with Gene
Ma p per IDX 1.2 soft war e for Anal ysi s11. Several
laboratories confirm that MiniSTRs® have a higher success
rate for DNA analysis of degraded DNA samples1.
The degraded tissue sample Contains enzymes, salts,
dNTPs, carrier protein, and 0.05% sodium azide. To enhance
the amount of the DNA, we amplify the selection through
AmpFlSTR® MiniFiler™ Master Mix to extract DNA from
the degraded tissue sample4; this kit Contains enzymes,
salts, dNTPs, carrier protein, and 0.05% sodium Azide.
Hence a good STR profile was presented.
RESULTS AND DISCUSSION
Serial no. Fetus in
formalin
In nanogram
Fetus in normal
saline
In nanogram
Fetus limb in
normal saline
In nanogram
Fetus toe
normal saline
In nanogram
Fetus toe in
formalin
In nanogram
1 0 2.06 2.96 3.02 1.52
2 0 1.36 1.96 2.08 0.56
3 0 0.62 0.56 3.86 1
4 0 0.42 3.56 1.52 0
5 0.32 3.45 1.13 2.6 0
6 0.52 1.96 1.98 4.5 0
7 0 2.36 2.96 3.56 0.32
8 0 1.06 3.45 4.5 0
9 0 2.96 4.56 5.06 0
10 0 4.58 3.22 2.06 0
MEAN 0.084 2.083 2.634 3.276 0.2088
STANDARD DEVIATION 0.183254529 1.309809571 1.212629833 1.207193255 0.534415569
Table 1: Real-Time PCR machine of DNA, mean and standard deviation of data
31
Fig 1: Screen representing running of samples on genetic analyser
Fig 2: DNA profiles of the samples
Fig 3: Mean value of all the samples as pie-chart representation
32
PROBABILITY PLOTS OF SAMPLES
Graph 1: Aboveprobability plots suggests that the datafollows a normal distribution
33
Graph 4: Comparison between different samples
(Graph 2) to obtain mean and standard deviation
COMBINED PLOTTING OF SAMPLES
Graph 3: Probability distribution plot of all the samples
to check mean and standard deviation
NORMAL DISTRIBUTION PLOTTING SAMPLES
Graph 2: Normal distributionplotsshow mean and
standard deviation of the observations13
34
In this comparative study,we took a sample of different
parts of fetal tissues like Fetus in formalin, Fetus in normal
saline, Fetus limb in normal saline, Fetus toe normal saline
&Fetus toe in normal formalin. All the above samples were
run through the instrument 7500 Real-Time PCR machine.
Hence, we ran ten samples, out of which two samples had
shown the result of 0.32ng & 0.52ng while the fetus is in
formalin3. The formalin preserved toe sample shows three
results 0.56, 1.52& 0.32. Based on the analysis of distribution
plot, histograms &standard deviation analysis, we found
that Fetus in normal saline shows maximum impact13.
The mean value was maximum in the sample where the
fetus toe is submerged in normal saline; this can be
identified from the pie chart and graph given above. The
distribution plot shows the normal distribution. The
histogram shows a sharp peak showsformalin’s fetus limb
graph. The rest has lower elevations, and the curve of the
fetus toe in normal saline is the most widely distributed in
the chart. The sharp peak is due to inadequate data as
DNA has been degraded by formalin, and no results came
during the experiment.
Previous work showed that Fetus in salinetoe samples
preserved in normal saline are the best DNA source10. We
plot a distribution plot and histogram of his data. We found
sharp peaks in the fetus limb, but uniform distribution is
seen in the fetus toe in saline.
After analysing previously available data1 0, newly
formed (Graph 6) and present data (Graph 3 and Graph 4),
we found that fetus toe in normal saline is 74.38% which is
more effective for DNA extraction than fetus limb in normal
saline.
CONCLUSION
As data acquired from the above analysis, we found that
preservative formalin for DNA extraction is not a good
option for DNA extraction,rather than saline solution will
ac t as an excell ent prese r vat i ve me thod for fe tus
preservation. This preservation technique will help in
extracting an adequate amount of DNA with minimum
wastage. The other fetal tissue will also provide DNA, but
if we focus on the most efficient part of the fetus for DNA
extraction, that will be the fetus toe in normal saline, as
seen in the above mathematical graphs.
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Graph 6: Histogram on previously published data10, showing wide distribution in foetus toe in normal saline
35
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