P-136
Are tetranucleotide microsatellites
implicated in neuropsychiatric diseases?
Jacewicz R1, Szram
S1, Gałecki P2, Pokora K1, Florkowski A2
and Pepiński W3
1Department of Forensic Medicine, Medical University of Lodz
2 Department of Psychiatry and
Neurosis Disorders with Crisis Intervention Ward, Medical University
of Lodz
3Department of Forensic Medicine,
Medical University of Białystok
Expecting
the significant breakthrough in the diagnosis of complex disorders of
neuropsychiatry background, intensive efforts are undertaken to establish
genetic markers associated with these disorders. It is known that neurological
diseases are correlated with disturbances of the catecholaminergic pathway. The
studies within genes involved in the synthesis, neurotransmission and
metabolism of dopamine, adrenaline and noradrenaline have not given
satisfactory results. Nowadays, great diagnostic expectations are related with
sequences of STR type, which are widespread throughout the genome. These
microsatellite repetitive sequences do not code proteins, but are supposed to
function as regulatory elements in processes of gene transcription and
expression. Association of di-, tri- or tetra nucleotide repeats with
neurological disorders has been reported earlier in different populations. We
have examined association between maniac-depression diseases such as
schizophrenia, bipolar and unipolar affective diseases and polymorphism of
several tetranucleotide genetic markers from different chromosome positions,
including those being candidate in main psychiatric diseases. Results of
statistical comparative analysis between neuropsychiatric patients from Poland and
their regionally matched healthy subjects are presented
Address for
correspondence:
Renata
Jacewicz, PhD
Head of Genetic Laboratory
Department of Forensic Medicine
Medical University of Lodz
91-304 Lodz, Sedziowska 18a
Poland
tel. + 48 42 654 45 36
fax + 48 42 654 42 93
e-mail: r.jacewicz@post.pl
Head of Genetic Laboratory
Department of Forensic Medicine
Medical University of Lodz
91-304 Lodz, Sedziowska 18a
Poland
tel. + 48 42 654 45 36
fax + 48 42 654 42 93
e-mail: r.jacewicz@post.pl
P-137
The association of polymorphic TH01 marker with schizophrenia in Poland.
Jacewicz R1, Szram S1,
Gałecki P2, Pokora K1, Berent.J1, Florkowski A2
and Pepiński W3
1Department of Forensic
Medicine, Medical
University of Lodz
2 Department of Psychiatry and
Neurosis Disorders with Crisis Intervention Word, Medical University
of Lodz
3Department of Forensic
Medicine, Medical
University of Białystok
TH01 locus, used for personal identification, is a
polymorphic microsatellite region located in the first intron of the tyrosine
hydroxylase gene (TH). This gene codes the enzyme limiting synthesis of brain catecholamines.
Disturbances in the synthesis and neurotransmission of dopamine and
noradrenaline are involved in the pathophysiology of psychiatric diseases such as
schizophrenia and affective disorders. The polimorphism in TH01 tetranucleotide sequence correlates
with quantitative and qualitative changes in binding by specific protein ZNF191
and may be involved in regulation of TH gene expression. The association
between these illnesses and polymorphism of TH01 marker has been reported in a
group of neuropsychiatric patients from France, Tunisia, Sweden and the UK (England).
Because of scarcity of the investigated samples former reports do not determine
unambiguously the case in question. We attempted our own population study to
compare distribution of allele frequencies in TH01 locus in a group of neuropsychiatric
patients from Poland and
their regionally matched healthy subjects. This report presents results of this
association study
Address
for correspondence:
Renata
Jacewicz, PhD
Head of Genetic Laboratory
Department of Forensic Medicine
Medical University of Lodz
91-304 Lodz, Sedziowska 18a
Poland
tel. + 48 42 654 45 36
fax + 48 42 654 42 93
e-mail: r.jacewicz@post.pl
Head of Genetic Laboratory
Department of Forensic Medicine
Medical University of Lodz
91-304 Lodz, Sedziowska 18a
Poland
tel. + 48 42 654 45 36
fax + 48 42 654 42 93
e-mail: r.jacewicz@post.pl
P-138
Evaluation
of the genetic affinity between populations based on the comparison of allele
distributions in two highly variable DNA regions
Jacewicz R1, Miścicka- Śliwka D2
1Department of Forensic
Medicine, Medical
University of Lodz
2Laboratory for Molecular and
Forensic Genetics, Medical
University in Bydgoszcz
Minisatellite DNA consists of
tandem repetitive 9 – 100 base pairs motifs of the length from few hundred to
over 20 000 base pairs. These non-coding sequences are the fastest evolving in
the genome due to comparatively high frequencies of mutation processes. The
investigation of diversity in these hyper variable loci proves to be a valuable
source of information ready to be used to characterize different human race and
populations, as well as to define their genetic affinity. The aim of this work
is to compare the distribution of alleles in the two highly polymorphic
mini-satellite DNA regions D7S21 & D12S11obtained from the Polish and other
world populations. To achieve this, we used the graphic analysis based on the
allele frequencies in the intervals of 100 base pair as well as the statistical
analysis. The analysis proved that the distribution of alleles in both the
Polish and other Caucasian populations of Europe
is similar. Moreover, it revealed significant differences in the structure of
distributions when we compared the investigated Polish population, representing
Caucasians, with Asian population and Afro-Caribbean population in particular.
Address for
correspondence:
Renata Jacewicz, PhD
Head of Genetic Laboratory
Department of Forensic Medicine
Medical University of Lodz
91-304 Lodz, Sedziowska 18a
Poland
tel. + 48 42 654 45 36
fax + 48 42 654 42 93
e-mail: r.jacewicz@post.pl
Head of Genetic Laboratory
Department of Forensic Medicine
Medical University of Lodz
91-304 Lodz, Sedziowska 18a
Poland
tel. + 48 42 654 45 36
fax + 48 42 654 42 93
e-mail: r.jacewicz@post.pl
P-139
Population
genetic study of the three minisatellites loci:
D7S21, D12S11 and D5S110 in Poland.
Jacewicz R1, Miścicka- Śliwka D2
1Department of Forensic
Medicine, Medical
University of Lodz
2Laboratory
for Molecular and Forensic Genetics, Medical
University in Bydgoszcz
The VNTR loci: D7S21, D12S11 and D5S110 are the highly polymorphic
markers of the human genome. Though they are used in the most difficult cases
of kinship analysis, a comprehensive database of these regions has not been set
up for the Polish population: such a database is essential for carrying out
analysis and performing calculations. The distribution of allele frequency as
well as evaluation of the Hardy and Weinberg equilibrium are the subject of
this report. The efficiency of forensic evaluation for investigated loci
in the population of Poland was
compared with similar data for other world populations. The combined values of
PD and PE for the three-locus profile in the investigated population were
calculated to be at 99.99997% and 99.996% respectively. Our practise indicates
that investigated loci are an invaluable help in resolving most difficult
forensic cases in kinship analysis, especially
when the alleged father or mother are not available, or when there is a
risk that the child’s father is the defendant’s close relative, or when we
analyse the relationship between any given people.
Address for
correspondence:
Renata Jacewicz, PhD
Head of Genetic Laboratory
Department of Forensic Medicine
Medical University of Lodz
91-304 Lodz, Sedziowska 18a
Poland
tel. + 48 42 654 45 36
fax + 48 42 654 42 93
e-mail: r.jacewicz@post.pl
Head of Genetic Laboratory
Department of Forensic Medicine
Medical University of Lodz
91-304 Lodz, Sedziowska 18a
Poland
tel. + 48 42 654 45 36
fax + 48 42 654 42 93
e-mail: r.jacewicz@post.pl
P-140
Study to compare three commercial
Y-STR testing kits
Johns LM, Burton
RE, Thomson JA
LGC, Queens Road, Teddington,
TW11 0LY
An evaluation study was carried out to test the
performance of three commercially available Y-STR DNA profiling kits for their
suitability to forensic case work. The three kits assessed were Reliagene’s
Y-PlexÔ 12 kit, Promega’s PowerPlexâ Y system and Applied
Biosystems’ AmpFℓSTR® Yfiler™ kit. Four experiments were devised to assess the
performance of the three kits. Allelic peak height data was used to measure the
reproducibility, sensitivity, male specificity and ability to discriminate male
mixtures of the three kits. Samples were processed following the manufacturers
recommended protocols. PCR products were run on 3100 electrophoresis platforms
and the resultant DNA profiles analysed using GeneScan and Genotyper analysis
software packages.
All three kits gave reproducible results with
concordant genotypes between replicates and kits. Average peak height data
showed the AmpFℓSTR® Yfiler™ kit to be the most reproducible kit during the
evaluation study. PowerPlexâ Y system was shown to be the
most sensitive kit during the evaluation study. All three kits gave full male
profiles for all samples processed in the specificity experiment. There was no
evidence of female artefacts in the PowerPlexâ Y and AmpFℓSTR® Yfiler™
samples however there was evidence of additional female artefacts in all Y-PlexÔ 12 samples. The AmpFℓSTR®
Yfiler™ kit showed the least degree of
variation in peak area ratio’s for the expected male mixture ratio’s and
therefore showed that it was able to discriminate male mixtures better than the
PowerPlexâ Y and Y-PlexÔ 12 kits during this
evaluation study.
P-141
Validation of QuantifilerÔ Human Quantification Kit for Forensic Casework
Johns LM, Thakor A, Ioannou P, Kerai J, Thomson
JA
LGC, Queens Road,
Teddington, Middlesex, TW11 0LY, UK
A study was carried out to test the suitability of
Applied Biosystems Quantifiler™ Human Quantification Kit and validate it for
forensic casework. The QuantifilerÔ assay was performed using an
Applied Biosystems 7900HT Real-time PCR system. The validation exercise
comprised five parts. (1) Reproducibility (2) Sensitivity (3) Effect of
bacterial DNA (4) Effect of reducing reaction volume (5) Back to back
comparison with PicogreenÒ quantification assay. DNA extracts
generated using a variety of extraction methods from different forensic sample
types were used for the validation exercise. After quantification the DNA
extracts were analysed using SGMplus amplification kits. The PCR products were
run on 3100 electrophoresis platforms and the resultant DNA profiles analysed
using GeneMapperID analysis software.
QuantifilerÔ gave reproducible results for
samples in the DNA concentration range of 0.1ng/mL - 5 ng/mL. The sensitivity of the
assay was demonstrated with DNA concentrations of down to 0.03ng/mL being detected. The presence
of increasing ratios of bacterial DNA had no effect on the specificity of the
assay. There was no significant difference in calculated DNA concentrations
when QuantifilerÔ was run at half the recommended reaction
volume. The back to back study demonstrated that QuantifilerÔ generated SGMplus profiles
which were on average of better quality than PicogreenÒ generated profiles. All
extracts for which no SGMplus profile could be obtained had QuantifilerÔ DNA concentrations of zero. The number of
times a samples requiring a second amplification before an acceptable profile
was obtained was three times lower for QuantifilerÔ samples compared to PicogreenÒ samples. The validation
exercise demonstrated the suitability of the QuantifilerÔ assay for forensic casework.
P-142
Application of
less primer method to multiplex PCR
Kane
M1,2, Masui S1,3, Nishi K2
1Forensic Science
Laboratory, Shiga Police Headquarters, Japan
2Legal Medicine, Shiga University
of Medical Science, Japan
3Legal Medicine, Osaka University, Japan
Multiplex
short tandem repeat (STR) analysis have been indispensable for the forensic
genotyping because it can use minute amounts of DNA and has a high degree of
discrimination. In the case of an imbalance from locus to locus, the
manufacturer recommends that reducing the number of PCR cycles and amplification
using less templates can improve the balance among loci.
In
order to obtain even PCR products as well accurate genotype analysis, reaction
conditions including concentrations of primer, amplification cycle number and
annealing and extension time were examined. The primer concentration (3 % of
commercially available kit, AmFLSTAR Profiler kit, Applied Biosystems) was set
at minimum required to the plateau below 8000 relative fluorescent units (RFU)
without pull-up phenomenon. Contrast to the conventional PCR product that
depends on amount of the template, less primer method has the upper limit. The
locus of higher efficient amplification is reached to the plateau during early
PCR cycles, the remaining PCR cycles employ to the production of lower efficient
locus. Therefore, PCR product in this method is almost constant in every
reaction and maintains the reproducibility and good balance among loci. Even if
it can not converge on the optimal amounts of PCR products, the sensitivity of
this method at 40 PCR cycles has increased more than one of protocol at 28 PCR
cycles. When a minute template that has not reached to the plateau is treated,
5% primer is more sensitive than 3% primer. The ordinary primer concentration
at 40 cycles results in non#8211; specific PCR because free primer lead to the
disordered reaction according to the increase in cycle number. Thus, the cycle
number in various kits is limited about 30 cycles. Less primer with higher
number of PCR cycles permits the specific amplification.
We think
that the annealing and extension time plays a key part because of few
opportunities to encounter between template and less primer. In conventional
PCR, the excess primer combines with the template immediately. It takes longer
to anneal between less primer and the template, likewise, compose of less
primer#8212; template and polymerase. The larger yield of low molecular locus
is produced at three minutes of the annealing and extension time and five
minutes promote dramatically the amount of PCR product of high molecular locus.
As molecular weight become higher, the template of locus reduces, especially in
degraded sample. Therefore, high molecular locus needs five minutes for both
annealing and extension. The accurate genotyping from degraded samples in this
method result from the upper limit and the specific amplification with high
number of amplification cycle.
P-143
DNA analysis as the
only solution for identification of remains found in secondary mass graves
Karija Vlahovic M, Furac I, Masic M, Marketin
S, Raguz I, Kubat M
DNA
Laboratory, Department of Forensic Medicine & Criminology, School of
Medicine University
of Zagreb
Killed soldiers and civilians, displaced and exiled
persons, missing people, destroyed homes and towns; those are consequences of
all wars. Unfortunately the same happened in Croatia. More than hundred mass
graves were found in Croatia
during past ten years after the war ended. For the identification of human
remains found in mass graves conventional forensic methods were used, as well
as DNA analysis. DNA analysis is the primary tool used for identification of
fragmented remains and for the re-association of individual fragments. Here we
present results of identification of war victims remains found in the secondary
mass grave. 19 civilians were killed and buried in 1991 in Eastern
Slavonia. In 1997 the 18 bodies were packed in seven large plastic
barrels and transported across the country where they were discovered in a mass
grave in the year 2000. After medical experts and antropologist finished the
preliminary identification, it was decided to use DNA profile analysis as the
final identification method since the body parts were commingled. 37 body parts
and 20 blood samples from relatives were analysed at sixteen STR loci using
PowerPlex 16 Kit. From 37 analysed samples we managed to obtain full STR
profile for 34 samples. DNA profile comparisons enabled us to sort the 34 typed
body parts into 17 individuals, as well as identifying the 16 victims for whom
reference samples were available.
P-144
Y-chromosome variation in
Swedish, Saami and Österbotten male lineages
Karlsson A1,
Götherström A2, Wallerström T3, Holmlund G1
1The National Board
of Forensic Medicine, Department of Forensic Genetics, University Hospital,
SE-581 85 Linköping, Sweden
2Department of
Evolutionary Biology, Uppsala
University, SE-725 36 Uppsala, Sweden
3Institute for
Archaeology and Ancient History, University
of Lund, SE-223 50 Lund, Sweden
We have analysed 383 unrelated
males from Sweden
(n=305), Saamiland (n=38) and Österbotten in Finland (n=40). Haplogroups were
determined using 16 different Y chromosomal binary markers (M9, Tat, 92R7, M17,
M35, M78, M89, M201, M170, M26, M223, SRY10831, M253, M269, YAP and 12f2). The
Y-chromosome single nucleotide polymorphisms (Y-SNPs) were typed using
Pyrosequencing™ technique. Nine Y-chromosome short tandem repeat loci (DYS19,
DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393 and the separation of DYS385
into DYS385a and DYS385b) were also analysed to get a more detailed view of the
variation.
A total of 13 different haplogroups were
identified. In Sweden
haplogroup I1a* was most frequent (37%), while N3 was the most common
haplogroup in both the Saami and the Österbotten population (45% and 68%,
respectively).
RST values were calculated, from
haplotype data, in order to analyse the genetic differences between the
populations. Using all haplotypes, RST values revealed that Swedes
are more closely related to Saami than to males living in Österbotten. It also
showed that Saami lineages are closer to Österbotten than to Swedes.
The Swedish sample consisted of males from
seven geographically different regions in Sweden. Västerbotten, a northern
Swedish county, was significantly different (P<0.05) from the other Swedish
regions both comparing haplogroup frequencies and RST values.
P-145
STR data for 15 AmpFLSTR Identifiler loci in a Tibetan
population (Nepal)
Kido A1, Dobashi Y2, Hara M3, Fujitani N4,
Susukida R1, Oya M1
1Department of Legal Medicine, Faculty of
Medicine, University
of Yamanashi, Yamanashi, Japan
2Scientific Crime Detection Laboratory,
Yamanashi Prefectual Police Headquarters, Yamanashi,
Japan
3Department of Forensic Medicine, Saitama Medical School,
Saitama, Japan
4Department of Biochemistry, Faculty of
Science, Okayama University, Okayama, Japan
Allele frequency data for 15
short tandem repeat (STR) loci, D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01,
D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA, were
determined in 122 Tibetan individuals living in Katmandu (the capital of
Nepal). DNA was extracted from serum
samples, which were stored at -20oC
for six years, by the QIAamp DNA Mini Kit (Qiagen). PCR amplification of the 15
STR loci was performed using the AmpFLSTR Identifiler Kit (Applied Biosystems)
according to the manufacturer’s recommended protocol. 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). Possible divergence from the Hardy-Weinberg equilibrium
was determined using the exact test. Some statistical parameters of forensic
interest such as heterozygosity, power of discrimination, mean exclusion chance
and polymorphic information content were calculated. Typing of STR loci was
impossible in some samples. This tendency was salient in the STR loci with the
long fragment. Amelogenin included in the AmpFLSTR Identifiler Kit was detected
in all the samples. The agreement with Hardy-Weinberg expectation was confirmed
for all studied loci with the exception of FGA. It appears that this departure
is caused by the small number of samples. Among the 15 STR loci, FGA showed the
highest power of discrimination and the highest mean exclusion chance. The
combined power of discrimination and the combined mean exclusion chance for the
15 STR loci were 0.9999999999999999902 and 0.9999988, respectively.
Contact: akido@yamanashi.ac.jp
P-146
Novel Sample
Preparation Tool Quickly and Efficiently Prepares Cell Lysates
to Facilitate
Forensic Genomic Research.
Kirsher S, Dorion
R, Chu S
Cartagen Molecular
Systems Inc., Seattle, USA.
Plant
and insects samples associated with crime scenes are gaining recognition as a
source of valuable information related to the overall forensic process. We present data on a novel sample preparation
tool for use by the forensic researcher when working with plant and insect
samples. The BioMasher sample
preparation device was developed by Nippi Inc. (Tokyo, Japan)
to prepare bovine brain cell lysates prior to testing for BSE (Bovine
Spongiform Encephalitis). We have found that the BioMasher is a versatile
tool well suited for preparing PCR ready cell lysates from plant and insect
samples. Several plant and animal species were evaluated by PCR using
samples prepared with the BioMasher. We compared direct PCR of cell
lysates to PCR of DNA isolated using standard genomic DNA
extraction methods. We found that the
BioMasher consistently provides efficient homogenization of both plant and
animal tissue for use in direct PCR analysis or as the front end to
commercially available genomic DNA extraction kits
contact:
skirsher@cartagen.com
P-147
Old friends revisited:
Physical location and linked genes of common forensic
STR markers.
Klintschar M1,
Immel U-D1, Kleiber M1, Wiegand P2
1 Department of Legal
Medicine, University
of Halle-Wittenberg, Halle, Germany
2 Department of Legal
Medicine, University
of Ulm, Ulm, Germany
Good practice in a
forensic DNA laboratory requires knowing the sequence of the alleles, the
allelic distribution, the chromosomal location and population genetic data.
Nevertheless, as STR markers of forensic interest are also used in medical
genetics, for many of the established loci further information, e.g. the exact
physical localization and potential gene and disease linkage, is available. One
example would be TH01, for which it is known that it is located in intron 1 of
the tyrosine hydroxylase gene on the short arm of chromosome 11 (11p15.5), and
closely linked to the insuline gene and the Harvey ras
1 oncogene. These facts have elicited further genetic studies which found that,
either by linkage to one of these genes or by direct influence on the gene
regulation, the allele 9.3 seems to be associated to diseases such as
hypertension and psychosis. As such phenotypic effects of STRs are highly
undesirable in forensic sciences, it appears to be worthwhile to investigate
the current extent of information about forensic STR loci in common genetic
databases.
To that end, for 16
loci for which only partial information is given in the forensic literature
(D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, D19S433,
D2S1338, D2S1242, D8S1132, D7S1517, D1S1656, D12S391 and D1S1171) an in silico
search in the UniSTS and the EMPOP databases was performed and information
concerning the exact physical location and closely linked genes was gathered.
As expected, none of
the markers was localized in a coding region. For all markers an exact physical
location was found.
Moreover, D3S1358
was found to be located in intron 20 of the leucyl-tRNA synthetase 2 gene and
is part of a region on 3p21.3 that is frequently deleted in various tumours.
D7S820 is located in intron 15 of the semaphorine 3A gene. D18S51 is located in
the B cell lymphoma 2 gene. Also the rarely used loci D1S1656 (Calpain 9 gene)
and D7S1517 (hyaluronoglucosaminidase 4 gene) were found to be located in
introns, whereas the remaining markers are located outside of genes.
A search in the OMIM
database for known linkage between diseases and markers revealed that D8S1179
was linked to Meckel syndrome (type 3) in an Indian family. D18S51 is linked to
polyostotic osteolytic dysplasia (McCabe disease). D1S1656 is linked to
Kenny-Caffey syndrome type 1 and to hypoparathyroidism-retardation-dysmorphism
syndrome in Arabs. D2S1338 is linked to familial pseudohyperkaliemia 2. All
these diseases are extremely rare inherited disorders, and linkage does not
necessarily allow the conclusion that typing these markers would infer the
undesirable diagnosis of an inherited disorder.
Our results allow
the reassuring conclusion that up to now for none of these 16 markers a
significant influence on the phenotype is known.
P-148
The risk of incorrect typing of D1S80 by unstable minisatellite expansion
R. Kobayashi1, 3 , N. Iizuka2，3 and Y. Itoh3
1Depatment of Microbiology, Tokyo Medical
University, Tokyo, Japan
2 Medico-Legal
Section, Criminal Investigation Laboratory, Metropolitan Police Department, Tokyo,
Japan
3Department of Forensic Medicine, Juntendo University School
of Medicine, Tokyo,
Japan
The D1S80 locus is very useful for personal identification in Japan. To analyze PCR amplification products at the D1S80 locus, DIG-labeled primer was used for PCR amplifications. After electrophoresis, the PCR products were transferred to a nylon membrane and detected with alkaline phosphatase labeled-anti-DIG antibody (AP-DIG Ab). Numerous extra bands were detected on the membranes, indicating that PCR amplification products at the D1S80 locus contain many extra products which cause the undesirable bands to appear during D1S80 typing. To obtain a correct genotype, it was necessary to perform Southern blotting using an oligonucleotide that includs an internal sequence of the amplification products as a probe.
Introduction: The minisatellite locus D1S80, (location; 1p35-p36), GenBank sequence accession #D28507), is a variable number of tandem repeat (VNTR) locus with a 16 base pair repeat size. With alleles defined by the number of repeat units, the D1S80 locus is highly polymorphic in Japan. However, it is well known extra bands frequently appear during typing. In this paper, we demonstrate that PCR amplification products at the D1S80 locus have numerous extra bands which may cause incorrect genotypes to be obtained and that Southern blotting using an internal sequence as a probe is very helpful to determine D1S80 genotypes.
Materials and Methods: The primer (MCT118F) was labeled with DIG-11-dUTP (Roche, USA) according to the manufacturer’s instructions (DIG-MCT118F). The probe (MCT118P: 5’-CTG CGT GTG AAT GAC CCA GGA GCG TAT C-3’) was designed and also labeled with DIG-11-dUTP (DIG-MCT118P). PCR amplification was performed as described by Kasai (1990). After electrophoresis of PCR amplification products with DIG-MCT118F and MCT118R using 2% agarose gel, DNA fragments were transferred to a nylon membrane and detected using AP-DIG Ab and NBT/BCIP. The PCR amplification products with unlabeled primers were also transferred to a nylon membrane and hybridized with DIG-MCT118P and detected with AP-DIG Ab and NBT/BCIP.
Results and Discussions: PCR amplification products at the D1S80 locus were analyzed using a DIG-labeled primer. Although only real bands of the products appeared under UV light after ethidium bromide staining, numerous bands were detected when using the DIG-labeled primer. This finding indicates that extra bands are produced under the regular PCR conditions and can be visualized when using the AP-DIG Ab and NBT/BCIP detection. These extra bands may be detected with ethidium bromide staining if additional amplification cycles are performed. This may cause incorrect genotypes to be obtained. However, Southern blotting using an internal sequence as a probe could isolate and detect the real bands. This finding indicates that Southern blotting may be very helpful to determine D1S80 genotypes.
Dr.
Y.Itoh Department of Forensic Medicine, Juntendo University School of Medicine,
Hongo, Tokyo
113-8421, Japan.Tel: +81-3-5802-1051 Fax:
+81-3-3814-9300 yitoh@med.Juntendo.ac.jp
P-149
Mutation typing in Patients
with Medium Chain AcylCoA Dehydrogenase Deficiency (MCADD) and PCR based
mutation screening in SIDS victims
Krause D1, Jachau K1,
Mohnike K2, Nennstiel-Ratzel U3, Busch U3,
Rosentreter Y1, Sorychta J1, Starke I2,
Sander J4,
Vennemann M5, Bajanowski T6, Szibor R1
1Institut für
Rechtsmedizin, Otto-von-Guericke-Universität Magdeburg, Germany
2Zentrum für
Kinderheilkunde, Otto-von-Guericke-Universität Magdeburg, Germany
3 Bayerisches
Landesamt für Gesundheit und Lebensmittelsicherheit
Oberschleißheim, Germany
4 Screening-Labor Hannover, Germany
5Institut für
Rechtsmedizin, Universitätsklinikum, Münster, Germany
6Institut für
Rechtsmedizin, Universitätsklinikum, Essen, Germany
In parts of this paper we
publish data on behalf of the GeSIDS Group*
We
investigated 80 MCADD patients in a German populations and found the following
frequencies of mutations: 985A>G
(81.9 %); 157C>T (3.1 %), 799G>A (3.1 %), 244-245 ins T (3.1 %), 362C>T (1.3 %)
and five rare mutations with frequencies below 0.6%. About 4.4% of the
mutations in our patients remained unidentified. After mutation typing
procedure we created rapid tests, which are based on the PCR / electrophoresis
technology and recognise the four most frequent mutations.(i. e. 985A>G ,
157C>T, 799G>A, 244-245 ins T ).
Using these screening tests we identified one MCADD case under 409 SIDS
victims. These investigations indicate that in very few cases MCADD may
contribute to SIDS.
*This
study was supported by the BMBF; we would like to thank all contributors of the
GeSIDS Group
P-150
Data analysis of SE33 allele
frequencies in the population of province Schleswig-Holstein (North
Germany)
Krause M, Heide K-G,
Labor für Abstammungsgenetik,
Kiel, Germany
Allele
and genotype frequencies for STR SE33 were determined in a sample of 1750
unrelated Germans for paternity cases. We found many “Variants”. No deviation
from Hardy-Weinberg equilibrium were observed in the population.
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