Paternity Disputes – Importance of Y DNA
Profiling in Mutation Cases
Deepak Y Kudekar1*, Vaishali B Mahajan2*, Bhausaheb P More3 and Krishna V Kulkarni4
1,2Assistant Chemical Analyser, India
1,2,3Deputy Director of Regional Forensic Science Laboratory,Nashik
44Director of Forensic Science Laboratory ,Maharashtra, India
Submission: May 22, 2019; Published:July 03, 2019
*Corresponding author:Deepak Y Kudekar, Assistant Chemical Analyser, Regional Forensic Science Laboratory, Maharashtra, India
How to cite this article: Deepak Y K, Vaishali B M, Bhausaheb P M, Krishna V K. Paternity Disputes – Importance of Y DNA Profiling in Mutation Cases.
J Forensic Sci & Criminal Inves. 2019; 12(1): 555829. DOI: 10.19080/JFSCI.2018.11.555829.
Now a day’s DNA fingerprinting is widely used to solve the paternal disputes. Since the application of DNA fingerprinting in forensic science, we see many cases related to paternity disputes where father denies his parentage towards child either in illegal relationship or in pregnancy due to rape. There is one more scenario where married man declines to accept his own child to seek divorce from his wife alleging her cheating on him. In such civil matters, court orders to do paternity testing for verification of the truth.
While DNA profiling, mutation is one factor that sometimes encounters. When there is a male child and mutation observes, Y DNA profiling is useful technique to establish the progeny and paternity as the Y DNA is only carried through male to male and it is same for child, father, uncle, brother which are from the same progeny.
In present scenario, two such cases received at forensic science laboratory, Nashik where in nuclear DNA profiling using AmpFlSTR®Identifiler® PCR amplification kit, mutation observed in one allele between father and child and as the child was male, we carried out Y STR DNA analysis using AmpFlSTR®YfilerTM PCR amplification kit, that resulted to the conclusion that the progeny was same for child and suspected Father
Keywords: PCR; Short tandem repeat; Identifier kit; Y-filer kit; Mutation; Paternity Dispute; Progeny
DNA fingerprinting is worldwide used technology in the field of forensic to solve crimes. It is valuable tool to solve paternity disputes including illegal relationships, pregnancy due to rape, suspicion on own child in civil matter and to prove maternity in which mother throws foetus to hide illegal relationship from society. Few million samples are run for purpose of paternity testing in country every year. While performing DNA analysis, mutation can be seen. There are several reasons for mutation. In view of mutation, paternity could not be established due to exclusion at single DNA locus. Generally, minimum 12 autosomal STR markers situated at least ten different chromosomes have to be examined, which is enough to reach a paternity probability of more than 99.9% in normal paternity cases or to find out more than three exclusions . Further, ‘two exclusion’ rule stated that if two genetic loci do not match between an alleged father and child, the alleged father cannot be excluded as being true father of child [2,3].
Now, routinely, 15 STRs are used to obtain exclusion or strong probability of paternity. In few cases, this number can be inadequate to solve the case particularly when the mother is
unavailable for the test or when the biological father is a close relative of the legal one or where only one or two exclusions are found . The number of different Y-chromosome markers were evaluated which gained a significant role in paternity testing with male children [5,6]. We analysed two such cases in which Y DNA analysis using AmpFlSTR®YfilerTM PCR amplification kit, proved the same paternal progeny of son and father in spite of mutation in Nuclear DNA analysis using AmpFlSTR®Identifiler® PCR amplification kit .
In case number 1, accused and victim were in ‘live- in’ relationship. Victim gave birth to male baby. Baby was only 8 days old. Accused father of the baby told victim that baby is ill. I will show him to Doctor. He took the baby and killed him, thrown his body in the river. Complaint was lodged by mother of baby in Police station. Our laboratory received sternum, femur bone and scalp hair of baby including blood sample of both parents. Nuclear DNA analysis shows mismatch of father with son at only one locus.
In case number 2, five accused persons took advantage of girl in their village and established sexual relationship with her at various times threatening to her. She became pregnant and
gave birth to baby boy, but everyone denied their crime. Initially
victim told name of one of them and later on she told names of
another four who took advantage of her. Police authority sent
the blood samples of baby and his mother including five accused
to forensic Laboratory. Out of five, four accused excluded at
multiple loci, but one accused excluded at only one locus out of
In above both cases, to ascertain the progeny, we performed
Y DNA profiling of baby and accused father. Y DNA profile of both
son and father matched in both cases. This confirmed that they
belonged to same paternal progeny
Extraction of DNA from Blood: Routine organic extraction
i.e. Phenol/Chloroform extraction approach is a sensitive method
for the recovery of DNA from wide variety of forensic samples .
This method was used for extraction of DNA from blood samples.
In this method, samples were lysed using Forensic Buffer (pH8),
Proteinase K and Sodium Dodecyl Sulphate. It was incubated for
2 hrs at 560C and Phenol: Chloroform: Isoamyl alcohol added.
The aqueous layer containing DNA separated and treated with 2 M Sodium Acetate and the DNA was precipitated using chilled
Isopropanol. Precipitated DNA finally dissolved in TE buffer
(pH7). This DNA was further used for PCR and STR Genotyping.
Simultaneous amplification of 16 STR Loci was completed and
analyzed [9,10]. DNA profiles were interpreted by comparing
with each other.
We had seen mutation at one allele among son and father in
above mentioned both cases. To confirm profiles re-extraction
carried on Automate Express machine using Prep Filer TM
Express DNA extraction kit. The Prep Filer TM Forensic DNA
extraction Kit (Applied Biosystems, Foster City, CA) enables
the isolation of DNA from a variety of biological samples that
contain small quantities of biological mater4ial in such a way
that substances that interfere with PCR are removed. The Prep
Filer TMK it was designed specifically to support both manual
and automated extraction of DNA from forensic samples [11,12].
Extraction of DNA from Bone: Extraction of DNA from
sternum and femur bone was carried out on Automate express
Machine using Prep Filer Express BTA kit. Lysis of sternum
and femur bone powder (50mg) was performed using 220μl of
PrepFilerBTATM Lysis buffer. Then 1 M DTT and 7 μl Proteinase
K is added. The Lysis mixture was incubated at 560C for 18 hrs
at 1100 rpm using thermoshaker. After lysis the tube containing
bone sample was centrifuged for 2 min at 10,000 x g and then
the supernatant was transferred to the PrepFilerTM sample tube.
Then DNA was extracted on Automate Express machine using
the Prep Filer BTATM instrument protocol.
Quantified DNA of all the blood and bone samples from both
the cases was processed for PCR using AmpFlSTR®Identifiler®
PCR amplification kit  and AmpFlSTR®YfilerTM PCR
amplification kit on Veriti Thermal Cycler of Applied Biosystems
(Figure 1 & 2). AmpFlSTRTMIdentifilerTM primers amplify the STR loci CSF1PO, D2S1338, D3S1358, D5S818, D7S820,
D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA,
TH01, TPOX, vWA and gender marker Amelogenin. Yfiler enables
simultaneous amplification of 16 Loci on the Y-chromosome,
namely DYS456, DYS3891, DYS390, DYS38911, DYS458, DYS19,
DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA
H4, DYS437, DYS438 and DYS448.
Master mix used for Polymerase Chain Reaction wasa)
AmpFISTR PCR reaction mix: 10.5 μl
b) AmpFISTR Primer Set: 5.5 μl
c) Polymerase: 0.55 μl and 0.8 μl for Y DNA profiling.
d) Volume of Master mix used: 15 μl
e) Volume of sample: 10 μl
f) After PCR amplification Denaturation carried out using
HiDi Formamide and Liz 600 size Standard.
PCR produces millions of DNA fragments of different sizes.
Amplified products were separated and detected using 3500
Genetic Analyzer  and analyzed using GeneMapper®
ID-X Software V 1.5 according to manufacturer recommended
procedure. The separation of different fragments of DNA
molecules on the basis of their size was achieved by capillary
electrophoresis. Simultaneous amplification of 16 STR Loci was
completed and analyzed [9,10]. DNA profiles were interpreted
by comparing with each other.
In case one The DNA extracted from blood sample and bone
was typed at 15 STR LOCI and gender specific Amleogenin locus
using PCR Amplification technique
Table 1 Shows the Nuclear DNA profile of accused, victim and
femur bone of baby. Out of 15 different genetic systems analyzed
with PCR, accused matched the obligate paternal alleles present
in femur bone of baby at 14 STR loci except one locus ‘vWA’ which
may be due to mutation. As per two exclusion rules in parentage
testing, we cannot exclude father of child as true father if he
excludes at two genetic loci. However, for mother matched at all
15 STR Loci.
In Table 2, case number 2, out of 15 different genetic systems
analyzed with PCR accused matched the paternal alleles with
baby at 14 STR Loci except ‘vWA’. Here also chances of mutation
cannot be ruled out. Mother matched maternal alleles at all 15
In above both cases, to rule out the paternity, we had taken
support of Y DNA profiling to prove whether the progeny of child and father is same. This type of analysis can establish paternal
lineage though mutation observed in nuclear DNA profile. In
both the cases (Table 3 & 4) Y DNA profile (male haplotypes)
obtained from biological sample of baby matched with Y DNA
profile (male haplotypes) obtained from blood sample of father
As in both the cases, mutation was observed at one locus
between baby and father, that locus was same in both cases
which was ‘vWA’ (Table 1 & 2). Being baby was male, we had
an option of Y-DNA profiling to confirm the mutation and it was
found valuable technique to come over the conclusion that the
baby belongs to same progeny of the father (Table 3 & 4). It
means the father cannot be excluded as biological father of baby.
Analysis of 15 autosomal STR markers in parentage issues may
not be sufficient for conclusive results in all the cases. So, the
analyst should either take the support of kits having more than
15 Loci or should use the kits like Y-STRs to ascertain the results.
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(2001) Applied Biosystems. AmpFlSTR®Identifiler® PCR amplification kit user’s manual, part # 4323291 Rev. B. Foster City, CA: Applied Biosystems.
Applied Biosystems 3500/3500XL Genetic Analyzer User Guide Applied Biosystems, Faster City CA.