P-013
Population study of
small-sized short tandem repeat in Japan and its application to
analysis of degraded samples
Asamura H, Tsukada K, Ota M, Sakai H, Takayanagi K,
Kobayashi K, Saito S, Fukushima H
Department of Legal Medicine, Shinshu University School
of Medicine
Analysis
of short tandem repeat (STR) is the most valuable tool for the clarification of
personal identity. Recently, several commercial multiplex STR kits are
regularly used in forensic practice. However, for some highly degraded samples,
analysis of STR by means of the commercial STR kits is practically impossible
due to DNA fragmentation and occurrence of PCR inhibitors. Some papers reported
that smaller–sized PCR products were effective in analyzing such highly
degraded samples. We previously performed multiplex PCR for the TH01, TPOX,
CSF1PO, and vWA loci using a newly designed pair of primers that yield smaller
fragment, and reported several successful analyses of the degraded samples. In
this study the six mini-STR loci (D1S1677, D2S441, D4S2364, D10S1248, D14S1434,
and D22S1045), which Coble et al. reported, were investigated in Japanese
population. As the result, analysis with use of the six loci demonstrated the
moderate degree of polymorphisms in Japanese population. Moreover, it was
confirmed that these six loci assays for typing degraded samples were more
successful than those of commercial STR kits. Consequently, it was considered
that combination analyses of the six mini-STR loci and the four loci, which we
previously reported, are highly beneficial in the context of Japanese forensic
practice from degraded DNA samples.
P-014
Allele distribution of two X
chromosomal STR loci in a population of Sicily
(Southern
Italy)
Asmundo A., Perri F., Sapienza
D.
Dipartimento
di medicina sociale del territorio, Sezione di medicina legale, Università di
Messina, Azienda Ospedaliera Universitaria
“Policlinico G. Martino”, Via Consolare Valeria 98125 Messina, Italy
Population genetic data for
two X-chromosomal STR loci (DXS7423 and DXS9902) were obtained by analysing a
population sample (n= 60 males and 60 females) and 43 family trios (showing a
probability of paternity and maternity > 99.9%) from Sicily (Southern Italy)
by using PCR and PAGE followed by silver staining. Five and four different
alleles of DXS7423 and DXS9902 loci were detected, respectively. The allele
frequencies of both ChrX markers were in good agreement with Hardy-Weinberg
equilibrium. The analysis of the family trios, based on the investigated
meiotic events, showed no mutation. The observed eterozigosity of DXS7423 and
DXS9902, together with other forensic parameters were determined, so
confirmating that these markers are useful tools for parentage testing, mainly
in deficiency paternity cases when the disputed son is female.
P-015
Forensic evaluation of three
closely linked STR markers in a 13 kb region at Xp11.23
Augustin C1, Cichy
R1, Hering S2, Edelmann J3, Kuhlisch E4,
Szibor R5
1Institute of Legal
Medicine, University of Hamburg,
Germany
2Institute of Legal
Medicine, Technical University of
Dresden, Germany
3Institute of Legal
Medicine, University of Leipzig,
Germany
4 Institut für Medizinische Informatik und
Biometrie, Technical University of Dresden, Germany
5Institute of Legal
Medicine, University of Magdeburg,
Germany
Searching for markers located in the Xp11 region the
sequence of the clone AF196972 was checked for its content of microsatellites.
Two tetranucleotide STRs and one trinucleodide STR were tested in respect of
their forensic efficiency and registered in the GDB as DXS10076, DXS10077 and
DXS10078.
DXS10076 is located 48 065.564 - 48 065.759 kb from
the Xptel. DXS10077 and DXS10078 are located further 7.701 kb and 12.879 kb
downstream, respectively.
The three STRs differ clearly in their
individualization capacity as could be shown in a population sample of 201 male
and 151 female Germans. Whereas at Locus DXS10076 10 alleles and at Locus
DXS10078 13 alleles could be detected resulting in PIC and HET values of 0.767
and 0.747 (DXS10076) and 0.811 and 0.861
(DXS10078), respectively, the trinucleotide STR DXS10077 consists of 5 alleles
leading to much lower PIC (0.492) and HET (0.507) values.
Two paternal mutations were detected at DXS10078 in
150 families with confirmed paternity while no mutations could be found until
now at the other two loci.
Theoretically, this cluster could give rise to 650 different
haplotypes in the German population. In fact, genotyping of 201 males revealed
the presence of 72 haplotypes. Due to their closely linked location the three
STRs form a cluster free of recombination. The stability of haplotypes was
tested in 90 three-generation families. Hence, the Xp11.23 STR cluster reported
here can contribute to solving complex kinship cases. Special aspects such as
linkage disequilibrium etc. will be discussed in detail.
P-016
Danger of false inclusion
among deficient paternity case
Babol-Pokora K, Jacewicz R, Szram S
Department of Forensic
Medicine, Medical University
of Lodz, Lodz, Poland
Deficient
cases, such as motherless cases, are more difficult then standard ones, but a
conclusion can still be derived, based on the types of the child and alleged
father. More complicated are cases in which the alleged father is unavailable
for testing – these are difficult to calculate. But the real problem is when
both mother and father are unavailable. Such cases can be burdened with danger
of false inclusion.
In our practice we had a deficiency case, in
which the alleged father was unavailable, so we had to test his parents. We
used Identifiler™ system and there was no exclusion in that case, when we typed
the child and his grandparents only. The probability of paternity that we
obtained was 99,9 percent. Mother’s typing, however, revealed exclusions in 3
STR loci among the Identifiler™ system. Further researches showed exclusions in
4 STR loci out of 21 tested STR loci.
P-017
IDENTIFILER™ system as an
inadequate tool for judging deficient paternity cases
Babol-Pokora K, Jacewicz R, Szram S
Department of Forensic
Medicine, Medical University
of Lodz, Lodz, Poland
Identifiler™
is known to be one of the most useful multiplex systems for standard paternity
testing. But in some cases the mother is unavailable and there can be a problem
with obtaining sufficient value of probability of paternity. We typed nine
hundred unrelated individuals from Central Poland
population (Lodz
region), in order to check the usefulness of Identifiler™ for analysis of
motherless cases. The most important thing was to compare the evidence value
between standard cases (trios) and deficient ones (duos). One hundred and fifty
excluding cases and one hundred and fifty including ones were analysed and the
results were estimated for trios and duos. Power of exclusion and paternity
index were analysed for each locus as well as for the entire set of the fifteen
STR markers. Our researches confirmed the usefulness of Identifiler™ system for
standard paternity testing, and showed that the minimal probability of
paternity that can be obtained, is 99,999 percent. In motherless cases however,
the average value of probability of paternity was as low as 99,9 percent. The
minimal number of excluding loci among trio cases was four, whilst among
duo ones there were events of exclusion in one locus only. That is why
Identifiler™ is proper for standard paternity cases, however motherless cases
need to be examined more widely.
contact: katarzynababol@wp.pl
P-018
Is SGM Plus™ the sufficient
system for paternity testing?
Babol-Pokora K, Jacewicz R, Szram S
Department of Forensic
Medicine, Medical University
of Lodz, Lodz, Poland
SGM Plus™ is
one of the multiplex systems commonly used in forensic genetics. It consists of
10 STR loci which can be useful for parentage testing. We present results of
typing in nine hundred unrelated individuals from Central
Poland population (Lodz
region), in order to check the usefulness of SGM Plus™ system for paternity
testing. One hundred and fifty excluding cases and one hundred and fifty
including ones were analysed in the range of 10 STR loci of SGM Plus™
system and the results were estimated for standard and motherless cases. Power
of exclusion and paternity index were analysed for each of ten loci as well as for entire SGM Plus™ set. Our researches showed that the SGM Plus™ is
not sufficient for parentage testing. The minimal number of excluding loci
for SGM Plus™ analyses was one among duo cases and two among trio ones and
there was an event of false inclusion, which was revealed after Identifiler™
analysis. Additionally the number of excluding loci among twenty seven percent
of duo cases was less than four. The
probability of paternity in almost sixty percent of trio cases was 99,99
percent and lower. Additionally the average value of probability of paternity
among duo cases was as low as 99,0 percent. That is why we consider SGM
Plus™ not to be sufficient for paternity
testing among deficient cases as well as standard ones.
P-019
The Beneficial Effect of
Extending the Y Chromosome STR Haplotype
Ballard DJ, Khan R, Thacker
CR, Harrison C, Musgrave-Brown E, Syndercombe Court D
Centre for Haematology, ICMS,
Barts & The London,
Queen Mary’s School
of Medicine &
Dentistry, UK
Y chromosome
testing is becoming a more frequently used technique both in criminal and
relationship cases. One drawback with
this method however, is the relatively low haplotype diversity when compared
with autosomal DNA profiles. The
consequence of this lower diversity is that individuals can present with the
same Y-STR haplotype even if they are not closely related.
During the development of Y chromosome
testing there was a scarcity of known Y-STR loci and it is only recently that
larger numbers of polymorphic markers have been discovered. Haplotype diversity is therefore partially
compromised in standard forensic Y chromosome testing protocols by the need to
use the established markers, not all of which are highly polymorphic. We have taken a number of the recently
discovered highly polymorphic markers and analysed them in addition to the
standard set of Y chromosome loci.
Presented here are the allele frequencies for these loci in the British
population along with the resulting increase in haplotype diversity associated
with their incorporation. Also detailed
is a relationship case that demonstrates the advantages that additional
informative Y chromosome loci can confer when used in forensic casework.
P-020
Application of Whole Genome
Amplification for Forensic Analysis
Balogh MK1,
Børsting C2, Sánchez Diz P3, Thacker C4,
Syndercombe-Court D4, Carracedo A3, Morling N2,
Schneider PM1, and the SNPforID Consortium
1 Institute of Legal Medicine, Johannes Gutenberg
University of Mainz, Germany
2 Institute of Legal
Medicine, University of Santiago de
Compostela, Spain
3Department of
Forensic Genetics, Institute
of Forensic Medicine, University of Copenhagen, Denmark
4Centre for
Haematology, ICMS, Barts and The London,
Queen Mary's School
of Medicine and
Dentistry, UK
www.snpforid.org
Fundamental to most forensic
analysis is the availability of genomic DNA of adequate quality and quantity.
To perform a multitude of genetic analysis and assays requires sufficiently
large amount of template. However, DNA yield from forensic samples is
frequently limiting. Whole Genome Amplification appears to be a promising tool
to obtain sufficient DNA amounts from samples of limited quantity. The WGA
method is based upon the “Strand-Displacement Amplification" approach used
in rolling circle amplification. The exponential amplification process
theoretically enables the amplification of DNA from one single cell up to a
million-fold. Therefore the main purpose of our study was to systematically
investigate its sensitivity, accuracy and suitability for DNA diluted with
quantities of 50, 100, 150, 250 and 500pg. We have performed the study using
diluted DNA from two cell lines, HepG2 and K562. The WGA reactions were
repeated five times, followed by STR PCR carried out twice for each cell line
and dilution. To generate sufficient data, to assess the sensitivity, accuracy
and suitability of the Whole Genome Amplification four laboratories were
included in this study.
WGA was found to be very
efficient, all sample dilutions amplified well, and the amplification yield
does not relate to the amount of input DNA. In general ~500ng/µl were obtained,
independently of the amount of target DNA. However, reliable STR amplification
was dependent on the DNA quantity used for WGA. Consistent and reliable STR
typing was only obtained using 500pg genomic DNA. Dropouts and allelic
imbalance started to occur at 250pg and more dramatically at 100 and 50pg.
Therefore the usefulness of
WGA in forensic casework is limited, however the method may be very useful for
saving rare samples provided that the DNA is of adequate quality.
P-021
DNA typing from 15 years old bloodstains
Barbaro A.1,
Cormaci P1 and Barbaro A.2
1 Department of Forensic Genetics, 2Director
of SIMEF
SIMEF - Reggio Calabria-ITALY
www.simef.com
- e-mail: simef_dna@tiscali.it
The aim of this study is to compare the efficiency of different
validated methods for DNA extraction on old bloodstains. The study has been
performed on bloodstains placed on a cotton surface, stored at room temperature
for 15 years. As reference were used liquid blood samples,stored at –20°C,
belonging to the same donors above. DNA has been extracted from all samples
using different procedures (chelex, paramagnetic silica particles, silica
membrane column, desalting procedure), then quantified in Real-Time PCR by the
Quantifiler Human DNA Quantification kit (Applied Biosystems) and
amplified by AmpFlSTR Identifiler kit (Applied Biosystems).
We've evaluated the ability of each method to
extract DNA, the quantity of human DNA extracted with each procedure, the
ability to perform multiplex STRs amplification and the reproducibility of
results obtained .
P-022
Multiplex STRs amplification
from hair shaft validation study
Barbaro A.1,
Cormaci P1 and Barbaro A.2
1 Department of Forensic Genetics, 2Director
of SIMEF
SIMEF - Reggio Calabria-ITALY
www.simef.com
- e-mail: simef_dna@tiscali.it
Mt-DNA analysis, that is
widely used in forensic genetics in case where the amount of DNA is very small
or degraded, is unfortunately a complex and time-consuming procedure, so, since
several years in other our previous papers, we've showed the possibility to
amplify in single-plex DNA extracted from hair shaft. Now in the present study
we've evaluated the ability to perform multiplex STRs amplification and the
reproducibility of results obtained.
In particular we
analysed 20 hair shafts beloging to known donors (2 male and 2 female) using
different DNA extraction procedures (fenol-clorophorm, paramagnetic silica
particles, silica membrane column,chelex).
Extracted DNA has been
quantified by Quantifiler Human DNA Quantification kit (Applied
Biosystems) using a 7300 Real-Time PCR System and amplified by AmpFlSTR
Identifiler and AmpFlSTR Y-Filer kits (Applied Biosystems).
Amplified samples have been
analyzed on an ABI PRISM 3130 multicapillary sequencer.
As reference were used saliva
samples coming from the same hairs donors.
We verified that in some cases
where there's a sufficient quantity and a good quality of medulla cells inside
the hair stem a multiplex amplification can be performed and this is very useful
for obtaining in a single step the typing of many loci avoiding the loss of
DNA.
The ability to identify STRs
markers in difficult samples as hair shafts gives a great opportunity to obtain
DNA profiles useful for any further comparison or searching in DNA database.
P-023
LCN DNA typing from touched objects
Barbaro A.1,
Cormaci P1 and Barbaro A.2
1 Department of Forensic Genetics, 2Director
of SIMEF
SIMEF - Reggio Calabria-ITALY
www.simef.com
- e-mail: simef_dna@tiscali.it
A married beautiful
woman received to her home, in different slots, 2 envelops containing
pornographic photos and indecent proposals from an anonymous persistent
admirer.
The woman sent the
material above to our laboratory for latent prints development and for
searching biological traces for DNA typing.
While latent prints
research gave negative results, we were able to found on the stamps some saliva
traces useful for DNA analysis. STRs typing showed that both stamps were licked
by the same male individual.
Since the husband
of the woman suspected a colleague, after some weeks from the analysis above,
he brought us two marking pens (one red and the other one black), that the man
was used to utilize at the workplace, for performing DNA typing from any
eventual sweat/skin residual found on them, with the aim to compare DNA
profiles obtained with the one from stamps.
We were able to
obtain from biological traces on the red marking pen a mixed DNA profile, while
from the black pen we had a partial DNA profile: all profiles found matched
with the one from the stamps.
So DNA analysis
confirmed the hypothesis: the husband colleague was the bother perpetrator.
This casework is a
further confirmation that it’s possible to type LCN DNA with very good results
if an appropriate collection and analysis of biological material is performed.
P-024
X-STRs typing for an
identification casework.
Barbaro A.1,
Cormaci P1 and Barbaro A.2
1 Department of Forensic Genetics, 2Director
of SIMEF
SIMEF - Reggio Calabria-ITALY
www.simef.com
- e-mail: simef_dna@tiscali.it
X-STRs have been
proven to be useful in case of deficiency paternity testing and
in effective mother-son kinship and father-daughter testing. Male individuals inherit their one X-Chr from
their mother, while female individuals receive one X from the mother and the
other one from the father. So, female individuals fathered by the same man
share their paternal Chromosome X.
Hence in case of
deficiency paternity in which the mother is available for typing, the possible
X alleles of the putative father can be determined and the paternal profile can
be reconstructed.
In the present
casework we used X-STRs for the identification of a biological material
supposed to be belonging to a girl disappeared from several years. In fact in
the house of a man (suspected to be the author of another woman murder) was
found a headscarf similar to a one obelonging to the girl and inside it some
hairs. In absence of any biological sample belonging to the disappeared girl we
verified the relationship between hairs above and the mother and the sister of
the disappeared girl.
In particular we
used Mentype® Argus X-UL that is a new kit commercialized by Biotype
for fast and reliable profiling of the following 5 unlinked X chromosomal STRs
markers DXS8378, DXS7132, HPRTB, DXS7423 and Amelogenin.
Additionally we
investigated in triplex DXS101, DX6789, HumSTRX1 and in duplex GATA1872D05,
DX7133 using MWG-Biotech primers and our own amplification protocols.
By comparison
between DNA profiles it was possible to identify in the woman, that was surely
daughter of the not available father, the paternal possible X alleles and then
to verify the presence in the questioned samples of maternal and paternal
X-STRs.
The present case
demonstrates the impact of additional X-STRs markers in special reverse
paternity case that cannot be solved using autosomal markers.
P-025
Study of 16 Y-STRs
in the population of Calabria
using AmpFlSTR Y-filer kit
Barbaro A.1,
Cormaci P1 , Falcone G. and Barbaro A.2
1 Department of Forensic Genetics, 2Director
of SIMEF
SIMEF - Reggio Calabria-ITALY
www.simef.com
- e-mail: simef_dna@tiscali.it
Y-STRs are very useful for
forensic laboratories to identify and analyse male DNA from evidence-containing
mixtures of male and female DNA (for example in case of sexual assault), in
difficult paternity analysis or for reconstruction of male lineage or
application in kinship analysis.
AmpFLSTR® Yfiler™ PCR
Amplification kit is the last commercial kit for Y-STRs analysis produced by
Applied Biosystems. It uses the 5-dyes chemistry for co-amplification, in a
single PCR reaction, of 16 Y-chromosome STRs (DYS456, DYS389I, DYS390,
DYS389II, DYS458 DYS19 DYS385 DYS393 DYS391 DYS439 DYS635 DYS392 YGATAH4 DYS437
DYS438 DYS635 DYS448), including the European Minimal Haplotype loci, the loci
recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM)
and 6 additional highly polymorphic loci.
In the present study we
analysed the distribution of the Y-STRs above in 3 populations from a Southern Italy region: Calabria.
In particular DNA was
extracted, by Instant Gene Matrix (Biorad) treatment, from blood/saliva samples
of male unrelated healthy donors (100 per each area), since 3 generations, at
least, belonging to the populations of Reggio Calabria, Catanzaro and Cosenza.
All samples were
quantified by the Quantifiler™ Human DNA Quantification Kit using a 7300 Real Time System and then amplified
according to the Yfiler™ kit protocol using GeneAmp PCR Systems
9600,9700,2400,2720 thermal cyclers (Applied Biosystems). Female and Male
Positive controls and negative controls were used during all amplification
steps.
Amplified products were
analyzed by capillary electrophoresis on ABI PRISM 310 and ABI PRISM 3130
Genetic Analyzers (Applied Biosystems) employing Genotyper and GeneMapper 3.2
softwares.
P-026
Male contribution in the constitution of the
Brazilian Centro-Oeste populations estimated by Y-chromosome binary markers
Barcelos RSS1,2,3, Ribeiro GGBL1,
Silva Jr. WA4, Abe-Sandes K4,5, Godinho NMO1,2,
Marinho-Neto F7, Gigonzac MAD7, Klautau-Guimarães MN1,
Oliveira SF1.
1Departamento de Genética e Morfologia, Universidade de
Brasília, Distrito Federal, Brazil; 2Superintendência de
Criminalística, Secretaria de Segurança Pública do Estado de Goiás, Brazil; 3Departamento
de Biomedicina, Universidade Católica de Goiás, Brazil; 4
Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade
de São Paulo, Brazil; 5Departamento de Ciências da Vida,
Universidade do Estado da Bahia, Campus I - Salvador, Bahia, Brazil.
Due to Brazil’s
large dimension, this country is divided in five geo-political regions: South,
Southeast, Center-West, North and Northeast. The Center-West region is the
subject of our study and is composed by three states - Goiás, Mato Grosso and Mato Grosso do Sul - and the Federal District. The settlement of this territory, which
was a result of the miscegenation among different ethnic groups, especially
Europeans, Africans and Amerindians, did not happen in a homogeneous way, which
reflects in the current genetic population composition. Meanwhile the Brazilian
colonization was initiated in XVI century, the Center-West region settlement
took place only after XVII century and the Federal
District, where is placed the Federal capital (Brasília), was
founded in the late 1950s. Differently from the others Brazilian’s regions, the
colonization of Center-West region was derived from internal migrations of
already mixed individuals from all others Brazilian regions. Another Brazilian
peculiarity is the directional mating between European males and Amerindian or
African females. Therefore, after consider these characteristics, could this
region be considered as the best representative population group of the
Brazilian population? How is the male constitution in the Brazilian
Center-West? Seeking answer these questions, we studied eleven unique-event
polymorphism (UEPs), located in the non-recombinant region of the Y chromosome,
in 200 unrelated men from Goiás state
and Federal District. The results showed that
the last population presented a greater genetic diversity than the first one,
which reflects in a low divergence between these two populations. The greater
genetic diversity of Federal District
corroborated the historic data of migrations from all regions of the country
and indicated this population as the most representative group of the Brazilian
population genetic constitution. The most common haplogroup in this survey,
P92R7, presents a wide geographic distribution. However, due to Brazilian
settlement history, its presence may reflect a European contribution. The
contribution estimated using European haplogroups was similar in both
population and is greater than the African one. It was also observed a little
male contribution from Amerindian to the constitution of both populations and
from Japanese only to Federal District
constitution. These results demonstrated a greater male contribution of
Europeans than Africans or Amerindians to the formation of both populations,
which corroborated the historic data of this region settlement.
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