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Research Article
Volume 14 Issue 2 - June 2020
DOI: 10.19080/JFSCI.2020.14.555882
J Forensic Sci & Criminal Inves
Copyright © All rights are reserved by Kareem Essam
Role of DNA in Paternity Testing
Kareem Essam1*, Mona Hamza2 and Ayman Diab3
1B.Sc from the faculty biotechnology at MSA University, Egypt
2Consultant in forensic DNA, MD. Medical Microbiology and immunology, Kasr El Einy
3Ph.D. in Biotechnology from Cornell University, USA and Dean of the faculty of Biotechnology at MSA University, Egypt
Submission: June 21, 2020; Published: June 29, 2020
*Corresponding author: Kareem Essam, Researcher in the faculty of Biotechnology at MSA University, Egypt
J Forensic Sci & Criminal Inves 14(2): JFSCI.MS.ID.555882 (2020) 0016
Introduction
By the end of 2018, there were about 17,000 divorce cases
in Egypt which marks a 13.4% rise in divorces. According to the
Central Agency for Public Mobilization and Statistics (CAPMAS),
the total number of divorces reached 199,867 in comparison to
180,244 in 2014. In Egypt, most women marry between the age
of 20 and 25 while most men marry between the ages of 25 to
30. Men between 60 and 65 years and women over 65 years
had the lowest marriage rate so that they had the lowest judicial
problems between them like the paternity issues. Paternity issues
always happen due to huge problems between married couples. It
happens when married women enter a sexual partner in her life so
the child will be not the son of her husband. There are also many
cases that lead to paternity and also lead the father to deny his son
or daughter and vice versa. The science has developed and played
important role paternity problems by solving these problems
through different techniques. The old one is ABO blood typing
and the latest one is STR. (Aswatmasriya.com, 2017).The aim of
this research is to identify the biological parents and to prove that
the case is inclusion or exclusion by extracting the DNA sample
from a blood sample or from a buccal swab in order to identify the
16 codes core STR loci with chromosomal positions. Egypt faces
many paternity cases that cause several issues for children who
grow up and didn’t know their biological parents. These cases
have a bad effect on the children’s psychology that may lead them
to fail in their future and it is also one of the main reasons for
children’s streets. Some estimates say that two million children
are living on Egypt’s streets. A quarter of street children are to be
less than 12 years old, two-thirds between the age of 13 and 16,
on the street for a variety of reasons, including family breakdowns
due to divorce and remarriage. Some of these Egyptian children
are victims for backward society and they are deprived of their
rights in education, health, and social care and especially the right
to family care so that the sciences present the STR polymorphism
in paternity analysis to solve the paternity cases and return the
right of these children in family care [1-5].
Abstract
Egyptian society suffers from increasing in the number of divorce cases which leads to different problems. One of them is the children’s
street that is a result of paternity issues. Paternity cases are caused due to huge problems between married couples because women may be
entering a sexual partner in her life so the child will be not the son of her husband. This study discusses the techniques that are used in paternity
cases in order to detect the biological father and prove which technique is more accurate compared to other techniques. Blood groups were the
repeat. This technique depends on 16 short tandem repeat loci founded on the chromosomes. It also depends on the length of each locus. This
study discusses 6 samples concerned with 2 paternity cases while in one case concerned father, mother and two children.
Keywords: Paternity Test; Short tandem repeats; Deoxyribonucleic acid; Polymerase chain reaction; Genetic analyzer
Abbreviations: CPI: Combined Paternity Index; RI: Relationship Index; KI: Kinship Index; LR: Likelihood Ratio; PI: Paternity Index; CAPMAS:
Central Agency for Public Mobilization and Statistics; STR: Short Tandem Repeat
Journal of Forensic Sciences & Criminal Investigation
How to cite this article: Kareem E, Mona H, Ayman D. Role of DNA in Paternity Testing. J Forensic Sci & Criminal Inves. 2020; 14(2): 555882.
DOI: 10.19080/JFSCI.2020.14.555882
0017
Modern DNA Typing Procedures
Nowadays, the most common technique that is used in
STR analysis. In the sample collection step, the DNA is extracted
from its biological source moreover it is measured in order to
evaluate the quantity of DNA recovered in the quantitation step.
and copied with polymerase chain reaction which is known as
PCR. Finally, in the STR analysis step, the Commercial kits are
commonly used to enable simultaneous PCR of 13 to 16 short
tandem repeat (STR) markers. STR alleles are interpreted relative
capillary electrophoresis and data analysis software [6].
DNA Quantitation Using RT-PCR
To ensure that DNA recovered from extraction is human
DNA quantitation is required. Only after DNA in a sample has
been isolated can its quantity and quality be reliably assessed.
Detection of the appropriate amount of DNA template to include
scale data and associated artifacts are the main purpose of DNA
too much DNA results in overblown electropherograms that make
interpretation of results more challenging and time consuming to
review. Too little DNA can result in loss of alleles due to randomly
in the sample. A number of DNA quantitation tests have been used
over the years to estimate the amount of total DNA or human DNA
present in a sample. Several DNA quantitation tests are used in
many approaches such as yield gels, Pico Green, end-point PCR,
real-time quantitative PCR, UV absorbance, and slot blot. UV
absorbance is the most common technique to determine DNA yield
easier. Absorbance measurement is simple, moreover, it requires
commonly available laboratory equipment. All that is needed for
the absorbance method is a spectrophotometer equipped with a
UV lamp, UV-transparent cuvettes depending on the instrument,
at 260nm (A260) where DNA absorbs light most strongly, and the
number generated allows one to estimate the concentration of
the solution. To ensure the numbers are useful, the A260 reading
should be within the instrument’s linear range (generally 0.1-1.0).
DNA concentration is estimated by measuring the absorbance at
260nm, adjusting the A26 measurement for turbidity (measured
by absorbance at 320nm), multiplying by the dilution factor, and
using the relationship that an A260 of 1.0 = 50µg/ml pure dsDNA.
Real-time PCR is known as quantitative PCR because it analyzes
is performed without opening the PCR tube and therefore can
be referred as a homogeneous detection assay. There are two
common approaches that are used in DNA quantitative either the
dye such as SYBER Green. Quantifying the DNA in a sample is
smallest volume required for the reaction ranges for, 0.5 to 1.0 ng
Figure 1.
Figure 1: Schematic of TaqMan (5’ nuclease) assay.
Journal of Forensic Sciences & Criminal Investigation
How to cite this article: Kareem E, Mona H, Ayman D. Role of DNA in Paternity Testing. J Forensic Sci & Criminal Inves. 2020; 14(2): 555882.
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0018
PCR Amplication
replicated over and over again to yield many copies of a particular
sequence. A copy of the target DNA sequence is generated for
every molecule containing the target sequence during each cycle.
These oligonucleotide primers are complementary to the 39-
ends of the sequence of interest. In the ideal reaction with 100%
region on the DNA template have been generated after 32 cycles.
Lower quantities of PCR products are produced due to a reduction
annealing [11]. The polymerase chain reaction (PCR) technique
has become the standard process for DNA paternity testing. PCR
technology allows scientists to amplify a very small quantity of
DNA in order to increase the amount of DNA up to billions of copies
of the same DNA that are made for testing and analysis. The PCR
machine also makes this process happen fairly quickly. Using PCR
technology, DNA relationship tests can be performed much more
easily and quickly. Buccal swab specimens are collected from each
tested party in a non-invasive manner in a standard DNA paternity
test today, to make it ideal even for newborn babies. As the
quantity of DNA required in the PCR based test is so small, genetic
DNA to determine paternity before the child is born (Figure 2) [6].
Thermal cycling typically entails 3 different temperatures that
are repeated over and over again 25 to 35 times. There are three
phases according to three different temperatures. Denaturation
occurs at 95 C to separate double-strand DNA. Annealing occurs
at about 60 C so the primers can anneal with their complementary
sequence in the target DNA sequence. The temperature rises to
72 C in the extension phase, Because Taq polymerase functions
optimally at this temp. and begins polymerization, adding
nucleotides at 3’end of each primer attached to a DNA strand. PCR
can use very small amounts of DNA template as little as a single
cell [12-16]. DNA degraded to fragments only a few hundred base
amplify contaminated DNA such as fungal and bacterial sources
However, three potential pitfalls are considered disadvantages of
PCR. The presence of PCR inhibitors in the extracted DNA may
not amplify the target DNA template. The sequences changes in
the primer- binding region of the genomic DNA template may fail
DNA samples is possible without careful laboratory technique and
prevent dangerous something to happen.
Figure 2: Thermal cycling temperature proles for PCR.
In order to prevent cross-contamination during liquid
transfers, Aerosol-resistant pipette tips should be used and
changed on every new sample. Reactions may also be set up in
should be worn and changed frequently. Equipment, such as
pipettes, and reagents for sitting up PCR should be kept separate
from other laboratory supplies, especially those used for the
analysis of PCR products and also Pre- and post-sample processing
areas should be physically separated. Usually, a separate room or
reactions. Importance of STRs in forensic DNA typing. DNA
Journal of Forensic Sciences & Criminal Investigation
How to cite this article: Kareem E, Mona H, Ayman D. Role of DNA in Paternity Testing. J Forensic Sci & Criminal Inves. 2020; 14(2): 555882.
DOI: 10.19080/JFSCI.2020.14.555882
0019
these DNA markers exhibit the highest possible variation or a
number of less polymorphic markers that can be combined in
order to obtain the ability to distinguish between samples STR
Project beginning in April 1996 and concluding in November
1997 involved 22 DNA typing laboratories and the evaluation of
just 16 candidate STR loci: D8S1179, D21S11, D7S820, CSF1PO,
D3S1358, THO1, D13S317, D16S539, D2S1338, D19S433, VWA,
TPOX, D18S51, Amel, D5S818 and FGA [6]. These STR loci are
located on the chromosomes [17-21].
Paternity Index
The paternity index (PI) compares between the possibilities
that a genetic marker (allele) and that the alleged father (AF)
passed to the child with the probability that a randomly selected
unrelated man of similar ethnic background could pass the allele
to the child. This is presented in the formula X/Y, where X is the
chance that the alleged father could transmit the obligate allele
and Y is the chance that some other man of the same race could
behave transmitted the allele. X is assigned the value of 1 if the
AF is homozygous for the allele of interest and 0.5 if the alleged
father is heterozygous. Ratio of the two probabilities. This ratio
hypothesis that the AF is the real father. The larger ratio is more
evidence that this man is the real father. Two possible results are
be this child’s father. In the exclusion case, there is no way that
the AF could be this child’s father when multiple genetic systems
are tested, a PI is calculated for each system. The genetic system
is inherited independently [22-25]. the product of system PIs is
the combined paternity index (CPI) the combined paternity index
is also determined by the multiplying the individual PIs for each
locus tested. This value is referred to as system PI. Likelihood
ratio (LR) is called the relationship index (RI) or kinship index
(KI). The main functions of the likelihood ratio are to describes
how strongly genotypes support one relationship versus the
other relationship and also to express the likelihood of obtaining
3-5. Each independent locus tested produces its own relationship
index, which can be multiplied by those of other independent loci
to calculate a combined relationship index (CRI).
a. CRI > 1 supports the numerator (claimed relationship)
b. CRI < 1 supports the denominator (alternative
relationship)
c. Larger CRI values provide more support for the claimed
relationship
Figure 3: Ratio of the two probabilities.
Figure 4: The DNA proles under two mutually exclusive hypotheses.
Materials and Methods
This study discusses two protocols in extracting DNA. Each
one is used according to the sample collection and also according
to the available kits that. These two protocols are extraction of
of DNA from buccal swab spin. After extracting protocols, the
codes core STR loci and the length of each locus is determined
by electrophoresis step which is done by using the 3500 genetic
founded in the case and prove that whether the case is inclusion
or exclusion. This methodology was applied to three paternity
cases. Each case consists of three individual which means that the
Journal of Forensic Sciences & Criminal Investigation
How to cite this article: Kareem E, Mona H, Ayman D. Role of DNA in Paternity Testing. J Forensic Sci & Criminal Inves. 2020; 14(2): 555882.
DOI: 10.19080/JFSCI.2020.14.555882
0020
Extraction of DNA from fresh blood using thermos
200 l of whole blood, 400 l lysis solution, and 20 l
proteinase k were put in a 1.5 ml tube and then the tube was
put in the vortex to mix well. The sample was incubated for 10
min at 56 °C in a shaking water bath. 200 l of 96% ethanol was
added and mixed well by vortexing spin. 820 l of the lysate was
transferred to a spin column and then the sample was centrifuged
through solution was discarded and a new collecting 2 ml tube
was placed. 500 l of wash I was added centrifuged for 1 min at
solution was discarded and placed a new collecting 2 ml tube. 500
l of wash II was added and centrifuged for 3 min at max speed
(14,000 rpm). The collecting tube was emptied and centrifuged at
max speed for 1min. the collecting tube was discarded containing
to a sterile 1.5 microcentrifuge tube. 170 l of Elution buffer was
added to the center of the spin column membrane to elute genomic
DNA. The sample was incubated for 2 min at room temperature
and centrifuged for 1 min at 10.000 rpm. The spin-column was
or stored at -20 °C [31-36].
Extraction of DNA from buccal swab spin
The buccal swab was placed in a 2 ml microcentrifuge tube.
400 l of cotton and DACRON swab was added to the sample. 20
l of QIAGEN protease stock solution and 400 l cotton DACRON
swab buffer AL were added to the sample and then mixed
immediately by vertexing for 15 sec. The sample was incubated
at 56 °C for 10 min.400 l of cotton or DACRON swab was added
to the sample, mixed again by vortexing, and then centrifuged
l of the mixture
was applied to the spin column in a 2 ml collection tube without
wetting the rim, the clap was cap and centrifuge at 6000 x g
(8000 rpm) for 1 min. The spin column was placed in a clean 2 ml
The previous step was repeated by applying up to 700 l of the
remaining mixture from step four to the spin column. The spin-
column was opened carefully and 500 l buffer AW1 was added
without wetting the rim. The cap was closed and centrifuged at
6000 x g (8000 rpm) for 1 min. the spin column was placed in
a clean 2 ml collection tube. The spin-column was opened and
500 l of buffer AW2 was added without witting the rim. The cap
was closed and centrifuged at full speed (20,000 xg; 14,000 rpm)
for 3 min. The spin column was placed in a new 2 ml collection
centrifuged at 20,000 xg (14,000 rpm) for 1 min. The spin column
was placed in a clean 1.5 microcentrifuge tube and the collection
was opened, and 150 l buffer AE was added. The sample was
incubated at room temperature for 1 min and then centrifuged at
6000 xg (8000 rpm) for 1 min [37-40].
Plus Kit
10l of Amp FISTR® ® Plus Master mix was
added to the tube and then 5 l of Amp FISTR® ®
Plus Primer Set was added per each reaction. The Am pFISTR®
® Plus Kit Master Mix and the Amp FISTR®
® Plus Kit Primer Set 11was thawed, then vortexed 3 seconds and
®
® Plus Kit Master Mix and the Amp FISTR®
® Plus Kit Primer Set was thawed, then vortexed 3 seconds
volumes of components were pipetted into an appropriately
sized polypropylene tube. The reaction mix was vortexed for 3
l of the reaction Mix was
dispensed into each reaction well of a micro Amp® Optical 96-
Well Reaction Plate or each Micro Amp® tube [41].
Prepared the DNA Samples
mix) is 25 l. The MicroAmp® Optical 96-Well Reaction Plate with
Micro Amp® Clear adhesive Film was sealed. The reaction mix
was vortexed for 3 seconds, then centrifuge the tubes at 3000 rpm
for about 20 seconds in a (Table 1-4) top centrifuge to remove
® in PCR
system 9700 with the gold-plated silver 96-well block. The plate
was loaded into the thermal cycler and closed the heated cover
and then Program was started to run One completion of the run,
6.
Figure 5: Independent loci to calculate a combined relationship index.
Journal of Forensic Sciences & Criminal Investigation
How to cite this article: Kareem E, Mona H, Ayman D. Role of DNA in Paternity Testing. J Forensic Sci & Criminal Inves. 2020; 14(2): 555882.
DOI: 10.19080/JFSCI.2020.14.555882
0021
Table 1: preparation of DNA sample, negative control and positive control.
DNA sample Prepared
Negative control 10 l of low TE buffer was added
Test sample
of 10 l. 10 l of the diluted sample was added to the reaction mix
Positive control 10 l of 9947A control DNA was added (0.1ng/ l)
Table 2: Program of the thermal cycling conditions.
Initial Incubation step Cycle (28 or 29) Final Extention Final Hold
Denature Anneal/extend
Hold Cycle Hold Hold
95°C for 11 minutes 94°C for 20 seconds 59°C for 3 minutes 60°C for 10 minutes 4°C
95°C for 11 minutes 94°C for 20 seconds 59°C for 3 minutes 60°C for 10 minutes 4°C
Table 3: Paternity case 1.
locus D8w1179 D21S11 D7S820 CSF1PO D3S1358 THO1 D13S317 D16S539 D2S1338 D19S433 VWA TP OX D18S51 Amel. D5S818 FGA
Father Alleles 13 15 28 30 11 13 11 12 15 18 7 9.3 11 13 9 11 17 18 12 14 15 19 8 12 14 19 X Y 12 12 20 21
Mother Alleles 13 13 29 31 11 12 11 11 15 16 7 11 12 12 12 12 17 22 12 14 16 16 8 8 12 15 X X 13 13 22 25
Child
(Female) Alleles 12 13 31 31 11 12 11 11 16 16 7 11 11 12 12 12 22 22 12 1442 16 17 8 8 12 15 X X 12 13 23 25
Table 4: Paternity case 2.
locus D8S1179 D21S11 D7S820 CSF1PO D3S1358 THO1 D13S317 D16S539 D2S1338 D19S433 VWA TPOX D18S51 Amel. D5S818 FGA
Father Alleles 11 14 30 31 7 9 12 14 14 17 6 6 11 12 9 13 17 25 12 14 16 17 9 9 16 17 X Y 10 12 21 26
Mother Alleles 11 13 31 31 11 11 11 12 17 17 8 9 12 14 9 12 18 21 14 14 15 19 11 11 19 19 X X 8 12 23 12
Child
(female) Alleles 11 15 31 31 8 11 11 12 17 18 8 9 12 14 9 11 18 21 13 14 15 16 9 11 16 19 X X 11 12 23 24
Figure 6: Thermal cycle.
Prepared samples for electrophoresis on the 3500
genetic analyzers
The volume of Hi-Di Formamide and GeneScan500Liz size
standard was calculated to prepare the samples according to 0.3
l Gene Scan 500 LIZ standard and 8.7 l Hi-Di formamide per
reaction. The required volumes of components were pipetted
into an appropriated sized polypropylene tube. The tube was
l of the Hi-Di-Formamide
and 1 l of PCR product or allelic ladder were added into each
Journal of Forensic Sciences & Criminal Investigation
How to cite this article: Kareem E, Mona H, Ayman D. Role of DNA in Paternity Testing. J Forensic Sci & Criminal Inves. 2020; 14(2): 555882.
DOI: 10.19080/JFSCI.2020.14.555882
0022
well of a Micro Amp® Optical 96well Reaction plate. The reaction
plate was sealed with appropriate septa, then checked the plate
to ensure that the contents of each well are collected the bottom,
and then. The reaction plat was heated in thermal cycler for 3
minutes at 95 °C. The plate was placed immediately on ice for 3
minutes. The plate was a prepared assembly on the autosampler.
Electrophoresis was started to run. Analysis of injected samples
was done according to gene mapper software analysis and
electropherograms are plotted and compared [42-45].
Results
The total number of 6 samples concerned 2 paternity cases.
Major cases included mother, father and child while in a single
case At D8S1179, D7S820 and CSF1PO locus, the alleles of female
child are 11: 15, 8:11and 10:12 respectively while the alleles on
the father of the same locus are 11:14, 7:9 and 12:14 respectively
and the mother 11:13, 10:11 and 11:12. This indicates that the
alleles 15, 8 and 24 are not shared from this father Figure 7-13.
Figure 7: DNA prole of the father in paternity case 1.
Figure 8: DNA prole of the mother in paternity case 1.
Figure 9: DNA prole of the female child in paternicty case 1.
Journal of Forensic Sciences & Criminal Investigation
How to cite this article: Kareem E, Mona H, Ayman D. Role of DNA in Paternity Testing. J Forensic Sci & Criminal Inves. 2020; 14(2): 555882.
DOI: 10.19080/JFSCI.2020.14.555882
0023
Figure 10: DNA prole of the father in paternity case 2.
Figure 11: DNA prole of the mother in paternity case 2.
Figure 12: DNA prole of the female child in paternity case 2.
Journal of Forensic Sciences & Criminal Investigation
How to cite this article: Kareem E, Mona H, Ayman D. Role of DNA in Paternity Testing. J Forensic Sci & Criminal Inves. 2020; 14(2): 555882.
DOI: 10.19080/JFSCI.2020.14.555882
0024
Figure 13: Dierencing loci between child and father.
Discussion
In order to identify that the case is whether inclusion or
compared with each other according to the length of short tandem
repeats of each locus. There are 16 candidate STR loci which are
D8S1179, D21S11, D7S820, CSF1PO, D3S1358, THO1, D13S317,
D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, Amel, D5S818
and FGA. In inclusion cases, the child shares the length of each
STR loci with his parents because each biological parent shares
23 chromosomes for their child. However, in exclusion cases, the
child’s length of STR loci differs between the father and mother. In
case 1 there is no difference between the length of short tandem
repeats in the child comparing to the short tandem repeats in his
mother and father because the child shares its loci with his father
and mother while in case 2, At D8S1179, D7S820 and CSF1PO
locus, the alleles of the female child are 11: 15, 8:11 and 10:12
respectively while the alleles on the father of the same locus
are 11:14, 7:9 and 12:14 respectively and the mother 11:13,
10:11 and 11:12. This indicates that the alleles 15, 8, and 24 are
not shared by this father. So DNA paternity testing is the more
accurate than blood groups testing because If DNA patterns
between mother, child, and the alleged father match on every DNA
probe, the likelihood of paternity is 99.9 percent, while if the child
and the alleged father do not match on two or more DNA probes,
so the alleged father can be totally excluded. To handle DNA
testing, either a blood or buccal swab, this provides a DNA sample
for testing. Children can be tested at any age [46,47]. Paternity
testing can even be done on an umbilical cord blood specimen at
birth. The accuracy of testing performed on cheek cells utilizing
the Buccal Swab is the same as an actual blood sample because
DNA is the same in every cell of the human body.
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DOI: 10.19080/JFSCI.2020.14.555882
