P-121
High throughput mitochondrial
DNA cloning in forensic and anthropological studies.
Hatsch D1,2, Amory S1,
Keyser-Tracqui C1,
Hienne R2,
Ludes B1
1EA 3428, Institut de Médecine Légale, Strasbourg, France
2Laboratoire CODGENE, Strasbourg, France
Mitochondrial
DNA is widely used in forensic and anthropological investigations. Therefore we
developed an in house high throughput mitochondrial DNA cloning method
targeting high speed at reduced costs.
A
home made T/A cloning vector was obtained after ddTTP tailing of a SalI
digested pUC19 vector. Due to this type of tailing, an extremely low background
is found. Amplified mtDNA fragments were directly cloned into this vector after
gel electrophoresis verification. PCR grade plasmid purification was performed
in 96-well blocks according to an adapted alkaline-lysis protocol. The obtained
plasmids were further sequenced on both strands and resulting sequencing
products were purified through ethanol precipitation.
This
method was applied in crime mixture analysis and in heteroplasmy determination
in ancient sample from Yakutia graves.
Such
a high throughput method is performed in the same time required by commercial
kits but with 20 times less costs. Thus it opens possibilities for its routine
use in forensic and anthropological laboratories.
Contact: didier@hatsch.net
P-122
Allele frequency data for 12
STR Loci in a population of North Germany
Heide K-G, Krause M
Labor für Abstammungsgenetik,
Kiel, Germany
12
Short Tandem Repeat ( STR ) loci TH01, vWA, D21S11, D18S51, FGA, D8S1179,
D3S1358, D7S820, D5S818, D13S317, D16S539, D2S1338 were analysed in a
Population geographically located in the north of Germany. We determined a sample of
3300 unrelated persons for paternity cases. Allele frequencies were calculated
for all 12 STR loci. No deviation from Hardy-Weinberg and genotype equilibrium
was observed.
krause@labor-krause.de
P-123
WHOLE GENOME AMPLIFICATION. A
USEFUL TOOL FOR THE INVESTIGATION OF FORENSIC SAMPLES?
Heinrich M1,2,
Brinkmann B1, Hohoff C1
1Institute of Legal
Medicine, University
of Münster, Münster, Germany
2present address:
Institute of Legal Medicine, University
of Freiburg, Freiburg, Germany
In
forensic genetics we are sometimes confronted with the fact that a sample
(e.g., a population sample) is running off, although several markers need to be
typed. The availability of commercially available whole genome amplification
(WGA) kits offer in principle the possibility to amplify the whole DNA in the
sample which in turns allows to type as many markers as necessary.
The
GenomiPhi kit (GE Healthcare, Freiburg,
Germany)
amplifies genomic DNA using the bacteriophage Phi29 DNA polymerase. The
theoretically exponential amplification of single- or double-stranded linear
DNA is performed in an isothermal strand displacement reaction. Due to the
proofreading activity of Phi29, the replication of the template DNA should be
extremely accurate.
We
have investigated whether the amplification of the whole human genome is
representative and analysed STR and SNP markers before and after WGA.
Data
on our experiences using cell lines, dilution series as well as artificial
stains will be presented.
P-124
A comparison of Y-chromosomal
binary polymorphisms in six populations from Germany, the Near and Middle East
Heinrich M1,2,
Nebelsieck H1, Alkhadam M1, Brinkmann B1,
Hohoff C1
1Institute of Legal
Medicine, University
of Münster, Münster, Germany
2present address:
Institute of Legal Medicine, University
of Freiburg, Freiburg, Germany
In comparison to short tandem
repeats (STRs) single nucleotide polymorphisms (SNPs) are more frequently found
sequence variations in the human genome and are thought to offer a lower
detection limit due to the possibility of creating very short amplicons. In
this study, we have investigated 29 binary polymorphisms on chromosome Y: 26
SNPs (M174, M45, Tat, M2, M170, M217, P25, M201, M304, M38, M207, M123, M35,
M128, P31, M216, M119, M173, M96, M122, M75, SRY1532, M168, M9, P2, M33), two
short insertions/deletions (INDELs: M17, M175) and the Alu-polymorphism
YAP.
Except for YAP
all markers were analysed in two multiplex reactions, comprising 10 and 18
markers, respectively. The amplification via PCR was followed by a purification
step using Exonuclease I and Shrimp Alkaline Phosphatase (SAP). Then, a
minisequencing reaction was performed using the SNaPshot kit (ABI, Darmstadt, Germany).
After an additional purification with SAP, the diagnostic fragments were
analysed using a 3100Avant Genetic Analyzer (ABI). The marker YAP was analysed by amplicon sizing using a native 8% PAA
gel with subsequent silver staining.
The six population samples are
as follows: one sample originated from North-Western Germany (Münster area),
three from the Eastern Mediterranean region of
Turkey
(Turkish and Arabian-speaking Eti Turks from Adana, Gypsies and Turks from
Kahramanmaraş area), one from Syria
and one from Afghanistan.
We investigated 100-120 as far as we know unrelated males in each population.
The haplogroup determination was performed according to M.A. Jobling and
C. Tyler-Smith (2003).
The haplogroup distribution within each population and a
population-genetic comparison including Y-STR data in the minimal haplotype
format of the six populations will be presented.
P-125
Pairwise
relatedness estimation: accounting for population substructure
Hepler A, Weir B
Department of Statistics, North Carolina State University,
Raleigh, North
Carolina, USA
The amount of
relatedness between two individuals has been widely studied across
disciplines. There are several cases in
which accurate estimates of this quantity are important in the forensic arena.
One common application is in the area of remains identification. In addition, there are several scenarios in
which pairwise relatedness estimates may be required in the courtroom. Many estimators of pairwise relatedness have
been proposed over the years, however none account for the potential effects of
population substructure. This could introduce an additional amount of
relatedness between the two individuals under consideration, which should be taken
into account when estimating pairwise relatedness. The objective of this research is to develop
a new maximum likelihood estimator of pairwise relatedness that accounts for
population substructure. We build upon
the foundation provided by earlier work in the area. A simulation study compares this new
estimator to the previous approach, using simulated populations with and
without inbreeding. We also evaluate the
new estimator using the CEPH family genotypes available online
contact: abhepler@stat.ncsu.edu
P-126
A cluster of six
closely linked STR markers: recombination analysis in a 3.6 Mb region at Xq12 –
13.1
Hering S1, Augustin
C2, Edelmann J3, Heidel M1, Dreßler J1,
Szibor R4
1Institute of Legal Medicine, Technical University of Dresden; 2Institute
of Legal Medicine, University of Hamburg; 3Institute of Legal
Medicine, University of Leipzig; 4Institute of Legal Medicine,
University of Magdeburg
Forensic use of X-chromosomal markers requires knowledge
about their linkage situation. Closely linked markers are inherited as
haplotypes. Searching for suitable tetranucleotide tandem repeats, we found a
cluster of three unutilized polymorphic markers located in the human X contig
NT_011669 (components AL049564 and AL049564) within 280 kb. These markers were
evaluated and submitted to the GDB and are now registered as DXS10079, DXS10074
and DXS10075.
To prove the stability of haplotypes within this
region of Xq12 we performed a recombination analysis. To obtain more
informative constellations, three known STR markers were included: DXS7132,
HumARA and DXS981 (STRX1). HumARA, which should not be used in forensic
casework, can be included in scientific research. Buccal swabs were collected
from 96 males with daughters and grandsons as anonymised samples. Primers were
designed according to GenBank information using the Primer3 software.
Amplification of the six markers was performed in two sensitive triplex PCR
assays. The resulting PCR products were resolved and detected by capillary
electrophoresis on the ABI Prism® 310 Genetic Analyzer.
In a German population study (693 males and 328
females) each locus of the three newly established STR markers exhibited 13
(DXS10079) and 14 (DXS10074, DXS10075) alleles by length, respectively.
Observed heterozygosity was 0.77 (DXS10079), 0.85 (DXS10074) and 0.67
(DXS10075). Since we cannot obtain any information on recombination in cases of
homozygous daughters, we included the three further STRs mentioned above: HumARA
is located about 50kb downstream of DXS10079, while DXS7132 and DXS981 are
outside the cluster which spans 3.6 Mb. Segregation of haplotypes involving the
six STRs mentioned is demonstrated in 96 trios consisting of grandson, mother
and grandfather. No recombination event was detected for the cluster at Xq12
investigated here. Hence, it can be concluded that the cluster DXS10079,
DXS10074 and DXS10075 segregates into stable haplotypes, providing a powerful
tool in kinship testing.
(contact: Sandra.Hering@mailbox.tu-dresden.de)
P-127
Further
sequence data of allelic variants at the STR locus ACTBP2 (SE33):
detection of a very short off-ladder allele
detection of a very short off-ladder allele
Hering
S1, Nixdorf R2, Edelmann J3, Thiede C4,
Dreßler J11Institute of Legal Medicine, Technical University of
Dresden;2Saxon State Criminal Investigation Office;3Institute
of Legal Medicine, University of Leipzig;4Department of Internal
Medicine, Carl Gustav Carus University Clinic, Technical University of Dresden
SE33 is one of the most
powerful STR markers in forensic use. A high number of length and sequence
variant alleles have been described, some of which may vary by as little as one
bp. The goal of this study is to add the sequence structure of some rare
variants to the known data, and examine a very short off-ladder allele which
has never been described before.Genetic characterization of more than 15,000
individuals (mainly Caucasians) was carried out using buccal cell swabs or
blood. Amplification of SE33 was performed either using the commercially available
multiplex PCR kits Nonaplex I and II (Biotype AG, Dresden, Germany)
or in a single PCR with the primer pair described by Polymeropoulus et al.
1992. Automated fragment analysis was carried out on the ABI PRISM®
310 or 3100 Genetic Analyzers. The direct Taq-cycle-sequencing method was
performed (following standard procedures).The study presents sequence
structures of regular alleles ranging from 8 to 38 in comparison with variant
alleles. The 120 bp 5’- flanking part and the 20 bp 3’- flanking part of the
central polymorphic region defined by Rolf et al. 1997 are included.A very
short off-ladder allele was found in a Somalian individual. Amplification with
Nonaplex II failed, indicating that there is a variation in the primer binding
region. Sequence analysis revealed a deletion of 15 tetranucleotide repeats in
the 5’ flanking region. A further allele originating from a Portuguese
individual with 28 bp deletion in the 5’ flanking region resulted in allele
length 9. The relatively frequent allele 6.3 was sequenced in four different
Caucasians showing an identical repeat structure. We found three classes of X.1
alleles: firstly, alleles ranging from 12.1 to 18.1 resulted from a single A
insertion between the AAAG repeats in the central region; secondly, two alleles
15.1* and 18.1* deviated in their structures by a deletion of AAA in the 5’
flanking region; and thirdly, by contrast, longer alleles 21.1 and 32.1
resulted from insertion of a single base pair (G or A) in the central repeat
region. We found that only half of the variant alleles have insertions or
deletions within the central region. Therefore, it is difficult to compare our
sequence structures with the existing data. However, although the short X.1 and
X.3 alleles are rare, accuracy in SE33 typing analysis is important for
distinguishing these from the common alleles.
References: Polymeropoulos
MH, Rath DS, Xiao H, Merril CR (1992) Nucleic Acids Res 20: 1432
Rolf B, Schürenkamp
M, Junge A, Brinkmann B (1997) Int J Legal Med 110: 69-72
(contact:
Sandra.Hering@mailbox.tu-dresden.de)
P-128
Allele frequencies for Penta D and Penta E in three
populations from Germany
and Hungary
C. Hohoff1, G. Nagy2, J. Bartsch1, I. Bajnóczky2, B. Brinkmann1
1 Institut für Rechtsmedizin, Universitätsklinikum Münster, Germany
2 Institute of Forensic Medicine, University of Pecs, Hungary
We here present the frequency
distributions of the autosomal STR systems Penta D and Penta E in samples from
unrelated 188 Germans (Münster area), 115 Hungarian Caucasian and 116 Hungarian
Roma (Pecs area).
Genomic DNA was extracted according to standard
techniques (e.g., Proteinase K / Chelex-100) and amplified utilizing different
amplification approaches (Powerplex16 or a Penta D/E duplex based on the
published Promega primer sequences). PCR products were separated by capillary
gel electrophoresis on an ABI PRISM 310 Genetic Analyzer and typed by
comparison against sequenced allelic ladders.
A variant allele
12.3 was observed in a Hungarian sample and characterized by sequencing after
cloning.
Both pentanucleotide STR systems are
highly informative markers in the three populations investigated, e.g., the
power of discrimination ranges from 0,897 (Penta D, Roma) to 0,977
(Penta E, Germans).
Address for correspondence
Prof. Dr. med. Bernd Brinkmann, Institut für
Rechtsmedizin, Universitätsklinikum Münster, Röntgenstrasse 23, D-48149
Münster, Germany, Fax: 00 49 (0) 251 8355158, eMail: remed@uni-muenster.de
P-129
Y-STR
analysis of Australian Aborigines
Carsten Hohoff, Ursula Sibbing and Bernd Brinkmann
Institut für Rechtsmedizin, Universitätsklinikum
Münster, Germany
We present the frequency distributions of
15 Y-specific STR polymorphisms (DYS19, DYS385, DYS389 I and II, DYS390,
DYS391, DYS392, DYS393, YCAII, DXYS156-Y, DYS437, DYS438 and DYS439) and the
frequency of the combination of these haplotypes in a population sample of male
Aborigines from Australia
(Adelaide
area).
DNA, that had been extracted from blood of 51 male Australian
Aborigines, served as template to amplify the Y-STR loci by means of different multiplex or
singleplex approaches. PCR amplicons were analyzed on an ABI PRISM 310 Genetic
Analyzer with GenoTyper software (Applied Biosystems) and sequenced allelic
ladders.
In the 51 samples, 40 different haplotypes
were observed. Of them, 32 haplotypes were unique and the others were shared by
2 or 3 persons.
A YHRD search revealed only 3 matches,
most likely due to the fact that until now no entries have been made for the
Aborigines population.
Address for correspondence
Prof. Dr. med. Bernd Brinkmann, Institut für Rechtsmedizin, Universitätsklinikum
Münster, Röntgenstrasse 23, D-48149 Münster, Germany, Fax: 00 49 (0) 251
8355158, eMail: remed@uni-muenster.de
P-130
Experiences from the ante mortem and post mortem DNA-analysis in Sweden for the identification of tsunami victims
Gunilla Holmlund, Iréne Lodestad, Helena
Nilsson and
Bertil Lindblom
The
National Board of Forensic Medicine, Department of Forensic Genetics, University Hospital,
SE - 581 85
Linköping, Sweden
After the tsunami catastrophe
in the Indian Ocean, December 26, 2004 more than 15 000
Swedish citizens were initially reported missing. Planning for DNA-analysis of
samples from relatives, ante mortem as well as of deceased, post
mortem, started just two days later.
The collection of reference
samples from relatives was started almost immediately and the first samples
were received on January the 4th. About 730 samples, of which 113
were from the PKU-bio bank were collected within a few weeks. After an official
request by the Swedish police the DNA analysis started on January the 12th.
550 samples from the genetically best references were analysed by mid February.
The number of persons missing was by then about 550.
At the beginning of March
several laboratories got an initial request, followed by an official at the
March 7th to participate in the analysis of post mortem
samples. We accepted to receive 500 – 600 samples, to be analyzed within 6
months after an initial quality test of 10 samples. Our quality was accepted
and on April 5th we got 600 post mortem samples to analyse.
Since the work is at best
going on we cannot here report results but hope to present an overview of our
participation in this work at the meeting.
Address
for correspondence: Gunilla Holmlund, The National Board of Forensic Medicine,
Department of Forensic Genetics, University Hospital, SE - 581 85 Linköping, Sweden.
E-mail: gunilla.holmlund@rmv.se
P-131
Y-SNP typing with the
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Hou YP, Shi MS, Liao LC, Yan
J, Zhang J, Wu J, Li YB
Department of Forensic
Genetics, Sichuan
University (West China
University of Medical
Sciences), Chengdu,
P.R.China
The single
nucleotide polymorphisms on Y chromosome (Y-SNP) were potential markers for
analysis of mixed biological stains in sexual assault cases and played a role
on forensic science. The purpose of our work was to establish a method for
analysis of Y-SNP based on the matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry. To explore the single nucleotide
polymorphisms on Y chromosome, a technique of primer extension was employed for
the analysis of the matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry. Our study showed that Y-SNP typing with the matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry yielded reliable
results. The results of our study implied that the analysis of Y-SNP was proved
to be suitable for forensic application and provided new genetic markers for
the forensic purpose
contact: forensic@mail.sc.cninfo.net
.
P-132
Molecular Evidence for the
Association of Persian Ethnicities
Massoud Houshmand, Arman
Ardalan, Mehdi Shafa Shariatpanahi, Mohammad Hossein Sanati.
National
Institute for Genetic Engineering and Biotechnology
Persia is an upland
long inhabited by miscellaneous tribes of many languages and cultures.
Linguistic remarks point out a close relatedness among majority of the core
population, as well as rather farther relations with the marginal aboriginals.
However, not only in a medical context accounting for genetic disorders, but
also from an evolutionary point of view there has been no adequate genetic data
to support this. In the first phase of a bigger project, we sequenced (at least
25 individuals of each ethnic group) the D-loop region of the mitochondrial DNA
(mtDNA) from 15 different ethnic groups (Pars [5different area], Kurd, Lor,
Bluch, Sistani, Gilani, Mazandrani, Turk, Armani, Jews, Arab, and Turkman).
Hypervariable nucleotide sequences were next aligned and compared through a
pairwise distance method. The neighbor-joining phylogenetic tree was drawn
using MEGA software package for the operational taxonomic units (OTUs) of the
average haplotypes in each group, together with some relevant GenBank
retrievals.
P-133
Population genetic analysis in
a Libyan population using the PowerplexTM 16 system
Immel U-D1, Erhuma
M2, Mustafa T3, Kleiber M and Klintschar M1
1Department of Legal
Medicine, University of Halle,
Germany
2Institute of Medical
Immunology, University of Halle,
Germany
3University Clinic of
Oral and Maxillo-Facial Surgery, University
of Magdeburg, Germany
Polymorphic
short tandem repeats (STRs) have become the markers of choice for forensic
purposes such as paternity testing and personal identification.
In
this study we present the results of a survey aimed at investigating the allele
and genotype frequency distribution of 15 loci amplified by the GenePrint®
PowerPlexTM 16 system (Promega) in Libya. DNA was isolated from blood
samples. 103 unrelated individuals were included in the database. Amplification
products were analyzed by capillary electrophoresis using the ABI 310® Genetic
Analyzer (Applied Biosystems).
Statistical
analysis was carried out using various statistical methods (Hardy-Weinberg-
Equilibrium, Mean Exclusion Power, Discrimination Power, etc.) to determine
allele frequencies and other population parameters of interest.
Address for correspondence:
U.-D. Immel, Institut für Rechtsmedizin, Martin-Luther University
Halle-Wittenberg, Franzosenweg 1, 06112 Halle/Saale, Germany, Email: uta.immel@medizin.uni-halle.de
P-134
Y-chromosomal STR haplotypes
in an Arab population from Libya
Immel U-D1, Erhuma
M2, Mustafa T3, Kleiber M and Klintschar M1
1Department of Legal
Medicine, Martin-Luther Universität Halle-Wittenberg, Halle/Saale, Germany
2Institute of Medical
Immunology, University of Halle,
Germany
3University Clinic of
Oral and Maxillo-Facial Surgery, University
of Magdeburg, Germany
Y-chromosomal
microsatellites (STRs) have been established in forensic practice for several
years. However, an in-depth evaluation of their population genetic properties
requires a large number of haplotypes from different populations. We therefore
analysed the Y-chromsome with eight Y-chromosomal STRs (DYS385, DYS19, DYS 389I
and II, DYS390, DYS391, DYS392, DYS393) in an Arabic population sample of 64
males from Libya.
DNA
was extracted from unrelated male blood samples according to standard Qiagen
procedures. Amplifications were performed using fluorescent dye labelled
primers according to Elmoznino and Prinz (//ystr.charite.de). The PCR products
were analyzed by capillary electrophoresis using the ABI 310® Genetic Analyzer
(Applied Biosystems).
The
results and the haplotype diversity were compared with data from other Arab
populations.
Address for correspondence:
U.-D.
Immel, Institut für Rechtsmedizin, Martin-Luther University Halle-Wittenberg,
Franzosenweg 1, 06112 Halle/Saale, Germany,
Email: uta.immel@medizin.uni-halle.de
P-135
Evaluation of Lewis genotyping by four PCR-based
methods
Y. Itoh1, K. Satoh1，2, K. Takahashi1，2, K. Maeda3, T. Tokura3 and R. Kobayashi1,4,
1Department of
Forensic Medicine, Juntendo
University School
of Medicine, Tokyo,
Japan
2 Medico-Legal
Section, Criminal Investigation Laboratory, Metropolitan Police Department, Tokyo, Japan
3Atopy Research Center, Juntendo University
School of Medicine, Tokyo, Japan
4Department of
microbiology, Tokyo Medical University, Tokyo, Japan
The antigenic epitope of CA19-9, i.e. sialyl
Lewis A antigen, has been used clinically as a tumor marker for pancreatic
cancer, colorectal cancer, and certain other malignancies. The synthesis of CA19-9, however, is complex
because there are three genes involved; Lewis genes encoding Le transferase (-1, 4-fucosyltransferase), secretor gene encoding Se
transferase (-1, 2-fucosyltransferase), and
the gene encoding sialyltransferase. Through the biosynthetic pathway, Le
transferase is thought to be a key enzyme.
The activity is genetically controlled by Lewis genotypes. Lewis phenotype Le(a-b+) or Le(a+b-) groups
have Le allele. The Le(a-b-)
group divided into two groups, genuine Le(a-b-) and non-genuine Le(a-b-) by the
results of Lewis genotypes. Genuine
Le(a-b-) groups have no Le allele, while non--genuine Le(a-b-) groups
have Le allele. Le is a
functional allele, and le1 is non-functional allele.
We developed PCR-based methods, confronting
two pair primers (PCR-CTPP) and sequence-specific-primers with PCR positive
control (PCR-SSPPC) to analyze a SNPs at nucleotide position 59 to reflect Le
transferase activity, which were analyzed by ABI PRISM® 3100 genetic analyzer. And we compared 4
kinds of PCR-based methods, sequence-specific-primers (PCR-SSP), restriction
fragment-length polymorphism (PCR-RFLP), PCR-CTPP and PCR-SSPPC. We found that all of these methods could be
applied to determine Lewis genotyping correctly. The frequencies of Le and le
alleles were 67.2% and 32.8% respectively.
Both PCR-CTPP and PCR-SSPPC for Lewis genotyping are simple, reliable and applicable for forensic and clininal
investigation.
Address correspondence to:
Dr. Y.Itoh
Department of Forensic Medicine, Juntendo University School
of Medicine, Hongo,
Tokyo 113-8421, Japan.
TEL: +81-3-5802-1051
FAX: +81-3-5802-1050
E-MAIL:yitoh@med.Juntendo.ac.jp
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