P-295
Sperm DNA
extraction from mixed stains using the DifferexTM System
Tsukada K, Asamura H, Ota M,
Kobayashi
K, Fukushima H
Department of Legal Medicine, Shinshu University School
of Medicine,0’
Asahi 3-1-1, Matsumoto, Nagano 390-8621, Japan
DNA typing is a
very important and powerful tool in criminal investigations, especially those
revolving around sexual crimes. Although nearly all specimens from crime scenes
are of blood or blood stains, in cases of rape many of the specimens are of
mixed stains, such as sperm with oral cells or sperm with vaginal cells. In
recent years, with the development of multiplex Y-STR PCR kits, it has become
possible to type many sperm loci within a short time. However, with autosomal
DNA typing of sperm from mixed stains, it is necessary to separate out the
sperm DNA from the mixed stain via a two-step method (two-step differential
extraction procedure). However, this two-step method requires a long time, at
least 1 to 2 days.
Recently, a new kit
named the DifferexTM System was newly supplied by Promega Co. (Madison, WI,
USA). The
DifferexTM System uses a combination of phase separation and
differential centrifugation for the separation of sperm and epithelial DNA. By
use of this system the time required for sperm DNA extraction from mixed
stains is greatly shortened (to approximately 2 hours) as compared with another
methods.
In this study,
mixed stains were created on pieces of cotton by mixing female epithelial cells with
sperm of various concentrations; we compared the extraction efficiency of the
DifferexTM System with that of the two-step method. The sperm DNA
extracted from the mixed stains was amplified using the AmpFlSTR
Profiler PCR Amplification Kit (AppliedBiosystems, Foster City, CA, USA). Electrophoresis was performed
using an ABI 310 Genetic Analyzer, and alleles were determined with GenoTyper
3.7 software.
(contact:
tuk-lab@mx1.avis.ne.jp)
P-296
Evaluation of an
Autosomal SNP 12-plex Assay
Vallone PM, Decker AE, Coble
MC, Butler JM
National
Institute of Standards and Technology, Gaithersburg,
Maryland, USA
SNPs have
potential to play a useful role in human identification testing. Small PCR amplicon sizes associated with SNP
typing technologies make SNPs attractive for typing degraded DNA or other low
copy number situations. SNP markers can
be useful in combination with STRs for resolving complex paternity issues (e.g.
incest), identifying victims of mass disasters where insufficient family
references are available and possibly inferring population of origin. Important considerations for SNP markers are
the larger number required to equal the discriminatory power compared to
traditional STRs, their inability to resolve complex mixtures, issues related
to databasing new loci, and the availability of a standard analysis platform. However, in appropriate situations SNPs can
be useful as a supplementary tool complementary to STR markers.
Various SNP
typing platforms exist, but at this time there is not a universally accepted
platform for SNPs and human identity testing.
Currently we are typing SNPs with multiplex allele specific primer
extension (ASPE) reactions. The assay is
comprised of an initial step of PCR followed by primer extension and subsequent
fragment separation and detection by capillary electrophoresis. ASPE multiplex panels can routinely type 6-12
SNPs in a single tube and have reported to go as high as 35 SNP markers. We
have recently developed a 12-plex SNP assay that has been used to type over 600
U.S.
population samples. The 12 markers are a
subset of 70 bi-allelic SNP markers that were previously typed in our
laboratory [1]. The amplicons range
between 62-110 base pairs. The 12-plex
assay has been used to successfully type DNA from shed human hairs. Samples typed by commercial and novel
multiplex STR panels allow for a direct comparison of SNP and STR markers.
Practical
and inherent characteristics of SNP markers will prevent them from replacing
traditional STR typing methods. However,
SNP markers can provide valuable complementary roles in human identity testing. Small autosomal panels of SNPs for typing
challenging DNA samples is an example of where SNPs can benefit the forensic
community. (contact: petev@nist.gov).
[1] Vallone, P.M., Decker, A.E.,
Butler, J.M. (2005) Allele frequencies for 70 autosomal SNP loci with U.S.
Caucasian, African American, and Hispanic Samples., Forensic Sci. Int. 149:
279-286
P-297
Haplotypes analysis of
the PowerPlex® Y System in northeast population from Italy
Turrina S,
Atzei R, De Leo D
Department
of Medicine and Public Health, Institute of Legal Medicine, University of Verona,
Verona, Italy
Y-chromosome
analysis is a useful tool in evolutionary study, paternity testing and personal
identification.
Every year an increased number of Y markers are being
reported in literature, nevertheless to evaluate their efficiency in forensic
science it is necessary to investigate a large number of different populations.
Recently, a new multiplex set of 12 Y-STRs loci
(PowerPelx® Y System, Promega) that includes the 9 Y-chromosome loci of the
European minimal haplotype (DYS19, DYS385 a/b, DYS389I, DYS389II,
DYS390, DYS391, DYS392 and DYS393) plus two
loci recommended by SWGDAM (DYS438
and DYS439) and DYS437 locus, was
commercially released.
In
the present study we evaluated allele frequencies and others statistical
parameters of the PowerPlex® Y System in a
population sample of 155 unrelated autochthonous healthy males from
northeast Italy. In the totally of the observed haplotypes,
there were 143 different haplotypes and among these, 134 were unique, while 9
haplotypes were observed more than one times. The haplotype diversity (HD) of
12 Y-STR multiplex was 0.9987.
Contact: deleodom@tin.it
P-298
Evaluation of an automated system for amylase detection in forensic samples
Da
Vela Ga, Bertino MGa, Ricci Ub
Azienda
Ospedaliera-Universitaria “A.Meyer”, Florence, Italy
a Laboratorio di Analisi Chimico Cliniche e
Microbiologia
b U.O. Genetica Medica
The amylase enzyme is present in human saliva and its
detection in forensic samples is a very important step for the identification
of the origin of a biological sample. The methods used in the forensic context
employ chromatic reactions with visualisation of a colour or with
spectrophotometric detection at an established wavelength. Here, we used an
automated system normally employed in the clinical chemistry laboratory of to
measure the activity of amylase in forensic samples. This method is perfectly
integrated with DNA typing. Samples with a known concentration of amylase were
tested with a manual system (BNP-Amylase test, Sclavo Diagnostics). Visual
detection and spectrophotometric detection at 405 nm were performed, in
comparison with the automated system. A calibration curve for sensitivity study
using a commercial preparation of amylase was also performed to verify the
linearity range. The automated method was employed for various samples
containing human saliva (cigarette butts, chewing gum, stamps, etc). We used
this detection system also on biological samples containing human saliva contaminated
with different materials commonly recovered in forensic casework (ground,
plaster, lipstick, glue).
The sensitivity of the system is superior to the other
systems and offers an objective evaluation of the amylase in forensic samples.
contact: u.ricci@meyer.it
P-299
South
Portugal
population Genetic analysis with 17 loci STRs
C. Vieira - Silva, C. Cruz,
T. Ribeiro, and R. Espinheira
National Institute of Legal Medicine, Forensic
Genetics, Lisbon Portugal
STRs
are the standard genetic markers mainly used in forensic cases. In routine
casework it is important to establish a population genetic database for further
reliable statistical analyses.
AmpF1STRâIdentifilerÔ (Applied
Biosystems) and Geneprint Powerplex 16â (Promega
Corporation, Madison WI, USA) are multiplex kits wich co-amplifie 17 STR - loci
including de segment of X-Y homologus gene Amelogenina routinely used in our
laboratory. 13 core short tandem repeat loci standardized under the combined DNA
Index System (Codis): CSF1PO, D3S1358,
D5S818, D13S317, D16S539, D18S51, D21S11, vWA, FGA, TH01, TPOX, two additional
tetranuceotide loci - D2S1338 and D19S433 – and two additional pentanucleotides
– Penta E and Penta D.
The purpose
of this study is to determine the allele distribution data of the 17 STR loci in 2445 caucasian unrelated
individuals from the south of Portugal, 176 unrelated individuals from Cabo
Verde and 102 unrelated individuals from Angola and compare it with the values
of the all the population resident in
the same area.
Allele
frequencies for each locus, observed heterozigoty, expected heterozygoty, power
of exclusion, power of discrimination and p values of chi square test for
departures from Hardy-Weinberg expectations were calculated.
contact:
genetica@dlinml.mj.pt
P-300
Evaluation of the 4-year test-period of the
Swiss DNA database
Voegeli P, Haas C, Kratzer A,
Bär W
Institute of Legal Medicine,
Forensic Genetics, University of Zurich, Switzerland
The Swiss federal DNA profile
information system (EDNA) is operational since July 2000, using the CODIS
Software provided by the FBI. The database holds DNA-profiles of suspects,
single stains and also mixed stains (presumably consisting of not more than 2
persons). The genetic criteria for entering profiles into the database are the
10 SGMplus loci for suspects, at least 6 SGMplus loci for single stains and at
least 8 loci for mixtures. At the end of the 4-year test-period (31 december
2004) the database contained 61'954 DNA profiles, 53'400 profiles from suspects
(89% male, 11% female) and 8'554 profiles from stains (90.7% single profiles,
9.3% mixtures). Thereof 548 profiles are from foreign countries. Stains which
are assigned to a suspect are removed from the database day-to-day.
During the test-period the
database provided excellent results. 6'825 stains could be assigned to a
suspect (offender hits), about 2'000 crime-sites could be connected (forensic
hits) and 35 criminal monocygotic twin-pairs were identified (offender
duplicates). 44% of the single stains that were entered into the database
resulted in an offender hit, allowing the identification of the unknown
perpetrator. About 50% of the mixtures revealed one offender hit and another
15% were solved with two persons.
The 6'825 offender hits can be
subdivided into the following crime categories: the major group with 85.2% was
burglary/theft/wilful destruction, followed by homicide/bodily harm with 4.2%,
robbery with 4%, sexual offenses with 2.4% and 4.2% other delicts.
Voluntarily the DNA profiles
of laboratory staff and involved police members could be entered into a
separate index, in order to detect contaminations. Thus, 36 stains could be
identified as contaminations. A special search mode (allowing 2 errors) helps
finding incorrect profiles. 33 additional hits were discovered using this
search mode.
P-301
The extent of substructure in
the indigenous Australian population and its impact on DNA evidence
interpretation.
Walsh SJ1, Mitchell
RJ2, Curran JM3, Buckleton JS4
1 Centre for Forensic Science, University of Technology, Sydney, Australia
2 LaTrobe University, Melbourne, Australia
3 University of Waikato, Hamilton,
New Zealand
4ESR, Auckland, New Zealand.
Indigenous Australians have a
unique evolutionary history resulting in a complex system of inter- and
intra-tribal relationships. Although
European colonisation has disrupted to a varying extent these and other
features of Aboriginal life, forensic DNA evidence has recently been called
into question with respect to the impact of this evolutionary past on issues
associated with population genetics and the estimation of DNA match
statistics. The extent of substructure
within the indigenous Australian sub-population raises two main questions
relevant the interpretation process; 1) what is the appropriate value of the
co-ancestry coefficient, theta or Fst?, and 2) what is the effect of
sub-population substructure on the performance of the sub-population
model? This paper describes research
that focuses on these issues. Research examining classical markers as well as
DNA SNPs has shown evidence of considerable heterogeneity within the indigenous
Australian population. The question is, to what extent do autosomal
microsatellites used in contemporary forensic testing show such structuring
effects? Autosomal microsatellite diversity within the indigenous Australian
population has been examined through the analysis of genotype data from a large
number of geographically distinct tribal groups and urban centres. Genotypes included highly polymorphic loci
from the Profiler Plus™ and Identifiler™ PCR systems. Autosomal STR Fst values have been estimated
from these data, and were found to be considerably lower than some values from
previous research that has focussed more often on SNPs or blood group and protein
loci. The performance of the sub-population model was
also investigated by simulation under circumstances where the assumption of
equilibrium in the sub-population is violated.
The results imply that departures from equilibria at the sub-population
level do very little to alter the inherent conservativeness of the model.
P-302
Analysis of single nucleotide polymorphisms and its application to a disputed paternity case
Wang X, Ito S, Sawaguchi A, Sawaguchi T
Department of
Legal Medicine, Tokyo Women’s Medical University, School of Medicine, Japan
There has
been recent progress in the areas of research and applied development in the
genetic analysis of the single nucleotide polymorphisms (SNPs) employing fluorescent
dye labeling technology. SNPs are places along the chromosomes where the
genetic code tends to vary from one person to another by just a single base.
They are estimated to occur about once every 1000 bases along the 3-billion-base
human genome. SNPs are an increasingly important tool for genetic and
biological research. SNPs analysis is becoming increasingly important studies
of drug resistance, evolution, and molecular epidemiology in mycobacterium
tuberculosis, human immunodeficiency virus, and other organisms. Although
current genomic databases contain information on several million SNPs and are
growing at a very fast rate, the true value of a SNP in this context is a
function of the quality of the annotations that characterize it. The most
common application of SNPs is in association studies that look for a
statistically significant association between SNP alleles and phenotypes, in
order to find pinpoint candidate causative genes. For this reason, large
databases of well-annotated SNPs have been developed, and are growing at an
ever-increasing rate. Data derived from analysis of SNPs are being applied in
many diverse fields, from medical studies of disease mechanisms and individual
drug response, to population genetics for tracking migration and mixing of
ancestral groups and also in forensic science for the identification of human
remains and identification of individuals from bodily samples.
In this study, we investigated
distribution of allele frequencies for 16 SNPs loci (G63767, G63754, G65359,
G63748, G65275, G65270, G65266, ss4019224, ss4947490, ss4974676, ss4974689,
ss4974729, ss4974915, ss5013903, ss6658727) in 120 unrelated healthy Japanese
individuals using multiplexed single nucleotide primer extension by ABI PRISM
SnaPshot Multiplex Kit and ABI PRISM 310 Genetic Analyzer (Applied Biosystems).
A total of 32 alleles and 48 genotypes were observed in these samples for 16
SNPs loci. The combined power of discrimination and the combined power of
exclusion for SNPs in 16 loci were 0.9954 and 0.92653, respectively.
We applied these databases to analyze a
case of special paternity testing. In this case, the putative father and the
child’s mother were decease. The DNA of the deceased putative father was only
extracted from a formalin-fixed liver tissue. In this inspection result of 16
SNPs, the paternal rights affirmative probability to the deceased putative
father’s child was 0.9912, and the possibility of existing related to the
deceased putative father and child was not denied in genetics. The result
demonstrated that analysis of 16 SNPs are an extremely effective method for the
diagnosis of paternity with formalin-fixed liver tissue in paternity testing of
a deceased parent. This study also indicated that if DNA fragment length is longer
than 100bp, that could be enough to using for analysis of SNPs from the liver tissue, even if the liver
had been fixed for a long time with formalin solution.
P-303
Analysis of Mitochondrial DNA Polymorphisms
based on Denaturing High-Performance Liquid Chromatography
Wang XD, Liao LC, Li YB, Wu J,
Hou YP
Department of Forensic
Genetics, Sichuan
University (West China
University of Medical
Sciences), Chengdu,
P.R.China
The purpose
of this study is to establish a novel method for the detection of polymorphism
of mitochondrial DNA (mtDNA) based on denaturing high-performance liquid
chromatography (DHPLC) and to explore the new mitochondrial DNA polymorphism in
coding region in order to improve the discrimination power of mtDNA in forensic
DNA typing. We explored the polymorphism of the sequence in the coding region,
which covered 1435bp. A total of seven pairs of primers for PCR were designed
to analyze the region of mtDNA, so that it was nominated as seven loci. To
explore the polymorphism of the region of mtDNA, a technique of sample pool was
employed for the analysis of DHPLC. All of seven loci were analyzed by DHPLC in
a Chinese population sample. Our study revealed that there were 53 haplotypes
at seven loci in the coding region with covering 1435bp and the haplotype
diversity was 0.8775 in our Chinese population sample. Among these, four loci
with higher diversity were proved to be suitable for forensic application and
provided new genetic markers for the forensic mtDNA typing
P-304
Linkage disequilibria between
6 STR loci situated in the HLA region on Chromosome 6
Wenda S, Dauber EM, Dorner G,
Reisacher RBK, Glock B, Mayr WR
Division of Blood Group
Serology, Medical
University Vienna, Austria
Six STR polymorphisms coded for by the HLA region of
chromosome 6p21.3 have been investigated: three tetranucleotide repeat loci
D6S389, D6S1051 (GenBank G08553) and D6S2822 (M2_4_25) situated in the HLA
Class II region (D6S2822 located between HLA-DQ and DP; D6S1051 4 cM
centromeric of HLA-DP and D6S389 1.3 cM centromeric of D6S1051), as well as two
tetranucleotide repeat loci, C1_4_4 (D6S2931) and C2_4_4 (D6S2939), and one
trinucleotide repeat locus C3_3_6 (D6S2906) located in the HLA class I region
(C3_3_6 0.4 cM and C2_4_4 1 cM centromeric of HLA-A, respectively; C1_4_4 0.2
cM centromeric of HLA-B). 284 haploytpes from 71 Austrian Caucasoid families
could be defined. The analysis of the linkage disequilibrum between alleles of
the 3 STR loci located in the HLA class I region (C1_4_4, C2_4_4, C3_3_6)
showed several significant values (the p values have been corrected by
multiplying them with the number of comparisons made). No linkage disequilibria
could be found between alleles of the 3 STR loci next to the HLA class II
region (D6S389, D6S1051, D6S2822) and between alleles of these 3 loci and of
the loci situated in the HLA class I region.
|
C1_4_4
|
C2_4_4
|
C3_3_6
|
χ2
|
Significance
Corrected p-value
|
|
|
*20
|
*17
|
34.88
|
p<0.001
|
|
|
*9
|
*12
|
27.36
|
p<0.001
|
|
*10
|
*9
|
|
153.13
|
p<0.001
|
|
*12
|
*11
|
|
83.29
|
p<0.001
|
|
*16
|
*16
|
|
25.74
|
p<0.001
|
|
*7
|
*16
|
|
24.86
|
p<0.001
|
|
*8
|
*10
|
|
23.24
|
p<0.001
|
|
*19
|
*10
|
|
20.85
|
p<0.001
|
|
*10
|
*10
|
|
19.27
|
p<0.01
|
|
*18
|
*17
|
|
18.69
|
p<0.01
|
|
*7
|
*17
|
|
14.29
|
p<0.05
|
|
*10
|
|
*12
|
31.34
|
p<0.001
|
|
*19
|
|
*9
|
15.05
|
p<0.05
|
Within the HLA class I STRs, only one haplotype with a
significant three-locus-disequilibrium could be observed: C1_4_4*10, C2_4_4*9,
C3_3_6*12. These 3 alleles are situated on the common Caucasoid superhaplotype
HLA-A1,B8,Cw7,DR3. The absence of a high degree of linkage disequilibrium
between the alleles of the HLA STRs is probably due to the fact that the STR
loci, in contrast to the phenotypically expressed HLA alleles, are not
subjected to selective forces. The lack of significant disequilibria between
the HLA class II STRs is also caused by the higher physical distance between
the loci.
References:- Y. Matsuzaka, et al., New
polymorphic microsatellite markers in the human MHC class II region, Tissue
Antigens 56 (2000) 492-500.- A. Foissac, et al., Microsatellites in the HLA region:
1999 update, Tissue Antigens 55 (2000) 477-509. Contact : sabine.wenda@meduniwien.ac.at
P-305
Validation and Evaluation of the
ABI 3100 Genetic Analyser for Use With STR Analysis of CJ Buccal Swabs - Systematic Differences
Between the AB13100 and ABI377
Warren T, Johnston I, Johns LM, Bardill SC
LGC, The Heath, Runcorn, WA7 4QX,
UK
For many
years ABI377 DNA sequencers have been used in the production of short tandem
repeat (STR) profile data for forensic human identification (HID) applications.
These instruments provided a 36-96 lane, slab gel, based electrophoresis
system. However accurate, using this system resulted in a relatively high level
of re-work due to the inconsistent nature of the polyacrylamide gels used. The
operation of the ABI377 was also labour intensive and hazardous due to the
requirements for gel production. The development of capillary electrophoresis
genetic analysers, e.g. ABI3100, suggested potential improvements in
sensitivity, reliability, and flexibility. In this study a comparison has been
made between the ABI 377 DNA sequencer and the ABI 3100 genetic analyser when
performing STR profiling of forensic Buccal swabs. The criteria used to
evaluate/validate the ABI 3100 are outlined and the assessments made are
described.
P-306
Variability of mitochondrial DNA mutagenesis in human
blood
von Wurmb-Schwark N1, Jelkmann I1,
Bruhn HD2, Oehmichen M1
1Institute of Legal
Medicine, University
of Kiel, Kiel, Germany
2Clinic for Internal
Medicine, University
of Kiel, Kiel, Germany
The 4977 bp deletion of
mitochondrial DNA (mtDNA) is known to accumulate with age in post mitotic
tissues. Meanwhile, this mutation can also be detected in tissues with a fast
turnover like blood or skin. From a forensic point of view, it is interesting
to elucidate the possible correlation between age and the amount of mutated
mtDNA (dmtDNA) in blood to estimate the age of an individual.
We
investigated mtDNA mutagenesis in blood from 10 persons (22-60 years) over a
time period of 6 month. During that time, we monitored exogenous factors that
might influence the integrity of mtDNA, like smoking habits, alcohol
consumption, and medicine intake. A blood sample was drawn from each proband
every other week, and the following criteria were investigated: 1. The amount
of total mtDNA/cell was measured using a real time PCR; 2. the occurrence and
relative quantification of deleted mtDNA was carried out in a Duplex-PCR with
subsequent fragment detection in an ABIPrism310; 3. a blood cell count was
done.
Real time PCR results showed
values between 1003 and 3275 mtDNA copies/cell (average 2127) with a very
strong variation within one individual from time point to time point. The
occurrence of the dmtDNA also varied considerably, showing ratios from 0 –
0,6621 (dmtDNA/undeleted mtDNA) in the same individual on different days.
Consequently, the quantity of
dmtDNA in blood is not a suitable measure to determine the age of an individual
for forensic purposes, since mitochondrial mutagenesis seems to be influenced
by too many exogenous factors.
P-307
Fast and simple DNA extraction from saliva or sperm
cells obtained from the skin or isolated from swabs
von Wurmb-Schwark N, Mályusz V, Fremdt H, Koch C,
Schwark T, Oehmichen M, Simeoni E
1Institute of Legal
Medicine, University
of Kiel, Kiel, Germany
The
forensic scientist often has to cope with problematic samples from the crime
scene due to their size and thus the amount of extractable DNA. The retrieval
of DNA from swabs taken from the surface of the skin, for example in cases of
strangulation, can be especially difficult.
We
systematically investigated swabs taken from the skin (to obtain a genetic
profile from the victim and also from a possible offender) and from sperm
containing swabs using two extraction kits: the Invisorb Forensic and the Spin
Swab kit (both Invitek, Germany).
DNA quality and quantity was tested on ethidium bromide containing agarose gels
and a highly sensitive Duplex-PCR which amplifies fragments of mitochondrial
and nuclear DNA. Absolute quantification was done using real time PCR. Samples
which were positive in the Duplex-PCR were also employed to genetic fingerprinting
using the Powerplex ES and the AmpFlSTRIdentifilerTM
kit. Additionally a 433 bp fragment of the mitochondrial HVI region was
generated and sequenced.
Our
study shows that the easy-to-use Spin Swab kit is very suitable for DNA
isolation from swabs taken from the skin and surprisingly also from sperm
cells. We will present modifications which greatly improve the isolation of DNA
and additionally simplify the whole process of trace handling and DNA
extraction.
P-308
Newly
designed multiplex amplification and genotyping system at four pentanucleotide
repeat STR loci useful for degraded mixed DNA specimens
Yamamoto T, Uchihi R, Ando Y,
Suzuki M, Yoshimoto T, Katsumata Y
Department of Legal Medicine
and Bioethics, Graduate
School of Medicine, Nagoya University,
Nagoya, Japan
Short tandem repeat (STR)
markers with tetranucleotide repeat are globally utilized for personal
identification and kinship analysis in forensic field, and give us very useful
information in almost practical cases. In case of mixed samples, however, the
‘stutter peaks’ sometimes make it difficult to interpret whether they are exact
‘stutter peaks’ or minor peaks originated from the another individual, because
of more than about 10 % of stutter percentage for tetranucleotide STRs which is
the ratio of the stutter peak height to a main peak height. In general, it is
said that the stutter percentages become lower with the more numbers of repeat
units. In the present study, we selected four pentanucleotide STR loci (Penta
E, Penta D, Penta B and D10S2325) to construct a multiplex PCR and genotyping
system with multicoloured fluorescently labelled primer sets newly designed for
degraded DNA samples, of which the amplicon sizes are smaller than about 200
bp. Sequence analysis was performed for all alleles at these all STR loci
observed in a Japanese population, and also the allelic ladder markers with
these alleles inserted into the plasmids in a commercially available kit were
constructed for semi-automated genotyping by making a template of Genotyper 2.5
software. Using the present multiplex system, 300 unrelated Japanese (Nagoya city) were
genotyped with written informed consent, and calculated the allele frequencies
at each locus. Three tests for Hardy-Weiberg equilibrium (HWE) were performed,
and the allele distributions at those four loci did not deviated from HWE. The
heterozygosities and the power of discriminations (PD) at all the four loci
were more than 0.80 and 0.93, respectively. The combined PD and MEC (mean
exclusion chance) were 0.9999962 and 0.9900966, respectively. The stutter percentages at
all alleles for these four loci, where stutter peak heights were more than 50
RFU, were calculated, and it was found that the stutter percentages were almost
directly proportional to the numbers of repeats at each locus, and that almost
all the stutter percentages were distributed within the regression lines from ±
3 times values of SD (standard deviation) of at each allele for each locus.
Accordingly, this could be a statistical standard to decide whether a
one-repeat small peak from a main peak is the stutter peak or the peak originated
from mixed individual sample. It was suggested that this system is one of the
useful multiplex typing system, especially for mixed DNA specimens.
yamachan@med.nagoya-u.ac.jp
P-309
STR
loci analysis of buccal cavity cells captured by laser microdissection
Yamamoto Y1, 2, Hara M2, Kido A3, Takada A2,
Saito K2
1Criminal Investigation Laboratory,
Saitama Prefectual Police Headquarters, Saitama,
Japan
2Department of Forensic Medicine, Saitama Medical
School, Saitama, Japan
3Department of Legal Medicine, Faculty of
Medicine, University
of Yamanashi, Yamanashi, Japan
In the present study, we have attempted to analyze
short tandem repeat (STR) loci for buccal cavity cells isolated from saliva.
The buccal cavity cells were captured from smear of saliva stained by Harris
Hematoxylin and Eosin by a laser microdissection method using PALM Microlaser
Systems (P.A.L.M). DNA was extracted from the buccal cavity cells using DNA
Extraction FM Kit (Wako). After the whole genome amplification of DNA extracted
from the buccal cavity cells carried out by the improved PEP PCR method, the 15
STR loci, D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539,
D2S1339, D19S433, vWA, TPOX, D18S51, D5S818 and FGA, as well as amelogenin
locus were amplified using AmpFLSTR Identifiler Kit (Applied Biosystems).
Amplified products were separated by denaturing capillary electrophoresis in
the ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The results were
analyzed using GeneScan Analysis v3.7 software (Applied Biosystems) and
Genotyper v3.7 software (Applied Biosystems). The 15 STR loci and the
amelogenin locus were determined from 20 buccal cavity cells, the 13 STR loci
and the amelogenin locus from 10 buccal cavity cells, the nine STR loci and the
amelogenin locus from five buccal cavity cells, and the five STR loci and the
amelogenin locus from two buccal cavity cells. This method is feasible for the
STR loci and the amelogenin locus analysis of a few buccal cavity cells. In further
studies, we should investigate the STR loci analysis of single cell from mixed
seminal/vaginal secretion stains and tissue slice specimens by technical
improvement.
Contact: CW4Y-YMMT@j.asahi-net.or.jp
P-310
ANALYSIS OF
SIX TETRANUCLEOTIDE POLYMORPHISMS OF THE
X-CHROMOSOME IN DIFFERENT SPANISH
REGIONS
Zarrabeitia MT1, Alonso A,1, Martín J2 , Gonzalez-Gay MA 3 , Martin-Escudero JC4 , Martinez de
Pancorbo M5, Martín P 6, Ruiz-Cabello F 7 and Riancho JA8
1 Unidad de Medicina Legal. Universidad de Cantabria.
Santander. 2 Instituto "Lopez Neyra", CSIC. Granada.
3 Complejo Hospitalario Xeral-Calde. Lugo. 4 Hospital
del Río Ortega. Universidad de Valladolid.
5 Facultad de Farmacia. Universidad del Pais Vasco.
Vitoria. 6 Instituto Nacional de Toxicología. Sevilla.
7 Hospital Virgen
de las Nieves. Universidad de Granada. 8
Departamento de Medicina. Universidad de Cantabria.
X-linked markers are
particularly useful in paternity deficiency cases, as all daughters of a father
inherit the same X chromosome. We studied 6 X-linked microsatellites in a large
group of Spanish individuals (n=614) from five different regions located in
northern, central and southern Spain.
All the markers had tetranucleotide repetition units (DXS9895, DXS9898,
DXS7130, DXS7131, GATA172D05, and DXS6789). They were amplified in two triplex
PCR. There were no significant sex- or region-related differences in allelic
frequencies, suggesting that general national databases can be adequate as a
reference in X-linked markers. Fst coefficients varied between 0.002 (DXS6789,
GATA172D05) and 0.066 (DXS7130).
The forensic efficiency
parameters are shown in the table.
|
|
DXS7130
|
DXS7132
|
DXS6789
|
DXS9895
|
GATA172D05
|
DXS9898
|
|
PIC
|
0.716
|
0.739
|
0.816
|
0.727
|
0.790
|
0.753
|
|
PD female
|
0.894
|
0.903
|
0.904
|
0.894
|
0.934
|
0.911
|
|
PD male
|
0.733
|
0.758
|
0.752
|
0.748
|
0.805
|
0.772
|
|
PE trio
|
0.699
|
0.719
|
0.718
|
0.705
|
0.777
|
0.735
|
PIC: polymorphism information content
PD: power of discrimination
PE: power of exclusion
The 6-locus combined analysis
in 316 males revealed 300 different haplotypes, 283 of which were found only
once. There was no evidence for statistically significant linkage
disequilibrium among the loci studied.
Therefore these markers are
quite polymorphic and useful for forensic purposes
contact: zarrabet@unican.es
Supported by a grant from
Fundacion Marques de Valdecilla-IFIMAV.
P-311
Estimating the postmortem interval by determining the age of fly
pupae:
Are there any molecular tools?
Zehner R, Mösch S, Amendt J
Institute of Legal Medicine,
J W Goethe-University Frankfurt, Germany
Forensic entomology, the use of insects in medicolegal
investigations mainly focus on the estimation of the postmortem interval (PMI)
by calculating the age of necrophagous specimens.
Usually the age of an insect developing
on a corpse is determined e.g. by the measurement of the length of the larvae
and using species-specific growth rates in consideration of the temperature
conditions at the scene of crime. The estimated age represents the minimal PMI
because the necrophagous insects do not oviposit before death. While staging
the age of the larvae is possible at a quite detailed scale, the age of the
pupae is not to specify without rearing up to the adult stage, which is
time-consuming or might be difficult.
However, the pupal stage represents
about 50% of the immature development time and the pupal age may serve as an
important tool in entomological PMI estimation if no larvae are present..
Our approach was to study gene expression patterns of
transcripts, which are differently expressed during pupal development and
estimate its usefulness in estimating the pupal age compared to morphological
characters of the pupae after removal of the puparium (outer shell).
Total RNA was extracted from single pupae of different
ages, reversely transcribed and subjected to differential display PCR. PCR
products were separated by PAGE and stained with silver. The banding profiles
from pupae of different ages were compared and evaluated with regard to their
use as age marker.
Using ddRT-PCR as a single tool, estimating the pupal
age is not more accurate than using morphological characters alone. The
significance of molecular techniques for entomological PMI estimation is discussed.
P-312
STR typing with High Performance Liquid
Chromatography
Zhu QF, Li YB, Liao LC, Wu J,
Hou YP
Department of Forensic
Genetics, Sichuan
University (West China
University of Medical
Sciences), Chengdu,
P.R.China
The purpose
of our work was to study the STR typing method with high performance liquid
chromatography and to comprehend the rules of STR typing with HPLC. Firstly,
all alleles of the STR marker at D10S2325 locus were sequenced and mixed to
construct an allele ladder. Secondly, the HPLC conditions to separate each
allele at D10S2325 locus were optimized. Thirdly, STR typing of D10S2325 was
carried out by comparing the retention time of the allele ladder on HPLC with
that of a sample. Our method was validated by typing same samples with the
polyacrylamide gel electrophoresis. Lastly, the sensitivity of this method and
the ability to analyze mixed samples were tested. The results showed that the
method of STR typing with HPLC established by us was successful. The results of
our study implied that it was important to consider both the conditions of
chromatography and the variation of retention time caused by the sample
concentration when STR typing was carried out with high performance liquid
chromatography
.
P-313
Haplotype distribution
of the mitochondrial control region in the native Canary
Islands population.
Zurita A1,
Hernandez A1, Sanchez J2, Cuellas JA1
1 National Institute of Toxicology and Forensic
Sciences, Canary Islands, Spain
2 Department of Forensic Genetics, University of Copenhagen, Denmark
Canary Islands constitute a crossroads
territory, with a considerable flow and settlement of different human groups,
mainly from Europe, North
Africa and South America. This
introduces a complex forensic scenario that makes often difficult the
interpretation of population genetic data and statistical models applied in
forensic genetics casework. To understand the genetic composition and origen of
the present-day Canary Islands population, we
studied the mitochondrial control region of a total of 210 voluntary native
islanders, i.e. individuals who were born in the archipelgo with at least two
known Canarian ancestor generations, in order to lessen the contribution of the
last fifty years immigration.
Both
HV1 and HV2 regions were sequenced by standard procedures and haplotype
frequency distribution was determined. From the data obtained some useful
forensic statistical parameters were inferred, like nucleotide diversity, gene
diversity or mean number of pairwise differences. Furthermore, the sequences
were compared with other population groups, like Europeans, North and
Sub-Saharians, and aborigens (from ancient samples), and phylogenetic
relatioships were established. Data were compared with those obtained with
chromosome Y SNPs.
Contact: ahernandez@canariastelecom.com
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