P-105
A multiplex PCR design for simultaneous
genotyping of X chromosome short tandem repeat markers
Gomes
I1,2, Carracedo A2, Amorim A1,3, Gusmão L1
1Institute
of Pathology and Molecular Immunology, University
of Porto, Portugal
2Institute
of Legal Medicine, University
of Santiago of Compostela, Spain
3Faculty
of Sciences, University of Porto,
Portugal
Short tandem
repeat markers (STRs) are extensively used as genetic tools in forensic and
population studies. Genotyping of STRs located on the autosomes and on the Y
chromosome (ChrY) has revealed significant information for application in these
fields, whereas X-chromosome STRs have so far played a slender role. However,
in the past few years, the human X chromosome has become object of studies that
focus on genetic markers suitable for population and forensic analysis. In
forensics, X-STRs can complement other STRs located on other chromosomes,
especially in complex kinship testing, as in the so-called paternity
missing cases, when the probant is female.
In this work, experimental
designs were conducted in order to develop a multiplex PCR amplification
strategy for X-STR genetic markers. ChrX loci were selected according to the
informative power of each locus described in previous population studies. A tetraplex system for the following X chromosome
genetic markers, DXS7423, DXS101, DXS8377 and HPRTB (human
phosphoribosyl transferase) was optimized in a
single PCR reaction. These short tandem repeat markers were typed in 65
individuals (29 female and 36 male samples) from a Galician population (Northern Spain) sample. Locus DXS7423 revealed 5 alleles
(alleles 13-17) and DXS101 12 alleles (alleles 17-29). For the DXS8377 marker,
17 alleles were found (alleles 40-57) followed by 12 alleles for the HPRTB
locus (alleles 9-16). In this genetic study of the Galician population, allele
frequencies were estimated for all loci. We compared our data to those obtained
in a German population study (Edelmann et al., 2001; Szibor et al., 2003) and
as expected, exposed similar results. The simultaneous typing of ChrX markers
for population and forensic studies is a pratical and simple method to obtain
large amounts of information as proven in many other studies using autosomal
and Y-chromosomal markers.
P-106
Y-chromosome
lineages from Portugal, Madeira and Azores record elements of Sephardim and Berber ancestry
Gonçalves R, Freitas A, Branco M, Rosa A,
Fernandes AT, Brehm A
Human
Genetics Laboratory, University
of Madeira, Campus of
Penteada, 9000-390 Funchal,
Portugal
A total of
553 Y-chromosomes were analyzed from mainland Portugal and the North Atlantic
Archipelagos of Azores and Madeira, in order
to characterize the genetic composition of their male gene pool. A large
majority (78-83% of each population) of the male lineages could be classified
as belonging to three basic Y chromosomal haplogroups R1b, J, and E3b. While
R1b, accounting for more than half of the lineages in any of the Portuguese
sub-populations, is a characteristic marker of many different West European
populations, haplogroups J and E3b consist of lineages that are typical from
the circum-Mediterranean region or even East Africa. Highly diverse haplogroup
E3b in Portuguese likely combines sub-clades of distinct origins. The present composition of the Y chromosomes
in Portugal
in this haplogroup likely reflects a pre-Arab component shared with
North African populations or testifies, at least in part to the influence of
Sephardic Jews. In contrast to marginally low sub-Saharan African Y
chromosome component in Portuguese, such
lineages have been detected at moderately high frequency in our previous survey
of mtDNA in the same samples indicating to the presence of sex-related gene
flow most likely mediated by the Atlantic slave trade.
P-107
DNA mixtures in forensic casework: report of
32 criminal
cases resolved
with autosomic STRs
Fabricio González-Andrade 1 • Dora Sánchez. 1 • Miguel Bolea 2 • Begoña Martínez-Jarreta 2
1 Laboratory of
Molecular Genetics, Metropolitan
Hospital (Ecuador)
2 Department of Legal
Medicine, University
of Zaragoza (Spain)
To assess the technical and judicial
consequences resulting from the practical application of DNA testing in
forensic research in the numerous sex crimes in Ecuador. When a sample contains DNA from more than one contributor, the
interpretation of its genetic profile becomes complicated. The incidence,
complexity, and importance of mixed profiles is increasing due to the sensitivity
of polymerase chain reaction (PCR) based typing methods. Mixed DNA samples
fromat least two contributors can be originated at the scene of crime, in the
course of sample-handling by investigating personnel or others, during forensic
examination,or in the laboratory. However, in casework, samples may be
degraded, unbalanced, contaminated, etc. thus overriding theoretical approaches
from programmed validation assays with laboratory samples. The aim of this work
is review our casework results obtained with the mixed genetic STR profiles
encountered in our laboratory. Occasionally interpretation
guidelines from validation studies are difficult to apply to real forensic
casework, especially in the case of mixed samples. Exogenous contamination, an
unknown number of contributors or unbalanced proportion of each one in the
sample and a varied degree of degradation of the biological materials,
contribute to the difficulties in the interpretation of sample profiles. In
this paper we have reviewed all the mixed genetic STR profiles encountered in
our laboratory and evaluated the problems in the interpretation of the results.
From 32 criminal cases with 53 samples typed, 36 showed a mixed profile.
Key words: DNA
mixtures • STR •
sex offences • Forensic casework •
PCR • Justice
Correspondence: fabriciogonzalez@usa.net
P-108
DNA typing in missing persons in Ecuador
Fabricio González-Andrade 1 • Dora Sánchez. 1 • Miguel Bolea 2 • Begoña Martínez-Jarreta 2
1 Laboratory of
Molecular Genetics, Metropolitan
Hospital (Ecuador)
2 Department of Legal
Medicine, University
of Zaragoza (Spain)
Relevant efforts have
been continuously made to identify cadavers and human remains after wars,
socio-political disturbances, and mass disasters. In many cases, the use of DNA
typing techniques offers a definitive answer for identification of victims and
thus a direct social benefit is realized. Although DNA analysis is a highly
discriminatory method, it is not self-sufficient and could not replace an
anthropological evaluation. Amplification and typing of DNA extracted from
compact bone of human remains could be useful in establishing the identity of a
person, as well as in excluding possible false identifications. Body identification
are making by using the results from relatives blood samples and information
gathered from family trees, to predict the genotype of the deceased family
member, in a paternity style analysis.
There
are two types of situations for DNA testing, called close and open studies.
Close studies are those in which the remains where a family member has
recognized a personal item they believe belonged to the individual the family
member claims is missing, or where some form of identification has been found on
or near the body, and there is a general agreement on physical characteristics
between ante mortem and post mortem data. In other words, when we know personal
identity a priori. An open study is when the identities of all the victims is
not known a priori. Open cases involve remains where there is a little or no
information as to identify individuals. We report 8 cases of missing persons
that we resolved by STRs technology.
Key words: DNA
typing • STR • Forensic casework • PCR • missing persons •
Justice
Correspondence: fabriciogonzalez@usa.net
P-109
Genetic data from Huaoranies Amerindian, the
last nomad population from Ecuador,
using Power Plex 16 and Power Plex Y
Fabricio
González-Andrade 1
• Dora Sánchez. 1 Miguel Bolea 2 • Begoña Martínez-Jarreta 2
1 Laboratory of
Molecular Genetics, Metropolitan
Hospital (Ecuador)
2 Department of Legal
Medicine, University
of Zaragoza (Spain)
Huaoranies,
Aucas or Jíbaros are last nomad Amerindians from Ecuador and from Amazonia
region. There are only two thousand individuals in small family groups, located
Between the Napo river in north, and Curaray river in the south. They speak Hao
Tiriro. Linguist studies have been demonstrated that there are not congeners
for this language. They are a people in extinction, like all diversity from Amazonia.
In a few years, Huaoranies to become extinct
by the oil industry. State of Ecuador has
recognized different indigenous nationalities, with own identity and language.
Ethnicity means cultural practices and moral values to distinguish groups and
communities. Individuals from ethnic group see themselves like different to
others social and native groups. This concept has two dimensions: cultural and
social characteristics (language, religion, faith, location, etc.) and a sense
shared of identity and tradition. Indigenous Nationality means a joint of
thousand-year old peoples before to Ecuadorian
State, that has an
historical identity, language and shared culture, that live in a certain
territory, among his institutions and traditional forms of social, economical,
political organization and practice of his own authority. Amerindians from Ecuador live
around all the country.
Adequate evaluation of the DNA forensic evidence need
of proper databases on STR polymorphisms distribution. In this paper, we report the
allele frequency distribution of STR loci (CSF1PO, TPOX, TH01, F13A01, VWA,
D13S317, D16S539, D5S818, D7S820, LPL, HPRTB, F13B), and Y Chromosome STR (DYS
19, DYS 385 a, DYS385 b, DYS, 389
I, DYS 389 II, DYS 390, DYS 391, DYS
392, DYS 393, DYS437, DYS 438, DYS 439) that have proven to be extremely useful
for forensic casework, human identification and population genetics in a
population sample of Amerindian Huaoranies from Ecuador.
Correspondence: fabriciogonzalez@usa.net
P-110
Sub-typing
of mtDNA haplogroup H by SnaPshot minisequencing
Grignani P1, Peloso G1, Alù M2,
Ricci U3, Robino C4, Fattorini P5, Previderè C1
1Dipartimento
di Medicina Legale e Sanità Pubblica, Università di Pavia, Italy
2Dip
Integrato Servizi Diagnostici di Lab e di Med Leg, Università di Modena e
Reggio Emilia, Italy
3UO di
Genetica Medica, Azienda Ospedaliera-Universitaria "A. Meyer",
Firenze, Italy
4Dipartimento
di Anatomia, Farmacologia e Medicina Legale, Università di Torino, Italy
5UCO di
Medicina Legale, Università di Trieste, Italy
Sequencing analysis of
hypervariable regions HVSI/II is the most common approach to mitochondrial DNA
(mtDNA) typing in the context of anthropological and medical studies. In the
forensic field, mtDNA analysis is particularly important in human
identification caseworks where the amount of genomic DNA recovered from samples
as skeletal remains and hair shafts is extremely reduced. The presence of
multiple copies of mtDNA in any cell can help in collecting a genetic result
where typing of conventional STRs fails or gives unreliable results. However,
the discrimination power of mtDNA typing is quite low also as a consequence of the maternal inheritance; in
fact, about 7% of the Caucasian population shares the same HVSI/II sequence. In
order to increase the discrimination of the common sequences for forensic
purposes, it could be useful to characterise other mtDNA polymorphisms, such as
single nucleotide polymorphisms (SNPs) of the coding region defining the most
common European haplogroups. Point mutation detection can be performed by PCR
amplification of a fragment containing the polymorphic site and restriction
fragment analysis (RFLP) on agarose or polyacrilamide gels. Recently, a
SnaPshot minisequencing assay based on ddNTPs single base extension of
unlabelled primers immediately adjacent to the polymorphic site was set up;
this provided the association of each individual to one of the nine major west
European haplogroups. In addition, the SnaPshot method was used to type 7 SNPs
allowing sub-typing of haplogroup H, the most common lineage in the European
population (about 50%).
In this study we analysed 197
individuals from North-Central Italy (Turin, Pavia, Modena
and Florence)
by sequencing the hypervariable regions HVI/II. MtDNA haplogroups were then
scored by RFLP typing. Haplogroup H was shared by 88 individuals (44,7%), in
agreement with the distribution found in other European population samples. The
SnaPshot minisequencing multiplex reaction set up by Quintans (FSI, 2004) was
then used to sub-characterise the Italian haplogroup H samples. Seven H
sub-haplogroups were found with the following frequencies: H*=47%, H1=28%,
H2=4.5%, H3=4.5%, H4=3.4%, H5=8%, H6=3.4% and H 7=1.2%. Data on Italian H
sub-haplogroups was then compared with the one calculated for the Spanish
(Galician) population sample analysed by Quintans and a statistical significant
difference (P<.0022) was found in the distribution of the frequencies,
probably reflecting a different population history. On the opposite, the 28
(14%) identical rCRS HVSI Italian samples showed the same distribution of H
sub-haplogroups, if compared with the Spanish ones. These results confirm the
utility of this SnaPshot minisequencing assay to increase the discrimination
power of HVSI/II sequencing analysis. In fact, the most frequent Italian mtDNA
haplotype (CRS,263G, 315.1C), shared by 8 individuals belonging to the same
haplogroup H, was discriminated by the SNPs analysis and subdivided in three H
sub-types (H*=3, H1= 4, H4= 1). The SnaPshot approach can be used as a rapid
screening method before sequencing, especially if many forensic or reference
samples have to be analysed. contact:
previde@unipv.it
P-111
Austrian
Caucasian population data of 15 STR loci complementing forensic core markers: A
highly discriminating set for paternity and kinship analysis
Grubwieser P, Zimmermann B, Niederstätter H,
Pavlic M, Steinlechner M, Parson W
Institute of Legal Medicine, Innsbruck Medical University, Austria
We
investigated 15 polymorphic STR loci (D1S1656, D7S1517, D8S306, D8S639, D9S304,
D10S2325, D11S488, D12S391, D14S608, D16S3253, D17S976, D18S1270, D19S253,
D20S161, D21S1437) which are not included in the standard sets of forensic loci
(ISSOL, CODIS). The loci were selected according to the complexity of the
polymorphic region: Seven of the 15 investigated loci showed a simple repeat
structure (D9S304, D10S2325, D14S608, D16S3253, D18S1270, D19S253, D21S1437),
three loci (D7S1517, D12S391, D20S161) consisted of compound repeat units and 5
loci (D1S1656, D8S306 , D8S639, D11S488, D17S976) showed a more complex
polymorphic region partly including different repeat blocks and incomplete
repeat units, which resulted in a relatively high portion of intermediate
alleles. A population study on a sample of 270 unrelated persons from Austria was
carried out. We did not observe significant deviation from Hardy – Weinberg
expectations. The combined PE for the 15 loci was 0.99999998. In combination
with the traditional set of STR markers included in commercially available kits
(no linkage was observed between these 15 loci and the PowerplexTM 16 System
loci) these markers approved as highly discriminating forensic tool, also
suitable for the analysis of difficult paternity and kinship constellations.
P-112
Genetic
analysis of autosomal and Y-specific STRs in the Karimojong population from Uganda
Gusmão L1, Sánchez-Diz P2, Gomes I1,2, Alves C1, Carracedo A2, Prata MJ1,3, Amorim A1,3
1 IPATIMUP, Instituto de Patologia e Imunologia da
Universidade do Porto, Portugal.
2Unidad de Genética Forense, Instituto de Medicina
Legal, Univ. de Santiago de Compostela, Spain.
3Faculdade de Ciências da Universidade do Porto,
Portugal.
The Karimojong are eastern Nilotic pastoral people of
northeastern Uganda.
They are the largest of a cluster of culturally and historically related
peoples, including the Jie, Teso, Dodoth, and Labwor of Uganda and the Turkana
of neighbouring Kenya.
They speak an Eastern Nilotic language of the Nilo-Saharan language family.
Many years ago, a number of groups of people referred to as the Nilotes
migrated from near the Nile valley in southern
Sudan
and Ethiopia
toward the south and west. Some of those groups took a south-westerly route,
passing through the region that is now Kenya, and they ultimately settled
on the high, dry plateau which is the Karamoja of today. The Nilotes are
spread in a region that corresponds to the fringe of Bantu migration route, and apparently
were not touched by the Bantu influence, keeping a Nilo-Saharan language
and maintaining a pastoral lifestyle. However, they still remain almost
genetically uncharacterized.
In this work, 90 individuals
living in Karamoja region were typed for 17 autosomal STRs (CSF1PO, D2S1338,
D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11,
FGA, TH01, TPO, VWA, Penta D and Penta E) and 40 males were also typed for 12
Y-STRs (DYS19, DYS385, DYS389I and II, DYS390, DYS391, DYS392, DYS393, DYS437,
DYS438 and DYS439).
Hardy-Weinberg equilibrium was
tested for each autosomal locus and no deviations from equilibrium were
observed. The only P value below 0.05 was found for CSF1PO (P=0.01236±0.00017)
but, if Bonferroni correction is used, the departure observed at this locus is
not significant.
For autosomal STRs, our sample
shows a combined matching probability of 1 in 6.5x1019 individuals
and a combined power of exclusion of 0.999999988. Haplotype diversity for Y-STRs was 0.9859
and 32 different haplotypes were detected out of the 40 samples analysed.
Our sample was compared with
available autosomal data from sub-Saharan African samples and significant
differences were found with Mozambique in 8 out of 17 loci; Cabinda (Angola) in
5 out of 17 loci; Equatorial Guinea in 4 out of 17 loci; and with Rwanda in one
out of 13 loci. Comparisons with the Y-STR data, revealed large genetic
distances between the Uganda
and Mozambique,
Cabinda and Equatorial Guinea (Rst=0.168,
Rst=0.195 and Rst=0.183, respectively).
The present work confirms the
high genetic heterogeneity between African populations and, therefore,
emphasizes the importance of using local forensic databases, for both autosomal
and Y-specific STRs.
Contact: lgusmao@ipatimup.pt
P-113
A new legal basis and
communication platform for the Swiss DNA database
Haas C1, Voegeli P1,
Hess M2, Kratzer A1, Bär W1
1Institute of Legal
Medicine, Forensic Genetics, University of Zurich, Switzerland
2Federal Office of
Police, Bern, Switzerland
The Swiss federal DNA profile
information system (EDNA) has been launched in July 2000 for a test-period of 4
years. Based on the "EDNA-Verordnung" DNA-profiles of persons who are
suspected of having committed a crime according to the Swiss crime catalogue
and DNA-profiles of stains from unknown perpetrators were entered into the
database. At the end of 2004 the Swiss DNA database contained 53'400 profiles
from suspects and 8'554 profiles from stains.
Since 1. January 2005 a new
legal basis for the Swiss DNA database is operational (DNA-Profil-Gesetz,
DNA-Profil-Verordnung). New criteria for entering a profile into the database
were established, no longer based on a crime catalogue. The following
DNA-profiles are newly entered into the database: suspects for crimes or
delicts, convicted offenders, dead persons, stains, not identified persons,
missing persons, relatives of dead or missing persons. Profiles of persons are
removed from the database on the basis of the new law.
At the same time the
administrative process - from the investigating police unit, to the DNA
laboratory, to the database, to the federal police and back to the
investigating police unit - has been improved. A new internet-based information
system (message handler) was implemented, which allows faster processing and
status information.
P-114
The AMOVA Analysis
of Pakistani Population Y STR Genetic Data
Hadi. S1 &
Goodwin, W. H.2
1,2
Department of Forensic&
Investigative Sciences, University
of Central Lancashire, Preston, UK
Pakistan is a large country
with a diverse population of 140 million consisting of four significant
population groups besides many smaller isolated populations. Male population samples were collected from
indigenous populations and the extracted DNA was used to amplify Y STRs. The haplotypes were developed for each
population and the results of the AMOVA analysis of the data will be discussed
in this paper. There were striking
results for these populations which even with smaller number of loci when
analysed led to historically relevant results.
The data shows that the populations are quite diverse as opposed to the
popular belief that cousin and other close relative marriages lead to high
degree of inbreeding in these populations.
The results also show that even with smaller number of loci significant
amount of genetic and phylogenetic information can be gained from the
data. AMOVA analysis of the population
data revealed significant correlations between the population groups and also
showed the effects of particular loci on the genetic diversity and ultimately
the discrimination power in each population.
Results show that Y STRs can be an effective tool to study micro
geography. The paper also discusses
autosomal & Y STRs in a rare Kalash population. Kalash live in the northwest of Pakistan. The genealogical history of both is quite
unknown and subject to mythical reports.
The data we present shows for the first time that at least the Kalash
population is closely related to other Pakistani populations. The history of Kalash suggested that there
would be significant amount of inbreeding and the Y data has shown that in
quite a strong way in at least one of the Kalash groups, though similar results
could not be detected when autosomal STRs were studied in this population.
contact: shadi@uclan.ac.uk
P-115
DNA
and the Innocence Project: Three separate 17 year-old rape cases from Georgia,
similar circumstances, different outcomes.
Hampikian, G
Department of Biology, Boise State
University, Boise, Idaho,
USA
Three men in Georgia
appealed to the Innocence Project for help to overturn their convictions. These separate cases had much in common: the
convicted men were all African Americans from Georgia, they had each served 17
years, each claimed innocence, and the physical evidence remaining form their
trials included smears obtained from the victims which were stored at room
temperature.
The author has co-written a book with one of
the men, Calvin Johnson (Exit to Freedom, Univ. of Georgia Press,
2003), and was part of the team that investigated the DNA evidence the other
two cases: Clarence Harrison and Joseph Lee Brown.
Johnson’s case was handled by Peter Neufeld and Barry Scheck of the New
York Innocence Project, and resulted in the first DNA exoneration in Georgia. However, it took several years to locate the
evidence, secure permission to test, and finally obtain a conclusive
profile. Finally, DQ alpha testing
excluded him at several loci in 1999.
The Calvin Johnson exoneration led directly to the Georgia DNA Evidence
law, and the formation of the Georgia Innocence Project (GAIP). The new law
requires the preservation of potential DNA evidence for 10 years, or until a
death sentence is executed.
Joe Brown was the first GAIP case, and the first
convicted offender to go to court requesting DNA testing under the new
law. After two rounds of STR typing, the
partial profile from the smear was consistent with Brown, and his case was
dropped by the GAIP (2004). Clarence
Harrison, the second GAIP case to request testing from the court, resulted in a
complete DNA profile from the 17 year-old slide which exonerated Brown, freeing
him within weeks of the test (2004). The
District Attorney in the Brown case, with collaboration from the victim who
still lives in the area, has pledged to investigate the case and submit the profile
to the CODIS database.
contact: greghampikian@boisestate.edu
P-116
Semi-automatic preparation of biological database samples for STR and SNP typing.
Hansen AJ, Simonsen BT,
Børsting C, Hallenberg C, Morling N
Department of Forensic
Genetics, Institute
of Forensic Medicine, University of Copenhagen, Denmark.
Application of laboratory
automation systems (LAS) for preparation of forensic crime-case and database
samples is necessary to support the increasing demand for fast, reliable and
cheap forensic genetic analysis. The LAS needs to guarantee a high level of
security of sample identity and chain of custody. Here, we present a validated
(ISO 17025) laboratory semi-automated system for STR and SNP analyses of
biological material immobilized on FTA-cards.
The
described LAS encompass registration of samples in a LIMS database, export of a
sample-file to a puncher, punching of FTA-cards by a BSD600-duet puncher,
electronic sample check by means of barcodes, wash of punches by the THEONYX
liquid-handler from MWG, PCR-amplification and electrophoresis of amplicons.
The system was tested using blood and saliva immobilized on FTA-cards as
sources of biological material.
The
designed LAS led to:
¾ Correct
STR and SNP typing of individuals with a quality equal to or higher than the
profiles produced using chelex-extracted DNA.
¾ A
reduction in the rate of sample-reanalyses by 0.03.
¾ A
reduced risk of mix-up of samples during the laboratory procedure.
¾ Fewer
sample transfers.
¾ A
reduction in the number of PCR-cycles by 4 compared to chelex-extracted DNA
from blood.
During
the validation process, we did not observe mix-up of samples, loss of FTA-card
punches, contamination from extern sources, or cross-contamination between
samples.
P-117
STR typing of
77-year-old umbilical cord in maternity test
Hara M1, Kido A2, Yamamoto
Y1, 3, Takada A1, Saito K1
1Department of Forensic Medicine, Saitama Medical
School, Saitama, Japan
2Department of Legal Medicine, Faculty of Medicine, University of Yamanashi,
Yamanashi, Japan
3Criminal
Investigation Laboratory, Saitama Prefectual Police Headquarters, Saitama, Japan
In Japanese it is customary to preserve umbilical cord,
which is presented to parents from maternity clinic, as a sacred materials. The
umbilical cord is sometimes available for parentage test and personal identification
because it has been preserved for a long time. We performed a maternity test
using DNA extracted from an umbilical cord preserved for 77 years. The 15 short
tandem repeat (STR) loci included in the AmpFLSTR Identifiler Kit were used for
DNA analysis. DNA was extracted from the umbilical cord of the putative mother
(already deceased) by ISOHAIR (Nippongene) and from the buccal swab of the
child who requested the examination, by the DNA Extractor FM Kit (Wako). Using
the AmpFLSTR Identifiler Kit (Applied Biosystems), the 15 STR loci, D8S1178,
D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA,
TPOX, D18S51, D5S818 and FGA, as well as amelogenin locus were analyzed. For the
amplification of the umbilical cord, PCR conditions were modified as followed;
denaturation at 94ºC for 1 min, annealing at 59ºC for 5 min and extension at 72ºC
for 5 min (40 cycles) using 3% primer. Amplified products were separated by
denaturing capillary electrophoresis in the ABI PRISM 310 Genetic Analyzer
(Applied Biosystems). The results were analyzed by GeneScan Analysis 3.7
software (Applied Biosystems) and Genotyper 3.7 software (Applied Biosystems).
The 15 STR loci and the amelogenin locus were determined from the umbilical cord.
A contradiction in the mother and child relationship was not observed in the 15
STR loci. This means that the 15 STR loci were correctly typed from the
77-year-old umbilical cord. The maternity probability was 0.999937 and the
exclusion probability was 0.999629. Preserved umbilical cord are available for
parentage test and personal identification using STR typing.
haramasa@saitama-med.ac.jp
P-118
The Effects of Cleaning Agents
on the DNA Analysis of Blood Stains Deposited on Different Substrates
Harris KA, Thacker CR, Ballard
D, Syndercombe Court
D
Centre for Haematology, ICMS,
Barts and The London,
Queen Mary's School
of Medicine and
Dentistry, UK
Potential evidential material at a crime scene is
often adulterated. Deliberate attempts to remove biological material (using a
variety of cleaning agents) is a problem faced by forensic scientists
routinely. The substrates on which the blood is supported can also have an
inhibitory role. It has been shown that complete DNA profiles can be obtained
from non-visible quantities of blood. Lemire et al (1) reported successful
analysis of 100ml of a dried 1:2560 dilution of blood. Sourcing such small quantities of blood at a
crime scene is aided by the sensitivity of the Kastle-Meyer presumptive blood
test.
Blood samples were obtained from 6 unrelated donors
and standardised using white cell count. Blood was applied to a number of
different substrates: denim jeans;
cotton shirts and carpet. The stains
were allowed to dry and were then cleaned with chlorinated bleach, soap or
disinfectant until there was no visible trace.
Chelex 100 (Sigma) was used to extract DNA from the cleaned areas. PCR
was performed at 28 and, if necessary, 34 cycles using the AmpFLSTRâ SGM Plusä PCR Amplification kit (Applied
Biosystems). In excess of 250 profiles were examined and characterised using
heterozygote imbalance (Hbx), split peak frequency (SPF) and stutter proportion
(SP). This information was used to assess the clarity of the electropherograms
and the ability to relate evidence and control suspect samples, over a period
of 15 days.
It was found that chlorinated bleach had the most
pronounced negative effects with respect to the characteristics considered. In
particular Hbx increased significantly over the 15 day trial. Such a change has
the potential of incorrectly confirming or negating a relationship between
evidential and suspect material. The SPF and SP satisfied the allelic
designation guidelines set out by Gill et al (2) for STR multiplex systems in
successful amplifications. Although dark
coloured fabric dyes are anecdotally considered to interfere with DNA
amplification, in these tests Khaki fabric (55% cotton, 45% polyester) was
found to be the most inhibitive of the materials examined. This may be related
to the effect of different chemical agents used in the manufacture of this
material.
(1) Lemire CE, Bieber F. Forensic aspects of trace
human blood evidence: from presumptive test to STR profile. URL:
http://www.promega.com/geneticidproc/ussympllproc/abstracts/lemire.pdf
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Sparkes R. Development of guidelines to designate alleles using an STR
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Address for Correspondence:Catherine R Thacker
Centre for Haematology- Institute of Cell and
Molecular Science
Barts and The London
Queen Mary’s School of Medicine
and Dentistry
4 Newark Street-London E1 2AT
United
Kingdom
E-mail: c.r.thacker@qmul.ac.uk
P-119
An Investigation in to the
Genetic Structure of a Barbadian Population
Harris KA, Thacker CR, Ballard
D, Harrison C, Musgrave-Brown E, Syndercombe Court D
Centre for Haematology, ICMS,
Barts and The London,
Queen Mary's School
of Medicine and
Dentistry, UK
The male-specific inheritance of the Y chromosome and
the maternal passage of mitochondrial DNA (MtDNA) allow the genetic features of
a population to be investigated. In this
study a total of 81 blood stains were characterized using twenty-nine
Y-chromosome specific single nucleotide polymorphisms (SNPs) and MtDNA. The ABI PRISM® SNaPshot™ Multiplex
System (Applied Biosystems) was used to genotype the Y-SNPs and enabled the
paternal characteristics of the population to be assessed. Sequencing of the control region of the
mitochondrial DNA loop was carried out using the BigDye® Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems).
Haplogroup frequencies and genetic diversity values were calculated and
compared to a less well defined UK-resident Afro-Caribbean population. It was found that the Barbados
population studied was similar to other populations of African ancestry and it
is proposed that further characterization may be possible using population
specific SNPs. Historically, Ghana and Nigeria
supplied Barbados
with black labour during periods of slavery and it may be useful to first
target these ancestral populations when attempting to further define the
genetic features of Barbados.
We would like to acknowledge advice given by Juan J
Sanchez (Department of Forensic Genetics, Institute of Forensic
Medicine, University
of Copenhagen, Denmark)
and Maria Brion (Institute of Legal
Medicine, University
of Santiago de Compostela, Galicia,
Spain)
with regard to the Y SNPs used in this study.
This part of the study was completed as part of the SNPforID
project.
We would like to thank the Forensic Sciences Centre, Barbados for
their help in the collection and supply of samples.
Address for Correspondence:
Catherine R Thacker
Centre for Haematology
Institute of Cell and Molecular Science
Barts and The London
Queen Mary’s School of Medicine
and Dentistry
4 Newark
Street
London E1 2AT
United
Kingdom
E-mail: c.r.thacker@qmul.ac.uk
P-120
A Sensitive Issue:
Pyrosequencing as a Valuable Forensic SNP Typing Platform
Harrison C1,
Musgrave-Brown E1, Bender K2, Carracedo A3,
Morling N4, Schneider P2, Syndercombe-Court D1,
The SNPforID Consortium
1 Centre for Haematology, ICMS, Barts and The London, Queen Mary’s School of Medicine and Dentistry, UK
2 Institute of Legal Medicine, Johannes Gutenberg University
Mainz, Germany
3 Institute of Legal Medicine, University
of Santiago de Compostela, Spain
4 Department of Forensic Genetics, Institute of Forensic
Medicine, University
of Copenhagen, Denmark
Analysing minute amounts of
DNA is a routine challenge in forensics often accompanied by a variety of
obstacles resulting in difficulties with analysis. To minimize these events it is important to
choose technological platforms with specific criteria in mind. With so many typing technologies available
for genotyping single nucleotide polymorphisms (SNPs), the selection of a
suitable platform to meet forensic requirements can be difficult; particularly
when questioning the sensitivity of an instrument and its ability to optimize
detection and the amount of information obtained from forensic samples.
Here we investigate the
Pyrosequencing (Biotage) method for genotyping SNPs. The Pyrosequencing method offers intrinsic
quantifying capabilities and uniform electropherogram peak heights making it an
ideal platform for sensitivity analysis.
Using normalised concentrations of DNA and testing five autosomal SNPs,
varied amounts of genomic DNA were added to the PCR with the resulting products
compared on the two available instrument models: the PSQTM 96MA and
PSQTM HS 96A systems. In
doing so, a detailed comparison of the two models was completed while
establishing a lower limit of detection on both instruments to give results
supporting the use of Pyrosequencing as a valuable forensic SNP typing
platform.
Address for correspondence:
Cheryl Harrison, Department of Haematology,
Institute of Cell and Molecular Sciences, Barts and The London, Queen Mary’s
School of Medicine and Dentistry, 4 Newark Street, London, E1 2AT
Email: c.d.harrison@qmul.ac.uk
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