P-089
Adoption of automated DNA processing for high
volume DNA casework:A combined approach using magnetic beads and real-time PCR
Frégeau
CJ1, Lett M1, Elliott J1, Bowen KL1,
White T2, Fourney RM1
1National
DNA Data Bank Royal, 2Evidence Recovery Unit, Canadian Mounted
Police, Ottawa, Ontario
In the past five years, the National DNA Data
Bank of Canada
has successfully processed over 75,000 blood, buccal or hair samples from
criminal offenders using a fully integrated and automated approach requiring
very little human intervention. As of April 18 2005, the number of offender hits (crime scene to
offender) recorded was 3161 and the number of forensic hits (crime scene to
crime scene) was 390. In February 2004, an initiative was created between the
RCMP Biology Operations and the National DNA Data Bank group to increase the
number of profiles contained in the crime scene index of the data bank in order
to enhance the number of hits established with serious unsolved crimes. A new
DNA extraction process was developed and adapted for our TECAN Genesis 150
Robotic Workstations but optimized to allow processing of break and enter
(B&E) samples from non-suspect cases. The magnetic bead extraction
technology from Promega (DNA IQ™) was evaluated on a variety of samples from
B&E cases (e.g. cigarette butts, chewing gums, swabs from manipulated
objects, bloodstains). Our initial work focused on the recommended protocol
from the manufacturer based on the company’s Lysis Buffer and binding
conditions. While blood swabs and trace swabs produced good DNA yields and
generated STR profiles, many potential crime scene samples such as chewing gums
and cigarette butts failed to produce results. Blood swabs prepared with
different types of soil also failed to produce results. Modifications to the
lysis step, the DNA binding step onto the magnetic beads, the resin wash and
DNA elution step were needed to optimize recovery of DNA using our robotic
workstations. Using our modified and automated protocol, as little as 0.003
ng/µl (0.10 ng total) from very compromised samples can be isolated using the
Promega DNA IQ™. The DNA yields obtained using the magnetic bead-based approach
were equivalent to conventional processes and 3-4 fold higher for samples
compromised with soil. Promega DNA IQ™ process produces better quality DNA than
organic extraction, and resulted in very balanced peaks across all STR loci
tested. To determine the amount of human DNA present in the B&E samples,
real-time PCR technology was used. The Quantifiler™ Human Quantification Assay
developed by Applied Biosystems 1) quantitates human DNA specifically by using
human specific primers for a single copy gene and 2) ascertains the presence of
PCR inhibitors in the DNA extract upon failure to amplify an internal PCR
control. The amplification assay set up has been incorporated at the end of the
extraction routine on our TECAN robotic workstation followed with actual
cycling and detection in an ABI PRISM® 7000 instrument. This assay
is extremely simple to automate yet is very sensitive detecting reliably down
to 0.003 ng/µl of DNA using the Promega K562 standards. The PCR setup of all
samples following their quantification is also carried out robotically using
the output file from the ABI PRISM® 7000 instrument. This automated
protocol combining Promega DNA IQ™ and ABI RT-PCR technology represents a
unique way to process B&E samples in a very efficient and cost effective
manner. A full batch of 84 samples plus controls (96 in total) can be extracted
in approximately two hours 15 min. following lysis overnight, quantitated in
approximately two hours as well (30 minutes to set up the reactions on the
robotic workstation and 1 hour 46 min for amplification and detection in the
ABI PRISM® 7000 instrument) and setup for STR amplification in
approximately 1 hour. The TECAN Genesis 150 Robotic Workstations used in our
process are equipped with non-disposable Teflon-coated stainless steel tips and
a stringent tip washing routine was developed to prevent cross-contamination. A
true 2% bleach wash was strategically incorporated within the extraction
process as well as after an extraction session using large volumes of system’s
liquid i.e. distilled water to remove any traces of DNA as well as any traces
of residual bleach from the line and tips. The use of the bleach within the
extraction does not have any adverse effect on the yield of DNA nor the quality
of the STR profiles produced.Our original Sample Tracking and Control System
(STaCS™) created for the National DNA Data Bank of Canada was amended to accept
B&E type samples. These modifications allowed us to keep the highest
standards of quality control while maintaining our capacity 1) to have a tight
control over B&E sample traffic and ensure that all samples are processed
error-free in the shortest possible time, 2) to batch process B&E samples
while maintaining the capability to customize processing conditions for each
sample (large scale versus small scale extraction), 3) to re-process B&E
samples at any step in the analytical process. This newly developed automated
protocol combining Promega DNA IQ™ and ABI RT-PCR technology is currently being
evaluated for other more challenging casework investigations (chantal.fregeau@rcmp-grc.gc.ca).
P-090
A novel DNA probe chemistry for
HyBeacons®: rapid genetic analysis
French D1, McDowell
DG1, Thomson JA1, Brown T2, Debenham PG1
1LGC, Queens Road, Teddington,
TW11 0LY, UK
2School of Chemistry, University
of Southampton,
Highfield, Southampton, SO17 1BJ UK
The analysis of single nucleotide polymorphisms (SNPs)
and short tandem repeats (STRs) has proven extremely valuable in both
healthcare and forensic analyses.
However, such analyses have historically been confined to the specialist
analytical laboratory both because of the processing and specialist nature of
the equipment required as well as the dependence on a skilled analyst for
interpretation.
In the field of genetic analysis, homogeneous PCR
offers much potential for simplifying the analytical process. However, the majority of such systems are
still confined to the specialist laboratory.
We describe here a novel homogeneous PCR probe system termed HyBeacons®
suitable for rapid genetic analysis.
HyBeacons are synthetic fluorescent oligonucleotides
which increase in fluorescence upon hybridisation without the need for quencher
moieties, secondary structures, multiple probe interactions or enzymatic
degradation. Analytical interpretation
is based on the generation of melting peaks post amplification and hence it is
the stability of the probe / target interaction which is the basis of the
result rather than any increase in fluorescence per se.
In SNP analysis, the presence of a base change within
the probe binding site can be highly destabilising depending on the nature and
position of the mismatch. With careful
assay design, melting peaks can be readily produced with delta Tms of 7-11ºC
which are easy to interpret.
Advantageously the same probe analyses both forms of the sequence and
hence both homozygotes and heterozygotes are easily called in a single tube
with each sequence effectively acting as a internal positive control for the
assay. Data will be presented for a
number SNPs commonly typed within the medical profession demonstrating result
direct from saliva in as little as 15-30 minutes.
For STR analysis, HyBeacons can be similarly applied
since melting temperature is affected by the length of hybridising sequence as
well as the presence of any mismatches.
Whilst less advanced than the SNP assays, we will again present data
indicating the potential for the analysis of STRs direct from saliva.
It is anticipated that HyBeacon based assays, in
association with a suitable analytical platform, could be configured for use
away from the specialist laboratory in primary healthcare or certain forensic
settings, significantly reducing the time to result. In consequence, rapid diagnosis and therapy
could be achieved in a healthcare setting, whilst for forensic applications,
data could be rapidly obtained to inform and prioritise further investigations.
Contact: Jim.Thomson@lgc.co.uk
P-091
Paternity Investigation in Father or
motherless cases: how to improve statistical analysis for missing kids DNA
databank?
Fridman C, Gattás GJF, Lopez
LF, Massad E
Department of Legal Medicine,
Bioethics and Occupational Health, Faculty of Medicine-University of São Paulo, Brazil
Paternity investigation of families where the trio
mother, child and alleged father is complete is almost well defined nowadays.
Different genomic DNA amplification kits are used and at least the genetic
markers recommended by the American CODIS are performed. Many times the
mother or the alleged father is absent or deceased, and one has to work
statistically with other family members or half brothers and sisters, trying to
deduct the genetic constitution of the parent. This
situation is frequent in DNA databank that is constructed in order to
investigate missing kids families. In these cases is necessary to do “reverse
paternity determination” where the number of genetic markers used must be
calculated in order to get the probability of paternity. Herein, we illustrate
this situation describing one fatherless and one motherless cases. In the first
one two sisters wished to know if a deceased man was their biological father.
The genotype of the unavailable alleged father was reconstructed based on
testing his other family that includes a daughter and son, and compared the
results with the genotype of the two sisters and their mother. In the second
case the
paternity investigation was done in a girl case having only the alleged father
and a maternal aunt. In both cases the DNA analysis was done using STR loci presented in
the AmpFLSTR® Identifiler® PCR Amplification Kit (Applied Biosystems) plus loci
HLADQA1, LDLR, GYPA, HBGG, D7S8, GC, F13A01, FESFPS, and D1S80 totalizing 25
markers. We discuss here the importance of the right selection of polymorphic
markers in special when we need to deal with similar situations in the DNA
databank that was elaborated to identify missing kids in Brazil in a
Project that calls “Caminho de Volta”. The first seven months of project (106
families) revealed a increased number of families where only one of the parents
is present (58% only mother and 16% only fathers) compared to only 13% of
families where biological material was collected from both parents.
Contact: cfridman@usp.br
Financial Support: FAPESP
P-092
mtDNA lineages in two Tunisian
Berber communities: comparing diversities between villages and towns
Frigi S1, Yacoubi B1, Pereira
F2,3, Pereira L2, Cherni L1, Amorim A2,3,
Elgaaied AB1
1Laboratory of
Molecular Genetic Immunology and Biotechnology, Faculty of Sciences, Tunis, Tunisia
2IPATIMUP (Instituto de Patologia e Imunologia Molecular
da Universidade do Porto), Porto, Portugal
3Faculdade de Ciências da Universidade do Porto, Porto,
Portugal
Haploid
markers are know to be more sensitive to genetic drift, bottlenecks and founder
events due to its effective size being ¼ relatively to autosomal. These effects
can be dramatic when samplings are carried out in small villages, where
inbreeding is very strong, as it has been the case of most studies conducted in
North Africa aiming to compare Berber and Arab
communities. We can ask, therefore, if this sampling strategy is suitable for
the construction of forensic database.
We
tried to evaluate the biases introduced by such a sampling strategy by
comparing the mtDNA haplotype diversities (HVRI and HVRII) between two north
Tunisian Berber communities: 47 individuals from the town of Sejenane (over
41,000 inhabitants); and 33 individuals from the small village of Takrouna (500
inhabitants).
The
sampling effort was considerably higher in the small village, where close
kinship was more and more difficult to rule out as the sampling proceeded, so
that at a certain point for all individuals not yet sampled a male relative had
been collected already.
As
expected, the diversity was higher in the town sample (haplotype diversity =
0.988 +/- 0.008; mean pairwise differences = 9.521 +/- 4.446) than in the
village (haplotype diversity = 0.907 +/- 0.024; mean pairwise differences =
4.625 +/- 2.328). The probability to find a haplotype match was much smaller in
the town (1.203%) than in the village (9.280%). And with respect to the
haplogroup distribution, the same higher diversity was observed for the town
sample (64% Eurasian; 32% Sub-Saharan; and 4% North African), comparatively to
the village one (97% Eurasian; 3% Sub-Saharan; and 0% North African).
We
assayed also if by pooling small Berber village samples we would get a similar
diversity to the town sample. This assay was limited to HVRI diversity because
this report will be the first one to describe HVRII diversity in North Africa. When we pooled 47 individuals from the
small village of Kesra with 33 from Takrouna we obtained
still a lower diversity (haplotype diversity = 0.897 +/- 0.028; mean pairwise
differences = 4.909 +/- 2.417) than the town sample (haplotype diversity =
0.979 +/- 0.012; mean pairwise differences = 6.141 +/- 2.973).
These
results claim some thought on the sampling strategy to be applied to the
construction of forensic databases, not only in Tunisia, but in the rest of North Africa and in other population coverages, where
similar sampling strategies are conducted that way.
Contact: fpereira@ipatimup.pt
P-093
The
opinion of the Spanish population regarding the procedural situation of the
owners of DNA profiles that would justify the inclusion of such profiles in a
National Data Base
Gamero JJ1, Romero JL1,
Peralta JL1, Carvalho M2, Vide MC2, Corte-Real
F3
1Faculty of Medicine.
University of Cádiz. Fragela s/n, Cádiz 11003. Spain
2Forensic Genetic
Service. National Institute of Legal Medicine. Largo da Sé Nova, 3000 Coimbra. Portugal
3National Institute of Legal Medicine. Largo da Sé Nova,
3000 Coimbra. Portugal
Different
questions must to be taken into account with regard to the procedural situation
of individuals involved in crime investigation whose DNA profile could be
included in a national DNA data base. Among these questions are the following:
1.- Should
the DNA profile of an accused individual be included in a national data base
only if found guilty in specific lawsuits?
2.- Should
the DNA profile of an accused individual be included in a national data base?
3.- Should
the DNA profiles of other individuals involved (suspects) or not in a crime or
offence be included in a data base?
The intention
of this paper is to add complementary information to previously studied aspects
of national DNA data bases, from an ethical and social perspective. The point
of view or criteria held by Spanish society regarding the procedural situation
which an individual must be in to justify the inclusion of their DNA profile in
a national data base will be analyzed. Likewise, opinion is also sought
concerning the criteria that should be taken into account in future regulations
affecting data bases in Spain.
P-094
Some social and ethical aspects of analyses and DNA profile
databases
Gamero JJ1, Romero JL1,
Peralta JL1, Carvalho M2, Vide MC2, Corte-Real
F3
1Faculty of Medicine.
University of Cádiz. Fragela s/n, Cádiz 11003. Spain
2Forensic Genetic
Service. National Institute of Legal Medicine. Largo da Sé Nova, 3000 Coimbra. Portugal
3National Institute of Legal Medicine. Largo da Sé Nova,
3000 Coimbra. Portugal
There is general agreement concerning the fact that research into
human genetics can affect the community as a whole, and for this reason it is
necessary for society, and not only scientists, to discuss and decide on what
they wish to accept and what they wish to reject. It thus seems clear that
there is a need in Spain
to examine and define the social and individual interests faced. In short, the
aim of this study, in accordance with the International Declaration on Human
Genetic Data, as well as the plan of action "Science and Society" of
the European Commission is to reveal the degree of information and criteria
society has with regard to a question that may affect it in specific
circumstances.
Indeed, it is of great interest to take into account the opinions
of different social groups before adopting legal decisions related with
biotechnology given that, in order to reach consensus, information should flow
in two directions, Society – Science.
In this paper, the
degree of information a representative sample of the Spanish population has
with regard to DNA profiles is analyzed, as well as the point of view this
population holds concerning the criteria of reliability, quality, precision and
security that must be established for the analysis and protection of stored
forensic genetic data. Finally, the population's opinion concerning other
questions relevant to this subject is also sought.
P-095
Haplotype distribution of four new
Y-STRs: DYS630, DYS631, DYS634 and DYS635 in a Chinese population
Gao Y1, He Y2, Zhang Z1,
Bian S1
1Department of Forensic Medicine, Medical School of Soochow University,
Suzhou, P.R.
China
2Deparmtent of Anatomy, School of Preclinical
and Forensic Medicine, Sichuan
University, Chengdu, P.R. China
In this study we analyzed the four new Y-STR loci, DYS630, DYS631, DYS634 and DYS635, investigated haplotype distributions of these
Y-STR loci in a Chinese Han population (eastern China), and sequenced alleles
of the four loci for clarifying the structure. Extracted DNA was amplified by
PCR and the PCR products were analyzed by non-denaturing polyacrylamide gel electrophoresis.
Alleles were sequenced on an ABI 3700 using a Dye Terminator Cycle sequencing
kit. During the genotyping procedure, no PCR products were found for all the 20
female specimens at the four Y-STR loci which indicated the male specificity of
the four Y-STR loci we studied. DYS630, DYS631 and DYS635
were found to be simple repeat systems, while DYS634 was complex repeat systems. Seven alleles at DYS630, four alleles at
DYS631, five alleles at DYS634 and seven alleles at DYS635 were observed in our
population sample.The gene diversities of DYS630, DYS631, DYS634 and DYS635 were 0.797, 0.418, 0.459 and 0.809,
respectively. A total of 50 different haplotypes was observed in 79 males. The
haplotype diversity for all the four Y-specific STR loci in Chinese population
was calculated to be 98.3% and the stand error (S.E) was calculated to be
0.3%.The results indicate that these four loci are useful Y-linked markers for
forensic applications.
(contact: yuzhengao@suda.edu.cn).
P-096
Distribution of Y-chromosomal
haplotypes in the Basque Country autochthonous population using a 17-locus
multiplex PCR assay
García
O1, Yurrebaso I1, Uriarte I1, Pérez JA1,
Peñas R1, Alonso S2, de la Rua C2, Izagirre N2,
Flores C3, Martín P4, Albarrán C4, Alonso A4
1Area de Laboratorio Ertzaintza, Larrauri Mendotxe 18,
E-48950 Erandio, Bizkaia, Spain
2Departamento de Genética, Antropología Física y
Fisiología Animal, Universidad del País Vasco, Bilbao, Spain
3Unidad Investigación, Hospital Univ. “Nuestra Señora de
Candelaria”, Servicio Canario Salud, Tenerife, Spain
4Instituto Nacional Toxicología y Ciencias Forenses,
Sección Biología, Luis Cabrera 9, E-28002
Madrid, Spain
Y-STR haplotypes were
determined from a sample of 168 unrelated males from the Basque Country autochthonous
population (individuals were considered autochthonous if the 8 surnames and
birthplace of their grandparents were of Basque origin) using the AmpFlSTR
Yfiler PCR Amplification kit (Applied Biosystems) that coamplifies 17 Y-STRs.
The panel of markers includes the 9-locus European minimal haplotype (minHT)
and the markers DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 (Y GATA
C4) and Y GATA H4.
Genomic
DNA was extracted by the standard phenol/chloroform extraction procedure. PCR
amplification was performed according to the manufacturer's recommendations. Samples
were denatured for 5 min at 95ºC and typed on an ABI310 sequencer.
Allele designations were made according to
recommendations of the DNA Commission of the International Society for Forensic
Genetics.
The number of alleles and
haplotypes, the gene diversities, the discrimination capacity and the
cumulative haplotype diversity were calculated and compared with results
obtained with the minHT-loci only.
contact: gobies01@euskalnet.net
P-097
Basque Country autochthonous
population data on D2S1338, D19S433, Penta D, Penta E and SE33 loci
García
O1, Yurrebaso I1, Uriarte I1, Pérez JA1,
Peñas R1, Martín P2, Albarrán C2, Alonso A2
1Area de Laboratorio Ertzaintza, Larrauri Mendotxe 18,
E-48950 Erandio, Bizkaia, Spain
2Instituto Nacional Toxicología y Ciencias Forenses,
Sección Biología, Luis Cabrera 9, E-28002
Madrid, Spain
Before a new marker system can
be introduced into forensic casework, a population database for the relevant
population must be established for statistical evaluation of the evidence.
Therefore, this report presents allele frequency data in a Basque Country
autochthonous population sample (n = 204) for 5 STR loci. The loci are:
D2S1338, D19S433, Penta D, Penta E and SE33.
Whole
blood was obtained from unrelated Basque autochthonous donors. Individuals were
considered autochthonous if the 8 surnames and birthplace of their grandparents
were of Basque origin. Genomic DNA was extracted by the standard
phenol/chloroform extraction procedure.
PCR amplification was
performed according to the manufacturer's recommendations using the AmpFlSTR
Identifiler PCR Amplification kit (Applied Biosystems) and the PowerPlex 16/ES
Monoplex Systems (Penta D, Penta E and SE33) (Promega Corporation). Samples
were denatured for 5 min at 95ºC and typed on an ABI310 sequencer.
Allele designations were made according to
recommendations of the DNA Commission of the International Society for Forensic
Genetics.
Statistical
evaluations were performed using the computer program GDA (Genetic Data
Analysis) and PowerStats. Analyses included the possible divergence from
Hardy-Weinberg expectations and other parameters of forensic importance:
observed and expected heterozygosities, mean exclusion chance, polymorphic
information content, discrimination power and the possible associations between
loci.
contact: gobies01@euskalnet.net
P-098
2004-2005 GEP proficiency testing programs: special
emphasis on the interlaboratory analysis of mixed stains.
García-Hirschfeld J1, Alonso A1,
García O2, Amorim A3 and Gómez J1
1Instituto Nacional de Toxicología y Ciencias Forenses,
Departamento de Madrid;
2Laboratorio de la Ertzaintza. Sección de Biología,
Departamento de Interior, Gobierno Vasco;
3Instituto de Patología e Imunología Molecular da
Universidade do Porto.
Both the 2004 and the 2005 GEP proficiency testing
programs consisted in a simulated paternity case and a simulated forensic
criminal case each including 4-5 reference samples (saliva or blood) and 2
forensic samples (mixed stains of semen and saliva or blood and saliva, and
clean or contaminated hair shafts).
Due to the widespread use of commercial STR kits, the
paternity test is no longer a problem apart from punctual discordances.
Nevertheless a theoretical challenge is also included in the paternity test and
the results obtained evidenced that rare alleles, mutations and possible silent
alleles are treated very differently among participating laboratories.
Moreover
the forensic tests have become the more fruitfully of the exercise. The results
showed that even in forensic labs, preliminary test are not always performed.
Samples management errors, transcription errors and missing a contributor in a
mix are also punctually observed.
In the
results of the 2004 forensic test (a mixture stain was analyzed consisting of
100 µl saliva from a female and 50 µl of a 1:20 semen dilution subsequently applied to a Whatmanâ
Bloodstain Card) apparently inconsistent results were observed between
autosomal STR profiling and mitochondrial DNA sequencing results. Additional
validation studies were planned by the GEP Working Group to progress in the
interpretation of mtDNA from different mixed stains.
In the
present year a new forensic challenge was proposed: an unbalanced mixture stain of saliva and blood (10 µl of
saliva and 30µl of blood) from two related contributors (sharing maternal and
paternal lineages). Also hair shafts contaminated with blood have been sent to
be analyzed.
As a
consequence of the high unbalanced presence of the saliva and the low DNA
content in this body fluid, no lab detected the minor component in the mixture
even when preliminary tests indicated the presence of saliva. This evidence the
fact that the detection of a minor contributor in a mixture is still a key
outstanding in forensic investigation.
Related
to the hair analysis also discussion is going to be generated because of the
specific extraction procedures applied at each laboratory and its influence in
final mtDNA results.
For
the first time in the 2005 exercise all labs were required to send
electropherograms and analysis data to better detect the errors source.
Contact:
julia.garcia@mju.es
P-099
A comparative study
of the sensitivity and specificity of luminol and fluorescein
on diluted and aged bloodstains and subsequent
STRs typing
Garofano L1, Brighenti
A, Romani F, Mameli A2, Marino A1, My D1, Festuccia N1,
Paolino S1 , Pizzamiglio M1
1 Raggruppamento Carabinieri Investigazioni
Scientifiche, Reparto di Parma, Italy
2
Raggruppamento Carabinieri Investigazioni Scientifiche, Reparto di Cagliari,
Italy
Luminol
and fluorescein are very important reagents for diluted and aged bloodstain
detection at crime scene. The aim of this study was to carry
out a comparative study of the sensitivity and specificity of these two
presumptive blood tests using a series of diluted bloodstains (from 1:10 to
1:10000000) on a large variety of substrates, as well as, to evaluate the
ability to type STRs on treated samples.
lugaro@tin.it
P-100
“Projeto Caminho de Volta”: a Brazilian DNA Program
for Missing Kids
Gattás GJF1, Garcia CF1, Fridman
C1, Neumann MM2, Lopez LF1, Barini AS1
, Souza APH1, Boccia TMQR1, Kohler P1,
Battistella LR1, Wen CL3, Massad E1
1Department of Legal
Medicine, Bioethics and Occupational Health, Faculty of Medicine-University of São Paulo
2 Centro de Pesquisa e Prevenção em Políticas Sociais, CEPESP, São Paulo
3 Telemedicine,
Department of Pathology- Faculty of
Medicine - University of São Paulo,
Brazil
The Brazilian missing kids´ project called "Projeto Caminho de
Volta" that means “coming home” is a program created by our group in
collaboration with the public security services of São Paulo State, including
molecular biology, genetics, psychology, bioinformatics and telemedicine
methodologies. Each year, 8000 kids and teenagers (under 18 years old)
disappear from their homes only in São
Paulo State.
In Brazil
the number is about 30.000 per year. The reason for this event is not well
addressed in our country and there is no epidemiological data about it, making
difficult to establish effective preventive programs. For this reason this
system was designed to follow three main goals: 1) to identify, through an
epidemiological study, why there is so many cases of missing kids; 2) to help
in the identification process of recovered missing kids after years (death or
alive) it was developed a DNA data base including biological samples of parents
(reference) to be compared to a DNA database of children and teenagers with
unknown families (questionable); 3) to give psychological support to missing
kids families during the entire process. Since September 13, 2004 this program
received 100 families, only in the city of São Paulo, totalizing 48 boys and 52
girls missing, with ages ranging from 2 to 17 years old (average = 11,5 yo).
The first analysis showed the mean problems are physical injury against
children (30%), domestic violence (20%),
alcoholism (20%), drug addiction (9%), and sexual abuse (5%) resulting in
running away of the kids (80%) that prefer to be on the street than at home. In
fact, about 40% of them are cases of more than one time of disappearance. For
the DNA database all the family members are being genotyped using AmpFLSTR®
Identifiler® PCR Amplification Kit (Applied Biosystems) that amplify 16 genetic
markers (including CODIS loci). The reference database (family members that
went to police to notify the disappearance) was mainly represented by only
mother DNA (58%) followed by only father (16%) or both (13%); the remaining
cases are from other family members like siblings (4,4%), grandmothers (3,5%),
aunts (3,5%), and uncles (1,6%). All families data are automatically included
in the online project database developed by our group. This program that allows
the rapid search and comparison between the genetic and epidemiological
information brings a new challenge in missing children identification in Brazil and
should provide data to establish future preventive public programs.
Contact: gfgattas@usp.br
Financial Support: Special Human Rights Secretariat - Federal
Government/FAPESP.
P-101
Validation of the Mentype® Argus X-UL kit
C. Gehrig and A. Teyssier
Institute of Legal Medicine, Geneva, Switzerland
With the aim of using X-chromosomal
polymorphic markers in Swiss crime cases (female DNA on a male background) and
particularly in kinship testing, a validation study of the Mentype® Argus X-UL
kit was performed. The Argus X-UL kit is a commercial multiplex
system which contains Amelogenin for gender determination as well as four
uncoupled X-chromosomal STR markers (DXS8378, HPRTB, DXS7423 and DXS7132). In
this study, we present the results of some forensic validation studies
including the following aspects : detection limit, evaluation of stutter bands,
analysis of female/male mixtures, frequency data from a swiss population study,
validation of our protocol consisting of blood on FTA cards and amplification
in a small PCR reaction volume (10 ml). The use of these
markers in a deficiency paternity case will also be shown.
Christian Gehrig, Institute of Legal Medicine, 9 av. de Champel 1211
Genève 4, Switzerland.
christian.gehrig@hcuge.ch
P-102
Genetic variability at eleven STR loci and mtDNA in NOA populations (Puna and Calchaqui Valleys)
Giménez
P1, Albeza MV2, Acreche N2, Castro JA1,
Ramon MM1, Picornell A1
1Laboratori de
Genètica, Universitat de les Illes Balears, Spain
2Facultad de Ciencias
Naturales, Universidad Nacional de Salta, Argentina
Human populations
from the Andean region of North Western Argentina (NOA), due to their origin
and their historical-demographic peculiarities, constitute an anthropological interesting
motive of study. There is little reliable information on the structure of these
populations before contact with Europeans in the late 15th century. In
addition, the lack of historical data for the post-contact period means that
the exact origin and/or the degree of admixture of the inhabitants of this
region is also unknown.In this Andean region two zones can be differentiated,
from an ecological and human point of view: the Puna and the Calchaqui Valleys.
The Puna region in the Province
of Salta (NW Argentina)
is a typical Andean plateau at high altitude, which is arid or semi-arid. The
populations in this region have extreme life conditions: low temperatures, low
oxygen pressure and poor soils. In addition, they are distanced from other
urban populations by poor and difficult roads. All these factors cause the
isolation of these populations. The settlement model in the Puna region, is a
dispersed one with a small population density. In this Andean area, San Antonio de los Cobres
(altitude 3,880m) is the most populated locality (approx. 3,000 inhabitants).
The Calchaqui Valleys are in the East of the Puna,
with an altitude between 1,700 and 3,000 m, occupying a band of approximately
200 km of extension in sense north-south (provinces of Salta, Tucumán and Catamarca). In the
pre-Hispanic epoch, these valleys were inhabited by the diaguitas and
these pre-Hispanic societies reached the highest socioeconomic and cultural
levels. The population dynamics of this zone is complex, as a consequence of
the invasion of the Incas, the European colonization and, finally, the polity
of estrangement of the rebels, from half of the XVIth century until the end of
the XVIIth, which supposed the disappearance of an important part of the
population. The current population (25,000 inhabitants in whole) has a low
density and is unequally distributed; the most populated localities are
Cafayate (approx. 9,000 inhabitants) and Cachi (6,000). This area has
characteristics different from the rest of the Argentine, which are similar to
those of the neighboring countries, specially Bolivia and Chile. It is a
region of ecological and cultural specific characteristics, combination of
Andean and Amazonian, because it was formed with peoples of both high and low
lands. Some demographic and only a few
genetic data for these populations, mainly based on blood groups and
cytogenetic techniques, have been published elsewhere. Also preliminary data on
STRs, but no data on mtDNA, have been published to date. The purposes of the
present study were: (i) to study the STR variation in Puna and Calchaqui
Valleys, (ii) to develop a mtDNA HVRI database from NOA individuals and, (iii)
to compare with Europeans and other American populations from the
literature.DNA samples from 161 unrelated individuals were analyzed: 106 from
different localities of the Puna (Salta) and 55 from Calchaqui Valleys. The STRs studied were: D3S1358, vWA, FGA, D8S1179, D21S11,
D18S51, D5S818, D13S317, D7S820 (AmpFlSTR Profiler Plus, PE Applied Biosystems),
HUMF13A1 and D12S391. The mtDNA region subjected to analysis was 15997-16400
(HVRI region). The RFLP motif –7025 AluI
was also analysed
in the samples to determine those that showed haplogroup H. In non-H samples,
every control region sequence was assigned to a haplogroup by using the
sequence motifs indicated by Richards et al. (Am J Hum Genet 2000, 67:
1251-1276).The eleven STRs studied in the 161 individuals from these
populations showed an heterozygosity of 0.762. The number of alleles observed,
between 7 and 17 (average 10.2), was high, taking into account that they are
small and inbreed populations, probably due to the Amerindian-European
admixture in these Andean populations. In mtDNA (HVRI) a total of 34 different
haplotypes in 99 individuals were observed, defined by 50 variable positions.
The incidence of unique haplotypes (13.1%) was very low. In relation to shared
haplotypes, 21 haplotypes were shared by two or more individuals, 17 within the
same population, and 4 between both populations. The gene diversity was
approximately 0.92, in both populations, and the random match probability was
10%. Amazingly, the haplogroup analysis showed that all the individuals in both
NOA populations had Amerindian haplogroups (A, B, C, D). Therefore, there is no
indication of European female contributions for these populations. Genetic
diversity of the Y-chromosome should be studied in order to estimate the
proportion of Amerindian and European genes, and the asymmetrical mating
according to sex and ethnic group, in NOA populations. apicornell@uib.es
P-103
Constituting
a Y chromosome Short Tandem Repeats loci database in Sicily
E.Ginestra (1), I. Ciuna (1) , Maria Guarnaccia(2), Antonella Agodi(2), D.Piscitello(1), C.Trapani (1), Giovanni Marcì(2), Gianluca Paravizzini(2), C. Romano(1), G. S.Travali (2) and L . Saravo(1)*
1 Laboratory of Molecular Biology –
Raggruppamento Carabinieri Investigazioni Scientifiche (RaCIS), Messina; Italy.
(2)Department of Biomedical Science,, University
of Catania, Italy
2 Department of Human Pathology, University of Messina,
and *Laboratory of Molecular Biology
Many Y chromosome Short Tandem
Repeats (STRs) have been studied and characterized over the past years. A few Y-STRs
multiplex kits have been placed on the market for forensic and population study
purposes. The aim of our work was to analyse a sample of Sicilian male
individuals to evaluate the allelic frequency and, thus, the possibility to
implement a genetic database. An 11 loci Y-STR Typing kit was used to yield haplotype profiles from male
DNA; amplification products were detected on ABI PRISM 310 Genetic Analyzer and
examined by Genemapper v 3.2 (Applied Biosystems). The population sample
subjected to the screening resulted to be very different with regards to
certain loci whereas other loci allelic profile was less variable. Despite a
minor discrimination power within the entire population, Y-STRs represent a
valid tool to simplify male/female DNA mixture interpretation which is a major
challenge when biological traces are found in case of sexual assault. As
regards to this, our results indicated that Y-STRs could be used to identify
male individuals with a reasonable accuracy.
Keywords: DNA STR typing;
Y STR-DNA database, Y aplotype
*Corresponding
author: rismebiologia@carabinieri.it
P-104
A
SNP-STR locus within the HLA class ii region:
sequence
and population data of D6S2822
Glock B, Reisacher RBK, Dauber EM,
Wenda S, Dorner G, Mayr WR
Division of Blood
Group Serology, Medical University of
Vienna, Austria
Polymorphic STR markers within
the HLA region can be used for a better characterization of HLA haplotypes,
determination of disease associations, recombination point mapping or even for
forensic testing if no linkage disequilibrium exists.
In this study, population
genetics of the tetranucleotide repeat locus D6S2822 (M2_4_25; GATA129G03;
GenBank G10435; UniSTS 239167, 464402, 464403; [1], [2]) situated nearby the
HLA class II region (6p21.3) were investigated in an Austrian Causasoid
population sample of 153 unrelated individuals in order to reveal its
specifications.
PCR amplification was performed
using the primers described by Matsuzaka et al. [2]. Typing of the
amplification products in comparison with a locus-specific allelic ladder
containing the most common alleles as well as cycle sequencing applying BigDye
chemistry were carried out using denaturing capillary electrophoresis on an ABI
Prism 310® Genetic Analyzer.
Sequencing of a total of 34
alleles from the population study and further samples, which were not included
into frequency data, revealed 7 different sized alleles ranging from 189 to 213
bp and showing a (TATC)9-14 (CATC)1-2 repeat pattern. No
incomplete repeats were found. Additionally, 17 bp upstream from the repeat
region (base 65 of the 5’-flanking region) a A/G SNP, with the minor allele G
(23.5%) and the major allele A (76.5%), was observed.
The resulting allele
frequencies (n=153) as well as further statistic data are shown below:
|
Allele designation*
|
Allele frequency
|
|
11
|
0.026
|
|
12
|
0.261
|
|
13
|
0.510
|
|
14
|
0.183
|
|
15
|
0.017
|
|
16
|
0.003
|
|
Rate of heterozygosity:
Power of exclusion:
Polymorphism information
content:
Power of discrimination:
Typical paternity index:
|
0.595
0.285
0.580
0.816
1.230
|
* the rare allele 10 was only found once in the additional samples and
thus not included into frequency data
No
deviation from Hardy-Weinberg equilibrium could be detected (0.4<p<0.5).
Furthermore no mutations were observed in 263 meioses in 71 families.
The
tetranucleotide locus D6S2822 showed an interesting sequence structure and an
average allele distribution. It might, apart from its applications concerning
the HLA system, due to its SNP nearby the repeat region, be a candidate for
phylogenetic investigations. Further work was carried out subsequently on the
linkage of D6S2822 with other HLA-STR loci, which will be displayed on another
poster.
References:
[1] Cullen M
et al. Immunogenetics 2003; 54: 900-910
[2] Matsuzaka
Y et al. Tissue Antigens 2000; 56: 492-500
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