P-184
Human DNA bank in Sao Miguel Island (Azores):
a resource for genetic
diversity studies
Mota-Vieira L1,3,
Pacheco PR1,3, Almeida ML2, Cabral R1,3,
Carvalho J2, Branco CC1,3, de Fez L1,3,
Peixoto BR1,3, Araujo AL2, Mendonça P2.
1
Molecular Genetics and Pathology Unit, Hospital of Divino Espirito Santo, São
Miguel, Azores, Portugal
2
Hematology Department, Hospital of Divino Espirito Santo, São Miguel, Azores,
Portugal
3Instituto
Gulbenkian de Ciência, Oeiras, Portugal.
The peopling of São Miguel Island in the 15th century was made by
Portuguese and settlers of foreign origin, (Flemish, Jews, Moorish
prisoners and black slaves), generating an admixture signature. Thus to unravel
São Miguel’s population genetic background and
to characterize its population’s polymorphisms, we decided to establish a human
DNA bank.
Here, we describe the
construction of the DNA bank, and analyse the information of 1000 samples
obtained from healthy blood donors. The bank follows the international ethical
guidelines, which include Informed Consent, confidentiality, anonymity of
personal data, and abandonment in case of expressed will. DNA was isolated from
blood samples, coded and immediately stored in a locked refrigerator. The
identifiable DNA bank has self-reported data concerning sex, age, birth,
current place of living, and parental birthplaces. The samples are
representative of all the island’s municipalities (r=0.995, p<0.01). The
majority (87%) of the participants are male, with mean age of 36.3 y (18-64y).
Birthplace analysis reveals that 902 (90%) have both parents born in São Miguel. Moreover, 477 (54%) have their parents born
in the same locality, confirming high rate of consanguinity in rural area.
To date, this DNA bank was
used to assess the Y-chromosome phylogeny and diversity in Azorean population (Pacheco
et al, Ann Hum Genet 69:145-156, 2005). Now, we are
analysing autosomal STRs for the better understanding of the gene pool and
genetic structure of the archipelago’s population
contact: lmotavieira@hdes.pt
Funded by DRCT, Azores.
P-185
Haplotype studies of germline
mutations in short tandem repeats using flanking markers
Mueller M1, Klintschar M2, Hohoff C1, Brinkmann B1
1Institute of Legal Medicine, University of Muenster,
Muenster, Germany
2Institute of Legal Medicine, Martin-Luther-University Halle-Wittenberg, Halle (Saale),
Germany
In our routine case work for
parentage testing we have observed more than 150 cases in which a de novo mutation
had occurred. The paternity was highly validated with a paternity value W of
greater than 99.99% including the mutation. Germline mutations occur in short
tandem repeats as deletion or expansion of repeat units. The following example
from STR system VWA shows the difficulty to assess the mutation's origin:
child: 14/17; mother 14/18; father 14/16. According to this, the child's allele
17 could be due a maternal expansion or a paternal deletion. With routinely
used STRs it was not possible to determine whether the mutation occurred in
maternal or paternal germline.
We here report our results
from 37 families with mutations at one of five different loci (i.e., D8S1179,
D18S51, D21S11, ACTBP2 and VWA) to specify the origin of the observed mutation.
We chose four to six
polymorphic flanking markers in each case and typed these markers by, e.g.,
amplicon sizing on a ABI PRISM 310 Genetic Analyzer.
The results of our study will
be presented and the consequences for the analysis of STR mutations will be
discussed.
P-186
Characterization of a novel
stutter product in the Y-STR marker DYS392 and a rare polymorphic variant in
the DYS456 homolog identified using the AmpFSTR® YfilerTM PCR Amplification
Kit
Mulero J, Chang C, Calandro L,
and Hennessy L
Applied Biosystems, Foster City, CA
94404
Y-chromosome short tandem
repeat (STR) markers yield a high degree of confidence that only the male
contributor is being analyzed in male-female mixtures. The AmpFlSTR ® YfilerTM
PCR amplification kit is a commercial multiplex system designed for the
simultaneous amplification of 17 Y-STR markers (DYS19, DYS385a/b, DYS389I/II,
DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458,
DYS635 (formerly known as Y GATA C4) and Y GATA H4).
A by-product of the
amplification of the trinucleotide repeat locus DYS392 is the formation of N-3
and N+3 stutter products. Sequence
analysis of the novel N+3 stutter band demonstrates that its sequence is one
TAT repeat longer than that of the corresponding main allele. Both N-3 and N+3 stutter percentages
increased as (1) the main allele repeat number increased, as (2) the magnesium concentration
was increased in the reaction or if (3) the initial amount of DNA template was
decreased. Since both stutter products
behave in a similar and reproducible fashion, we propose that the same rules
that apply to the interpretation of N-3 stutter products could be applied to
N+3 stutters.
During an extensive population study conducted
using the AmpFlSTR ®
YfilerTM PCR amplification kit, we identified a 71-bp FAMTM-labeled
fragment in 2.1% of the samples analyzed.
We determined that the DYS456 primers amplified the fragment. Direct sequencing of these fragments
indicated a T to G single nucleotide polymorphism (SNP) in the primer binding
site of the affected individuals. The
SNP is located within a X-Y homologous region on chromosome Xq21.31 and was
observed with the highest frequency within the African American population
(7.7%). This fragment is outside the Y STR allele size range and does not
interfere with allele calls. In
addition, we demonstrate that the AmpFlSTR ® YfilerTM kit is capable of yielding full
profiles of the minor male contributor (male: female=1:4000) even in the
presence of female DNA containing the variant G.
P-187
Use of
Fluorescence In Situ Hybridisation and Laser Capture Microdissection to isolate
male non-sperm cells in cases of sexual assault
Murray C, McAlister C, Elliott K
The Forensic
Science Service, Trident Court,
Birmingham Business Park, Birmingham, B37
7YN, UK.
In cases of rape where vaginal swabs taken from the
victim test positive for the presence of semen, but no sperm can be found (i.e.
the semen is azoospermic), other cells originating from the perpetrator may
still be present. These could include cells from the urethral tract, white
blood cells and epithelial cells from the penis. However, lysis of the cell
harvest is likely to yield only the victim’s profile due to the large number of
vaginal cells present on the swab.
Analysis of Y chromosome markers could be carried out, however a profile
with which to search the National DNA Database may be preferable in some cases.
Here
we describe a method to identify male (non-sperm) cells using Fluorescence In
Situ Hybridisation (FISH) and subsequently isolate them using laser capture
microdissection. Cell harvests from post-coital vaginal swabs were fixed onto
glass microscope slides and fluorescently labelled probes were hybridised to
the X and Y chromosomes. The slides were searched and any cells containing both
X and Y signals were collected using laser capture microdissection. DNA was
extracted from the collected cells in a direct lysis procedure and then amplified
using Low Copy Number conditions.
In this study, fifteen samples have been tested, where
time since intercourse (TSI) ranged from 1 hour to 24 hours. Positive results
(at least 75% of the male profile) were obtained from 10 of these samples, with
16 hours being the highest TSI to give a result. The remaining 5 samples had
too few male cells present to produce a profile.
Contact:
Caroline.Murray@fss.pnn.police.uk
P-188
Mixture interpretation using
SWaP SNPs and non-biallelic SNPs
Musgrave-Brown E1,
Anwar N2, Elliott K2, Phillips C3, Syndercombe
Court D1, Carracedo A3, Morling N4, Schneider
P5 and McKeown B2
1Centre for Haematology, ICMS,
Barts and The London, Queen Mary’s School of Medicine and Dentistry, UK
2Orchid BioSciences
Europe Ltd, Abingdon, UK
3 Institute of Legal Medicine, University of Santiago de Compostela, Spain
2 Institute of Legal
Medicine, Johannes
Gutenberg University
Mainz, Germany
4 Department of Forensic
Genetics, Institute
of Forensic Medicine, University of Copenhagen, Denmark
Improved analysis of degraded samples, increased
throughput, and a wider choice of typing platforms are some of the significant
advantages offered by single nucleotide polymorphism genotyping over current
short tandem repeat (STR)-based systems.
However, DNA mixtures present a considerable problem to SNP analysis as
there is currently no generally accepted method that allows recognition of the
presence of a mixed profile or identification of the individual contributors.
We describe a multiplex approach to solving this
problem that is based upon the use of two rare subsets of SNPs: SWaP™ SNPs and
non-biallelic SNPs. The SWaP SNP
technique relies upon the use of modified PCR primer tails to generate ‘mirror’
copies of the SNP under analysis so that the resulting amplicon contains a real
SNP that is flanked by two copies. The
mirror copies are generated in known ratios, so that comparison of peak height
or peak area ratios following SNaPshot enables an estimate of the relative
contributions of each allele to the mixture to be made.
An assay comprising eight such SWaP SNPs combined with
three non-biallelic SNPs is described and its value for forensic mixture
analysis is discussed.
Address
for correspondance: 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: e.musgrave-brown@qmul.ac.uk
P-189
Introducing a highly
polymorphic STR at the D12S391 locus valuable for use in forensic application
Massoumeh Nadji, Zahra
Lashgary, Hadi Namazi, Massoud Houshmand.
Legal Medicine Organization
Different
ethnic groups live in Iran,
among which Farsis, Kurds, Lors, Balooches, Bakhtiaris, Azari Turks, Taleshes,
Turkamans, Qashqais and Arabs may be pointed out. Smaller ethnic groups also
live in Iran.
In this study we have investigate allel frequency distribution for D12S391
locus of 354 Persian different ethnic groups. PCR and ALFexpress DNA sequencer
was used as methods in this study. Allele’s number was found from 2-15 in our
study for D12S391 locus in total 708 chromosomes. The results were compared by
means of statistic to evaluate confirmation to Hardy-Weinberg predictions.
Statistical investigation showed that this polymorphic STR with its simple
structure can be used as valuable STR locus for forensic purposes in Iranian
Population.
P-190
Analysis of the HVI, HVII and
HVIII regions of mtDNA in 400 unrelated Japanese
Nagai A, Nakamura I, Bunai Y
Department of Legal Medicine, Graduate School of Medicine, Gifu University,
Gifu, Japan
Sequence
polymorphism of the hypervariable regions HVI, HVII and HVIII of mitochondrial
DNA (mtDNA) was analyzed in a sample of 400 unrelated Japanese individuals
living in Gifu Prefecture (central region of Japan) by PCR
amplification and direct sequencing.
A
total of 306 different haplotypes resulting from 199 polymorphic positions was
found in our Japanese population sample. The most common haplotype (16129A, 16223T, 16362C,
73G, 152C, 263G, 309.1C, 315.1C, 489C) was shared by
10 individuals. The genetic diversity and the genetic identity were calculated
to be 0.9975 and 0.0050, respectively. The length heteroplasmy in the
homopolymeric C-stretch regions located at nucleotide positions 16184-16193 in
HVI, at positions 303-315 in HVII and at positions 568-573 in HVIII was observed
in 26.1%, 8.6% and 4.1% of individuals, respectively.
Contact: anagai@cc.gifu-u.ac.jp
P-191
Interpreting DNA
evidence isolated from a self made firearm in a
homicidal case
Nagy G1, Angyal M2, Czömpöly T4, Nyárády Z3, Bajnóczky I2
1 Institute of Forensic Medicine, University of Pécs, Hungary
2Forensic Examiner Unit,
Baranya County Police Department, Pécs, Hungary
3 Department of Oral
and Maxillofacial Surgery, University
of Pécs, Hungary
4 Institute of Immunology and
Biotechnology, University of Pécs,
Hungary
A woman was brutally murdered. She was
shot in head but no bullet was found in the head. Later the Baranya County
Police Department arrested a man and charged to murder the woman. A self made
firearm was found and secured for further investigation. The firearm works with
blank cartridge which lunches a hollow steel spike out of the firearm’s tube.
After the shoot the steel spike was jerk back to the tube by a spring device.
The weapon used to slaughtering pigs. After the murder at least six pig were
slaughtered with the weapon.
From the cavity of the hollow steel spike
biological material were secured from different depth. From the samples genomic DNA was extracted
according to standard techniques (Chelex-100 method) and amplified utilizing different
amplification approaches (AmpFlSTR SGM Plus produced by Applied Biosystem). PCR
products were separated by capillary gel electrophoresis on an ABI PRISM 310
Genetic Analyzer and typed by comparison against standard allelic ladders.
With the firearm ballistic tests were also made by the
Forensic Examiner Unit of Baranya County Police Department which proves that
the firearm could be the perpetrator’s weapon. From the biological samples the
victim’s full DNA profile was detected.
The suspect was sentence to prison for 25 yeas based
on the DNA and ballistic evidences.
Address for correspondence
Prof. Dr. med. István Bajnóczky, Institut of Forensic Medicine, Medical Faculty, University of Pecs, Hungary, Szigeti u. 12, H-7624 Pécs, Hungary, Fax: 00 36 72 536242, eMail: gergely.nagy@aok.pte.hu
P-192
Allele frequencies for 15 STR loci in two populations
from Hungary
Nagy G1, Nagy Zs1, Nyárády Z2, Bajnóczky I2
1 Institute of Forensic Medicine, University of Pécs, Hungary
2 Department of Oral
and Maxillofacial Surgery, University
of Pécs, Hungary
To enlarge our understanding of genetic
variation in Hungarian population, a population genetic study was carried out
on unrelated 115 Hungarian Caucasian and 116 Hungarian Roma (South-West Hungary area).
We here present the frequency distributions of 15 highly polymorphic autosomal
STR systems (CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S539,
D16S539, D18S51, D19S433, D21S11, FGA, THO1, TPOX, VWA) from the two
population.
Genomic DNA was extracted according to standard
techniques (Chelex-100 method) and amplified utilizing different amplification
approaches (Powerplex16 produced by Promega Corp. and AmpFlSTR SGM Plus
produced by Applied Biosystem). PCR products were separated by capillary gel
electrophoresis on an ABI PRISM 310 Genetic Analyzer and typed by comparison
against standard allelic ladders.
The overall pattern of allél frequencies
was similar to many Caucasian and Indian (compare to Roma) populations and
heterozygosity varied from 57% (TPOX, Caucasian) to 92% (FGA, Caucasian). For
all fifteen, no deviations from the Hardy-Weinberg equilibrium hypothesis were
detected. The mean exclusion probability ranged from 25% (TPOX, Caucasian) to
83% (FGA, Caucasian). All tetranucleotide STR systems are highly informative
markers in the three populations investigated, e.g., the power of
discrimination ranges from 0,744 (TPOX, Caucasian) to 0,969 (D18S51,
Caucasian).
The results suggest the usefulness of these
loci for anthropogenetic, paternity and forensic investigations in Hungarian
populations.
Address for correspondence
Prof. Dr. med. István Bajnóczky, Institut of Forensic Medicine, Medical
Faculty, University of Pecs, Hungary, Szigeti u. 12,
H-7624 Pécs, Hungary, Fax: 00 36 72 536242, eMail: gergely.nagy@aok.pte.hu
P-193
Y chromosome haplotypes in Roma and Caucasian
populations from Hungary
Nagy G1, Nagy Zs1, Nyárády Z2, Bajnóczky I2
1 Institute of Forensic Medicine, University of Pécs, Hungary
2 Department of Oral and
Maxillofacial Surgery, University of
Pécs, Hungary
To enlarge our understanding of genetic
variation in Hungarian population, a population genetic study was carried out
on unrelated 50 Hungarian Caucasian and 50 Hungarian Roma male (South-West
Hungary area). Eleven Y
chromosome STR polymorphisms (DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391,
DYS392, DYS393, DYS437, DYS438 and DYS439) were analyzed in the samples.
Genomic DNA was extracted according to standard
techniques (Chelex-100 method) and amplified utilizing different amplification
approaches (PowerplexY produced by Promega Corp). PCR products were separated
by capillary gel electrophoresis on an ABI PRISM 310 Genetic Analyzer and typed
by comparison against standard allelic ladders.
A general STR allelic frequency pattern in the Roma and Caucasian
population from Hungary
corresponds to other European populations. Thirty six haplotypes were observed
in single copy. Twenty one Hungarian haplotype were not previously observed in the Y STR
Haplotype reference Database among the set of European populations.
Address for correspondence
Prof. Dr. med. István Bajnóczky, Institut of Forensic Medicine, Medical Faculty,
University of Pecs, Hungary, Szigeti u. 12, H-7624 Pécs, Hungary, Fax: 00 36 72
536242, eMail: gergely.nagy@aok.pte.hu
P-194
Characterization of mtDNA SNP
typing using quantitative real-time PCR for forensic purposes with special
emphasis on heteroplasmy detection and mixture ratio assessment
Niederstätter H1,
Coble MD2,3, Parsons TJ2, Parson W1
1 Institute of Legal Medicine, University of Innsbruck, Müllerstrasse 44, 6020 Innsbruck
2 Armed Forces DNA Identification Laboratory, Armed Forces Institute of Pathology, Rockville, MD, USA
3 Biotechnology Division,
National Institute
of Standards and
Technology, Gaithersburg, MD, USA
The analysis of mitochondrial DNA (mtDNA) has proven
to be highly useful in forensic casework, especially when samples comprising
degraded DNA are analyzed which are not amenable to the typing of the highly
informative nuclear short tandem repeat markers (STRs). However, unlike
autosomal STRs, mtDNA testing does not provide definitive identification of
individuals because all members of a matriline are expected to match each other
due to maternal inheritance of the mitochondrial genome and the lack of
recombination. Therefore, the principal limitation associated with forensic
mtDNA typing is the low power of discrimination that is obtained when common
mitochondrial types are present. Current mtDNA testing typically targets the
variable base positions in the non-coding control region of the mitochondrial
genome by sequencing both strands, and most laboratories restrict their
investigations to one or two hypervariable regions (HV1 and HV2), comprising
approximately 600 base pairs (~3.6 % of the entire mitochondrial genome). In
the last couple of years it has become increasingly recognized that assays targeting
single nucleotide polymorphisms (SNPs) are well suited for efforts to gain
additional information in mtDNA testing. We investigated the forensic
applicability of real-time detection PCR using TaqMan probes targeted to the
highly discriminatory mitochondrial control region SNP 16519 T/C for several
reasons: 1) it’s large linear dynamic range in terms of target-molecule input
number allows – along with the short amplicons that are obtained - the analysis
of a broad spectrum of samples differing in DNA quantity and quality with a
single protocol, 2) the homogeneous format of the assay avoids potential
cross-contamination of samples with PCR products and makes it easy to
automatize, 3) the quantitative information that can be obtained aids the
formulation of objectively-based criteria for distinguishing between authentic
signal and contamination and 4) the multicolor capability of real-time PCR
instruments enables the simultaneous interrogation of both base-states of the
SNP under investigation. The last point is particularly important because of
the potential of mtDNA to manifest heteroplasmic mixtures in continuously
varying proportions. The results of a study on 135 paternity trios with known
control region sequences showed that 16519 can be reliably typed with the
TaqMan approach without a need to run samples in replicates. For both alleles
the linear dynamic range was at least 5 orders of magnitude with a lower end
sensitivity of approximately 10 double stranded target molecules. The apparent
single-cycle PCR efficiencies during the exponential phase of the amplification
were close to 100% for complex genomic DNA as well as for non-linearized
plasmids used as templates. Defined mixtures of plasmids containing 16519T and
C could be detected and quantitated reliably down to the 5% level for either
variant with a lower end sensitivity of a total of 100 - 200 double stranded
target molecules. Finally, the estimated mixture ratios for three heteroplasmic
and two homoplasmic paternity DNA samples were consistent with the results
obtained by typing approx. 300 bacterial colonies/sample containing PCR
amplified mitochondrial control regions.
P-195
Comparison
of six DNA quantification methods
Nielsen K1,
Mogensen HS1, Eriksen B1, Hedman J2, Parson W3,
Morling N1
1Department of
Forensic Genetics, Institute
of Forensic Medicine, University of Copenhagen, Denmark
2Swedish National Laboratory of
Forensic Science, Linköping,
Sweden
3Institute of Legal
Medicine, Medical University of
Innsbruck, Austria
Six commercial preparations of
human genomic DNA were quantified using six quantification methods, including
UV spectrometry, SYBR-green dye staining, slotblot hybridization with the probe
D17Z1, and three TaqMan real time PCR assays: Quantifilerä Human DNA
Quantification kit, Quantifilerä Y DNA
Quantification kit, and RB1 rt-PCR. In general, all methods measured higher DNA
concentrations than expected based on the information by the suppliers of the
human DNA preparations. The Quantifilerä Human DNA Quantification kit
gave the highest measures of the DNA concentrations of five of the six human
DNA preparations compared to the other five quantification methods. With the
Quantifilerä Human DNA
Quantification kit, the ratio of measured DNA/expected DNA ranged from 1.9 to
2.8. When the Quantifiler human DNA standard was replaced by a different
commercial human DNA preparation (G147A, Promega) to generate the DNA standard
curve in the Quantifilerä Human DNA
Quantification kit, the DNA quantification results of the human DNA
preparations were comparable to the results of other DNA quantification
methods. The ratio of measured DNA/expected DNA ranged from 1.1 to 1.6. Human
DNA preparations were quantified by the Swedish National Laboratory of Forensic
Science in Linköping, using the Quantifilerä Human DNA Quantification kit.
The results confirmed the Quantifilerä results obtained
in Copenhagen.
Samples of the same human DNA preparations were quantified by the Institute of Legal Medicine in Innsbruck, using RB1 rt-PCR, and the ratio of
measured DNA/expected DNA ranged from 1.1 and 1.5. The quantification results
using RB1 rt-PCR were comparable to those obtained with the other
quantification methods.
The results indicate a
calibration problem with the Quantifilerä human DNA standard for its
use with the Quantifilerä Human DNA
Quantification kit. The possible reasons of the problem are discussed, and a
solution is suggested. The results emphasise the need for standard reference
DNA material and standard methods for DNA quantification.
P-196
Effect of soil environment on detectability of SGM profiles in selected tissue samples
Niemcunowicz-Janica A 1, Pepinski W 1,
Janica JR 2, Skawronska M 1, Janica J 1,
Koc-Zorawska E 1, Soltyszewski I 3
1 Department of Forensic
Medicine, Medical University of
Bialystok, Poland
2 Department of Radiology,
Medical University of Bialystok,
Poland
3 Central Forensic Laboratory of
Police, Warsaw, Poland
Processes of autolysis and decomposition have always
been a concern to forensic specialists. In cases of decomposed bodies,
estimation of time of death – very crucial for evidential reasons – is often
impossible due to effect of various environmental conditions. The authors
attempted to assess capability to type AmpFlSTR SGM Plus (Applied Biosystems)
loci in tissue material stored in sand, garden soil and peat in view of
estimation of time of death. Tissue material was collected during autopsies of
five persons aged 20-30 years with time of death determined within the limit of
14 hours. Heart muscle, liver and lung specimens of dimensions 2x2x2cms were
placed in 40ml containers filled with sand, garden soil or peat and stored at
21°C. DNA was extracted by organic method from tissue samples collected in
7-day intervals. Recovered DNA was quantitiated fluorometrically and by
hybridization with human DNA-specific probe (QuantiBlot) with chemiluminescent
detection. DNA quality was assessed by 2% ethidium bromide agarose gel
electrophoresis. 2-10ng target DNA was amplified according to the
manufacturer’s instruction. ABI 310 and reference sequenced ladders were used
following the manufacturer’s instructions. As a threshold value a signal of ≥150
was assumed. Storage of liver specimens in garden soil for more than 14 days
resulted in allelic drop-out and after 21 days no profiles were typeable. Heart
muscle specimens were typeable in all SGM systems after 35-day storage in sand,
while allelic drop-out and subsequent lack of profiles were noted after 14 and
35 days, respectively. Lung specimens stored in garden soil exhibited allelic
drop-out and subsequent lack of profiles after 7 and 21 days, respectively. All
SGM loci were typeable in the latter material stored in sand up to day 35 with
gradual decline of longer amplicons (D2S1338, D16S539 and D18S51).
P-197
Effect of water environment on detectability of SGM profiles in selected tissue samples
Niemcunowicz-Janica A 1, Pepinski W 1,
Janica JR 2, Skawronska M 1, Janica J 1,
Koc-Zorawska E 1, Soltyszewski I 3
1 Department of Forensic
Medicine, Medical University of
Bialystok, Poland
2 Department of Radiology,
Medical University of Bialystok,
Poland
3 Central Forensic Laboratory of
Police, Warsaw, Poland
Processes of autolysis and decomposition have always
been a concern to forensic specialists. In cases of putrefied bodies,
estimation of time of death – very crucial for evidential reasons – is often
impossible due to effect of various environmental conditions. The authors
attempted to assess capability to type AmpFlSTR SGM Plus (Applied Biosystems)
loci in tissue material stored in water environment in view of estimation of
time of death. Tissue material was collected during autopsies of five persons
aged 20-30 years with time of death determined within the limit of 14 hours.
Heart muscle, liver and lung specimens of dimensions 2x2x2cms were placed in
40ml containers filled with pond water and sea water (0.8% salt) and stored at
21°C. DNA was extracted by organic method from tissue samples collected in
7-day intervals. Recovered DNA was quantitiated fluorometrically and by
hybridization with human DNA-specific probe (QuantiBlot) with chemiluminescent
detection. DNA quality was assessed by 2% ethidium bromide agarose gel
electrophoresis. 2-10ng target DNA was amplified according to the
manufacturer’s instruction. ABI 310 and reference sequenced ladders were used
following the manufacturer’s instructions. As a threshold value a signal of
≥150 was assumed. Liver specimens were typeable in all SGM loci within 100 days
of storage in pond water with gradual allelic drop-out at D18S51 in sea water.
Heart muscle specimens stored in pond water exhibited allelic drop-out at THO1,
FGA and D18S51, while all loci were typeable in sea water stored samples. For
lung specimens allelic drop-out was noted throughout the profile. The authors
conclude that the course of complex postmortem processes is variable. Dynamics
of tissue and internal organs decomposition in an intact corpse is different
than that in tissue specimens placed in a water environment which delays
decomposition processes promoted by bodily and microbial enzymes.
P-198
DNA quantity variation in shed
hairs, plucked hairs and contact traces
Nilsson M, Andréasson H, Allen
M
Department of Genetics and
Pathology, Rudbeck Laboratory, Uppsala
University, Sweden
Different categories of
forensic evidence materials are known to vary considerably in their DNA
content, affecting the probability of a successful identification analysis. In
this study, the mitochondrial and nuclear DNA content were quantified in 46
head hairs, 61 body hairs and 76 contact trace samples using a previously
reported real-time PCR method. In this TaqMan assay, two specific probes,
labelled with different dyes, enables quantification of the nuclear
Retinoblastoma 1 gene and the mitochondrial tRNA Lys
gene.
DNA quantification in the roots
and distal sections of plucked head hairs revealed large variations in DNA
content between the root and the shaft. Furthermore, large intra- and
inter-individual variations were found among hairs. The overall difference in
mean mtDNA content in the first cm between shed and plucked hairs was 74-fold.
In the first cm of shed hairs no nDNA copies were detected whereas plucked
hairs contained an average of 25,800 nDNA copies. Furthermore, variation in
mtDNA content was evaluated at different lengths of the hairs. The decrease in
average mtDNA content per cm was 2-fold between the first cm and the following
1-4 cm part in shed hairs. In contrast, an 80-fold decrease in the average
mtDNA content per cm was observed between the first cm and the following 1-4 cm
of plucked hairs. This large difference is only seen for the root part as the
first cm of shed hairs contain approximately equal average mtDNA amounts per cm
as the second part (1-4 cm) of plucked head hairs (45,700 and 41,700 mtDNA
copies per cm, respectively). Thus, most of the DNA content difference between
shed and plucked hairs is observed in the first cm and is likely to reflect the
different growth phases of hair. In other types of plucked body hairs
evaluated, beard contained on average 2-fold and 6-fold more nDNA compared to
eyebrows and arm hair respectively.
In addition, DNA content was
estimated in epithelial cells collected from fingerprints and accessories. The
quantification of epithelial cells on various items or from fingerprints also
displayed large variations, with some material categories containing large
amounts of nuclear DNA while no detectable nuclear DNA and only limited amounts
of mitochondrial DNA were seen in others. Fingerprints visualised by black
powder contained slightly more mtDNA and nDNA compared to magnetic powder
treated prints. The quantification results illustrate that some prints contain
up to 700 nDNA copies and the majority of the prints contain sufficient DNA
amounts for an mtDNA analysis. The quantification analysis of DNA collected
from accessories showed that earrings contained most DNA, 144,400 nDNA copies
on average while rings, bracelets, necklaces and charms contained the lowest
amounts of DNA with 80-300 nDNA copies on average. Samples collected from
glasses and watches contained on average 4,200 and 1,800 nDNA copies
respectively. In addition to the large variations seen between different sample
categories the DNA content were shown to vary largely between samples taken on
the same category.
In conclusion, the use of
real-time DNA quantification in this study has revealed several important
insights regarding DNA content in various forensic materials. Information regarding inter- and intra- individual
variation, variation in content within plucked and shed hairs at
different lengths, the average DNA content in different types of hairs as well
as in other materials is highly relevant for forensic applications.
Contact: martina.nilsson@genpat.uu.se
P-199
Sensitive forensic DNA analysis
using the Pyrosequencing technology
Nilsson M, Styrman H, Andréasson
H, Divne A-M, Allen M
Department of Genetics and
Pathology, Rudbeck Laboratory, Uppsala
University, Sweden
DNA from casework samples are in some cases
degraded and not in sufficient amounts for a routine STR analysis. Analysis of
mtDNA or reduced size PCR fragments of nuclear targets is often necessary for a
successful analysis of these samples. We have developed several sensitive,
rapid, flexible and easy-to-use mtDNA, SNP and STR typing systems based on the
Pyrosequencing technology. This is a non-electrophoretic, single-tube
sequencing-by-synthesis method in which a cascade of enzymatic reactions yields
detectable light. If a nuclear DNA analysis is permitted on degraded or limited samples,
STR analysis is preferred due to the large number of alleles at each locus. As
a complement to the routinely used STR assays, a pyrosequencing-based analysis
of short PCR fragments covering only a few bases outside the actual repeat
unit, have been developed. Ten widely used autosomal STR loci with short repeat
units with short maximum allele lengths have been analysed using
pyrosequencing. Since no size separation based on overlapping fluorescence
spectra is necessary using this method, all amplicons have been kept short,
between 66-175 bp. Discrimination of different alleles is possible by the use
of a termination-recognition base (TRB) and a sequence-directed dispensation
order. The TRB represents the first occurring downstream base in the template
that is not part of the repeat unit. For heterozygous genotypes, the signal is
reduced by half when the shortest allele terminates while no signal reduction
is observed for homozygous genotypes. Pyrosequencing was found suitable for
analysis of short simple repeat markers and a few markers could be interpreted
using DNA input concentrations of 25 pg.For analysis of mixtures, eight commonly used Y-STR
markers have been analysed in PCR fragments between 72 bp and 233 bp. The Y-STR
markers were easily interpretable and all markers could be analysed using 100
pg of input DNA, while half of the markers could be analysed at 25 pg input
DNA. When the Y-STR analysis fails in degraded or limited samples, SNP analysis
is likely to be more successful as the amplicons can be designed very short. A
system for analysis of 17 SNP markers on the Y-chromosome has been developed.
The PCR products are between 50 and 96 base pairs in size and the most
informative markers have been optimised to allow analysis in triplex PCR and
pyrosequencing reactions. The Y-SNP analysis could be performed on 10 pg of
input DNA for some markers. For severely degraded DNA samples an mtDNA analysis
system has been developed. Two PCR fragments covering the HVI and HVII regions
in the D-loop are analysed rapidly in eight pyrosequencing reactions.
Furthermore, 17 fragments in the coding region of the mitochondrial genome can
be used for additional discrimination. Each fragment covers multiple
polymorphic SNPs with an average read length of 74 nucleotides in the
pyrosequencing reactions. In order to save valuable material multiplex PCR and pyrosequencing
reactions are under development. Although it will be possible to analyse STR
markers in duplex pyrosequencing reaction by further developments, the
multiplex capability is the major limitation of pyrosequencing. Consequently,
the pyrosequencing method is more suited for analysis of a limited set of
markers in challenging samples allowing short amplicons on degraded DNA samples
rather than analysis of a large set of markers. Since the actual sequence is
determined rather than the repeat length in this assay there is a possibility
to achieve additional information such as the nature of a mutational event.
Furthermore, a rapid compilation of population databases and evaluation of
novel less complex STR markers can be performed. Pyrosequencing is a robust and
flexible system that can handle SNP analysis, STR analysis or sequencing of
short stretches of DNA with two hours post-PCR handling.
Contact: martina.nilsson@genpat.uu.se
P-200
Projeto
Paternidade Social
1 NOGUEIRA, G. C.; 1
MONTEIRO, E. H. G; 1 SILVA, L.S ; 1.NASCIMENTO,
D.S.; 1 TOMMASI, B. O;
1 Instituto Tommasi;
The main
objective of the present work is the familiar reorganization of children and
adolescents of low income. In each year, about 2,5 million of births are
registered in Brazil.
Of these, an average of 750 thousand is made without identification of the
father. As consequence, these children do not have officially father declared,
what frequently represents a serious emotional, social and economic problem.
This work counts with a multidiscipline team formed by biochemists, biologists,
biomedicine doctors, judges, lawyers, promoters and psychologists aiming to
help 132 families in 12 months. In this research, paternity investigation are
done analyzing 16 markers of type STR and for the psychological assistance are
being used focal psycotherapy and the clinical methods of Piaget. Until now 110
cases for DNA had been carried through, and the molecular examinations had
proven paternity with 99,999 % of probability in 82 cases (74,55%) and had
excluded paternity in 27 cases (25,45%).
Tommasi Institute and Canadá Embassy support this work
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