Validation of a 21-locus Autosomal SNP Multiplex for
Forensic Identification Purposes
Dixon LA, Murray CM, Archer EJ, Dobbins AE, Koumi P,
Gill P
The Forensic
Science Service, 2960 Trident
Court,, Solihull
Parkway, Birmingham, UK
A single nucleotide
polymorphism (SNP) multiplex has been developed to analyse highly degraded and
low copy number (LCN) DNA template, i.e. <100pg, for scenarios including
mass disaster identification. The
multiplex
consists of 20
autosomal non-coding loci plus Amelogenin for sex determination, amplified in a
single tube PCR reaction and visualised on the Applied Biosystems 3100
capillary electrophoresis system.
Allele-specific
primers tailed with
shared universal tag sequences were designed to speed multiplex design and to
balance the amplification efficiencies of all loci through the use of a single
reverse and two differentially labelled allele
denoting forward
universal primers. As the multiplex is
intended for use with samples too degraded for conventional profiling, a
computer program was specifically developed aid interpretation. Critical factors taken into account by the
software include empirically determined extremes of heterozygote imbalance (Hb)
and the drop-out threshold (Ht) defined as the maximum peak height of a
surviving heterozygote allele, where its partner
may have dropped
out. The discrimination power of the
system was estimated at 1 in 4.5 million, using a White Caucasian population
database. Comparisons using artificially degraded samples profiled with both
the SNP
multiplex and
AMPFlSTR SGM plus (Applied Biosystems) demonstrated a greater likelihood of
obtaining a profile using SNPs for certain sample types. Saliva stains degraded
for 147 days generated an 81% complete SNP
profile whilst STRs
were only 18% complete; similarly blood degraded for 243 days produced full SNP
profiles compared to only 9% with STRs. Reproducibility studies showed
concordance between SNP profiles for
different sample
types, such as blood, saliva, semen and hairs, for the same individual, both
within and between different DNA extracts.
Contact:
DNAPGill@compuserve.com
O-02
Multiplex
genotyping of 22 autosomal SNPs and its application in forensic field
Turchi C1, Onofri V1,
Alessandrini F1, Buscemi L1, Pesaresi M1,
Presciuttini S2, Tagliabracci A1
1Institute of Legal
Medicine, Università Politecnica delle Marche, Ancona, Italy
2Center of
Statistical Genetics, University of
Pisa, Italy
The sequence of the
human genome, within the framework of the Genome Project, has revealed the
existence of a new class of DNA polymorphisms involving one single base-pair,
called SNPs (single nucleotide polymorphisms), constituting the most abundant
form of genetic variation. This new class of markers offers interesting
prospects in the forensic field, given their
abundance and low mutation rates. Moreover, SNPs can be analyzed using
throughput technologies and, most importantly, they can be used when DNA is
highly degraded because they can be tested in short amplicons. In such
instances, they could be used with proficiency in association with classical
markers. On the other hand, the number of SNPs
required to achieve a significant discrimination power is higher than the number of STRs commonly used. It has been estimated that nearly 60 SNPs are needed to match the
power of the CODIS STRs set.
The aims of this
study were to set up multiplex PCRs of 22 autosomal SNPs suitable for forensic
purposes to assay their discrimination power in a population sample, and to
compare it with the already known power of STRs commonly used in forensic work
22 binary polymorphisms, with
an allele frequency of 0.5 in at least two different Caucasian population
studies, were extrapolated from the “SNP Consortium” database (http://snp.cshl.org). One SNP was chosen for
each autosomal chromosome. Three multiplex PCRs were constructed with primer
pairs designed to produce amplicons in a range between 56 and 151 bp. 1
nanogram of DNA template, extracted from 50 healthy Italian subjects, was
submitted to amplification reactions. SNPs typing was performed by
fluorescently labelled dideoxy single-base extension of unlabelled
oligonucleotide primers using the ABI PRISM SNaPshot™ Multiplex Kit (Applied
Biosystems). The extension products were electrophoresed in an automated ABI
310 5-colour sequencer (Applied Biosystems).
O-03
Development of a multiplex PCR assay with 52 autosomal
SNPs
Sanchez JJ1,
Phillips C2, Børsting C1, Bogus M3, Carracedo
A2, Court DS4, Fondevila M2, Harrison CD4,
Morling N1, Balogh K3 and Schneider P3
1 Department of Forensic Genetics, Institute of Forensic Medicine, University
of Copenhagen, Denmark
2 Institute of Legal Medicine, University of Santiago de
Compostela, Spain
3 Institut für
Rechtsmedizin, Johannes
Gutenberg University,
Mainz, Germany
4 Department of Haematology, ICMS, Queen Mary's School of Medicine and Dentistry, London, UK
An efficient method that can
be used to simultaneously amplify a set of genetic loci across the genome with
high reliability can provide a valuable tool for single nucleotide polymorphism
(SNP) forensic genotyping. A crucial element is the number of individual
biochemical reactions that must be performed. The SNPforID consortium
(www.snpforid.org) was established in 2003 with the principal goal of
developing a SNP-based system of DNA analysis that would have comparable
discrimination power and ease of use to existing short tandem repeat (STR)
based techniques. Here, we describe a strategy for amplifying 52 genomic DNA
fragments, each containing one SNP, in a single tube, and accurately genotyping
the PCR product mixture using two single base extension reactions. This
multiplex approach reduces the cost of SNP genotyping and requires as little as
0.5 ng of genomic DNA to detect 52 SNPs. We used a multiple injection approach
for sequencers that can effectively detect all the SNPs amplified in a single
electrophoretic run. We present SNP data for 709 unrelated individuals from 9
populations. Statistical interpretation of the results and comparisons between
the 52 SNP multiplex and commercial STR kits are discussed.
contact:
juan.sanchez@forensic.ku.dk
O-04
Application
of Nanogen Microarray Technology for Forensic SNP Analysis
Balogh MK, Bender K, Schneider
PM and the SNPforID Consortium
Institute of Legal Medicine, Johannes Gutenberg
University of Mainz, Germany
www.snpforid.org
The NanoChip® Molecular Biology Workstation (developed by Nanogen Inc.)
using electronic microarrays is a particularly attractive microarray approach
for rapid and high throughput analysis of SNPs. This instrument is fully
automated and uses a proprietary semiconductor microchip for electronic
addressing of capture probes to specific array sites followed by electronic
hybridisation of the single stranded PCR products, and passive hybridisation of
fluorescently labelled reporter oligos. Discrimination is achieved by applying
thermal stringency to denature the mismatched reporters. Allele calling is
carried out immediately using a built-in laser-activated fluorescence detection
system.
The main purpose of the performance assessment was to evaluate the
sensitivity, the accuracy and the multiplex capability of the platform by using
24 autosomal SNPs chosen from 52 non-coding SNPs, previously selected for the
SNPforID project. In the initial phase of the project, the amplicon down
assay was used for addressing the biotinylated amplicons directly to the
surface of the microarray, followed by hybridisation of the labelled reporter
probes, separately for all the 24 SNPs. However, forensic typing requires rapid
multiplex analysis from limited samples under high throughput conditions.
Therefore, the capture down assay is more suitable, since fragment specific
capture probes are bound to the array and the PCR amplicons are captured
simultaneously by electronic hybridisation, followed by passive hybridisation
of all labelled reporter probes in a single reaction.
24 SNP assays have already been designed using a modification of the
capture down assay which applies a “touch down” strategy to obtain the best
reporter probe discrimination. Overall the Nanochip platform appears to be
suitable for SNP multiplex typing and presently, an additional 24 SNPs are
under evaluation to be combined into a 48-plex.
Balogh MK, Institute of Legal Medicine, Johannes Gutenberg
University of Mainz,
Am Pulverturm 3, 55131 Mainz,
Germany
contact: balok000@students.uni-mainz.de
O-05
Fluorescence
labelling and isolation of male cells
Anslinger K1, Mack
B2, Bayer B1
1 Institute of Legal
Medicine, Ludwig-Maximilians-University, Munich
2 Department of Head
and Neck Surgery, Ludwig-Maximilians-University, Munich
Laser
capture microdissection (LMD) is a relatively new technique for the isolation
of single cells. In forensic science for example, LMD is used to select
spermatozoa out of Haematoxylin / Eosin stained vaginal smears. In particular
in cases with low numbers of sperm and in cases with aspermatozooic perpetrator
or men, which have undergone a vasectomy, it could be profitable to isolate
male cells in general, instead of focussing on the sperm only. Similarly, the
specific detection of male cells in a female/male epithelial cell mixture, for
example male saliva on female skin, could be of real advantage. The aim of this
study was to find a staining method, which allows the identification of male
cells from different origins for isolation via LMD. Therefore, we used a
fluorescence-in-situ–hybridisation kit from Vysis (Downers Grove, IL),
which includes probes for the X- and the Y-chromosome. The X-specific probe
hybridises to multicopy alphoid DNA located at the centromere and was labelled
with a red fluorescence dye. The Y-specific probe hybridises to
Satellite-III-DNA located on Yq12 and was labelled with a green fluorescence
dye. The simultaneous detection of the X- and the Y-chromosome could be seen as
an internal positive control for the success of the hybridisation. Different
mixtures of male and female cell samples were stained, and the male cells were
isolated via SL µCut LMD system from Molecular Machines & Industries AG
(MMI, Glattburg, Zurich, Switzerland). In comparison with
other LMD systems, the SL µCut doesn’t works
with glass slides. The samples are spread on a membrane, which is placed on a
special metal holder. The laser cuts the membrane around the cells of interest
and they are securely removed with an adhesive film technology.
DNA
was isolated from the LMD separated cells and a STR profiling was performed
using different multiplex PCRs. In parallel we determined the overall content
of male DNA of the different mixtures using the Quantifiler Human and
Quantifiler Human Male DNA Quantification Kits (AB, PE Corporation, Forster City, CA).
Taken together, the results of our study revealed that the staining method in
combination with LMD seems to be a real advantage when dealing with
unfavourable male/female cell mixtures. In cases where every single cell is
important for a successful STR profiling of the male component, this technique
can definitely increase the amount of male material that could be extracted via
LMD. Moreover it’s suitable to select male cells out of male/female mixtures
with identical cell types.
Contact:
Katja.Anslinger@med.uni-muenchen.de
O-06
Low
Volume PCR (LV-PCR) for STR typing of forensic casework samples.
Proff C, Rothschild MA, Schneider
PM
Institute of Legal Medicine, University Clinic Cologne, Germany
Commercial multiplex STR typing kits are often used with reduced PCR volumes. A volume reduction of 30-50% normally does not result in a significant loss of quality regarding signal intensity, allele balance etc. This is especially true for reference samples extracted from blood or buccal swabs where sufficient DNA of good quality is available. But even in low copy number (LCN) amplifications reproducible results can be obtained. This may be due to the assumption that DNA in a lower PCR volume could get into better contact with primer or polymerase molecules because the overall amount of DNA is less diluted than in a higher volume. Otherwise the volume of extracted DNA that can be used for the PCR assay is limited.
In the days of nanotechnology everything is getting smaller. In this
case commercial PCR chips (Ampli Grid® A60, Alopex, Kulmbach,
Germany; originally designed for diagnostic single cell PCR) have been
developed where multiplex PCR can be performed in a 1 µL-PCR volume on a 60
well glass chip in microscopic slide format. Circular hydrophilic wells are
separated by hydrophobic regions to ensure that the liquid PCR components do
not get into contact with each other and stay in a drop form comparable to the
'lotus effect'. The fluids are then covered by mineral oil to prevent
evaporation before the slide is put on a suitable in-situ PCR adapter that fits
into a common 96-well thermocycler. Using this technology, it is possible to
obtain a full 16-locus DNA profile in a 1 µl volume consisting of 0.5 µl DNA
sample and 0.5 µl PCR reaction master mix.
We have tested
LV-PCR with DNA from typical forensic casework samples using different
commercial STR typing kits with a variable number of STRs from different
manufacturers. The following criteria have been considered for this study:
sensitivity, reproducibility, contamination risk, total DNA amount and relative
DNA concentration, PCR cycling protocol, Taq polymerase, LCN amplification,
allele balance, allelic dropout, and signal intensities.
Address
for correspondence:
Dr.
Carsten Proff, Institute of Legal Medicine, Melatenguertel 60-62, 50823 Cologne,
Germany;
phone +49 221 47886623, fax +49 221 4783496,
phone +49 221 47886623, fax +49 221 4783496,
contact: carsten.proff@uk-koeln.de
O-07
Forensic Response Vehicle:
Rapid analysis of evidence at the scene of a crime.
Hopwood A, Fox R, Round C,
Tsang C, Watson S, Rowlands E, Titmus A, Lee-Edghill J, Cursiter L, Proudlock
J, McTernan C, Grigg K, Kimpton C.
Forensic Science Service, Trident Court, Solihull Parkway, Birmingham
Bus. Park, B’ham B37 7YN
The first hours of a criminal
investigation can be the most important. A suspect arrested soon after a crime
has less time to remove evidence from their person, possibly allowing stronger
forensic ties between the individual and the crime scene.
We have developed a mobile
laboratory with designated work areas for the searching of small items and pre and post PCR work.
An SGM+ profile can be
produced and compared to the National DNA Database in approximately 5 hours,
potentially providing the police with valuable intelligence early in the
investigation of a crime.
The DNA analysis process
utilises off the shelf equipment and consumables for the most part but a novel
instrument for the separation and detection of fluorescently labelled STR
amplicons has been developed from which data is directly imported to I3 expert
system software to provide an automated solution to profile designation.
The vehicle also carries the
capability for the interrogation of electronic items such as mobile phones, and
satellite communication systems allow direct connection with the FSS computer
network allowing images of fingermarks and footwear marks to be searched
against the appropriate databases.
Contact: Andy.Hopwood@fss.pnn.police.uk
O-08
The evolution of European national DNA databases –
conventional STRs, mini-STRs or SNPs?
Peter Gill and
Lindsey Dixon
Forensic Science
Service, Trident Court,
2960 Solihull Parkway,
Birmingham, UK
Recently, an EDNAP
exercise was instigated to compare the efficacy of conventional high molecular
weight STR systems compared to low molecular weight (mini-STR) loci and
SNP-plex of 21 loci, to analyse highly degraded stain material. We also assessed
the relative statistical attributes of SNPs v. STRs and present a computer
model that simulates DNA degradation. We concluded that the evidence suggests
that substantial improvements can be expected by moving to multiplex systems that
analyse smaller fragments of DNA than those in commercial kits that are
currently in common use. To improve existing STR multiplexes, we propose that
loci are re-engineered to produce smaller amplicons. In addition, 3 new
European loci that have specific low molecular weight characteristics have been
suggested for universal adoption – namely D10S1248, D14S1434, D22S1045.
The substantial
difficulties associated with preparing large multiplexed reactions suggests
that for routine stain work where the size of the sample is very limited,
mini-STRs are the best option, whereas SNPs are an option when there is effectively
unlimited sample, such as bone, that is available for analysis because several
different multiplexes can be successfully utilised. If new loci are introduced
into routine casework use it will be important to coordinate throughout Europe. To encourage this change, it will be essential to
facilitate the process via international collaborative groups such as EDNAP and
ENFSI.
Contact: DNAPGill@compuserve.com
O-09
Characterization
and Performance of New MiniSTR Loci for Typing Degraded Samples
Coble MD1, Hill CR1,
Vallone PM1, Butler
JM1
1Biotechnology
Division, National Institute of Standards and Technlogy, Gaithersburg, Maryland
A number of studies have demonstrated that successful
analysis of degraded DNA specimens from mass disasters or forensic evidence
improves with smaller sized polymerase chain reaction (PCR) products (1).
Forensic DNA analysts often perform short tandem repeat (STR) typing on highly
degraded biological material and then turn to mitochondrial DNA testing, which
is less variable but more likely to obtain a result due to higher copy numbers
in cells, if many or all of the STRs fail. The commercially available kits for
multiplex amplification of the 13 CODIS (FBI’s COmbined DNA Index System) STR
loci usually exhibit allele or locus-dropout for larger sized loci with
degraded DNA or samples containing PCR inhibitors.
By moving PCR primers closer to the STR repeat region,
we have demonstrated that it is possible to obtain fully concordant results to
the commercial kits while improving successful analysis of degraded DNA with
smaller PCR products or “miniSTRs” (1). However, many of the CODIS core loci
have large allele ranges (e.g., D21S11 and FGA) that make it impossible to
create small PCR products. We have
examined a battery of new potential STR loci that can be made less than 100 bp
in size (in most cases) and would therefore be helpful in testing highly
degraded DNA samples. These new STR loci are being put together into novel DNA
testing assays and evaluated across more than 600 samples representing the
three largest populations in the U.S.: Caucasian, African American,
and Hispanic. A set of six non-CODIS
markers have been characterized and published (2). More markers that have been recently
developed will be presented.
We have shown that the selection of STR loci that have
a narrow allele range (e.g., less than 50 bp) and can be made smaller than 100
bp works well with degraded DNA samples, such as shed hairs and old bones. The
successful typing of even a small number of nuclear loci from shed hairs can
greatly increase the forensic discrimination of the sample compared to mtDNA
testing alone, especially where a significant number of common types are
present in the population.
(1) Butler, J.M., Shen, Y.,
McCord, B.R. (2003) The development of reduced size STR amplicons as tools for
analysis of degraded DNA. J. Forensic Sci., 48(5): 1054-1064. (2) Coble,
M.D. and Butler,
J.M. (2004) Characterization of new miniSTR loci to aid analysis of degraded
DNA. J.
Forensic Sci., 50(1): 43-53.
contact: michael.coble@nist.gov
O-10
Development of a new multiplex assay for STR typing of telogen hair roots
Bender K1 and
Schneider PM2
1 Institute of Legal
Medicine, Johannes
Gutenberg University
Mainz, Germany
2 Institute of Legal Medicine, University Clinic
of Cologne, Germany
We have developed a new strategy in which 10 STR systems plus amelogenin were simultaneously amplified with maximal fragment sizes smaller than 270 base pairs. This method can be used for the amplification of DNA from casework samples from which only limited amounts or highly degraded DNA can be isolated, for instance when DNA is isolated from telogen hair roots.
The multiplex amplification reaction includes six STR loci from the European standard set of loci (ESS) for DNA databases (D3S1358, D8S1179, D21S11, THO1, FGA and VWA) as well as four additional STR systems selected for their robustness and short amplicon sizes (D2S1338, D12S391, TPOX and D5S818) together with the sex-specific locus amelogenin. After PCR amplification, the multiplex reaction is splitted into two sets of STR multiplexes.Using streptavidin-coated Sepharose beads five STR systems are separated from the other six systems prior to being analyzed in two different runs on a capillary gel electrophoresis instrument.
To verify the specificity of
the new STR multiplex undegraded human DNA samples from blood were amplified at
least twice, separated and analysed by capillary gel electrophoresis. The
results were compared to the typings with the SGM Plus™ (Applied Biosystems) or
PowerPlex® 16 (Promega) kits and the single amplification of the D12S391 STR
system. Furthermore DNA samples from artificially degraded DNA and from real
case work were analyzed.
Dr.
Klaus Bender, Institut Institute of Legal Medicine, Johannes Gutenberg
University of Mainz, Am Pulverturm 3,
D-55131 Mainz, Germany. Tel. 00 49 (0)6131 3932733;
Fax: 00 49 (0)6131 393183
O-11
Evaluation of methodology for the
isolation and analysis of LCN-DNA before and after dactyloscopic enhancement of
fingerprints.
Peter Leemans, Nancy
Vanderheyden, Jean-Jacques Cassiman and Ronny Decorte*
Laboratory for Forensic
Genetics and Molecular Archaeology, Department of Human Genetics, K.U.Leuven, Belgium
Several studies have
demonstrated the feasibility of using latent fingerprints for forensic DNA
analysis. The analysis of these LCN-DNA samples is however not trivial and
leads frequently to no results, partial results or the recovery of mixed DNA
profiles. As this kind of evidence material is increasingly be submitted by the
police for DNA analysis, we wanted to evaluate if current methodologies of
sampling by the police and DNA methods (mainly DNA extraction) in the
laboratory are optimal for DNA analysis of latent fingerprints. Fingerprints
were applied by 6 different donors onto clean microscope glass slides and the
fingerprints were recovered by using either cellotape, cotton swabs wetted with
physiologic water, cotton swabs wetted with ATL-lysis buffer (Qiagen) or cotton
tissue wetted with physiologic water.
Four different methods were used for DNA extraction: QIAamp DNA Mini kit
(Qiagen), QIAamp PCR Purification kit (Qiagen), a combination of both kits were
the flow-through of the QIAamp DNA Mini kit columns was applied to the QIAamp
PCR Purification kit columns, and the CST Forensic DNA Purification kit
(Invitrogen). Four different methods for the enhancement of fingerprints were
applied: white powder, black powder, cyanoacrylate fuming and enhancement of
cyanoacrylate with basic yellow. The DNA extracts were evaluated quantitatively
and qualitatively, respectively with the Quantifiler Human DNA Quantification
kit, and AmpFlSTR SGM Plus and Profiler (Applied Biosystems). In addition, a
multiplex of Y-SNPs (De Maesschalck et al., in preparation) was applied in
order to evaluate the analysis of SNPs
in LCN-DNA. The following conclusions could be drawn from the results of these
experiments: (1)Cotton swabs showed to be the preferable method for the
collection of latent fingerprints. The amount of DNA recovered with the
cellotape was significantly (4 times) lower than with the other methods. (2)No
difference was observed between the use of physiologic water and ATL-buffer for
collecting the fingerprints. Only when the swabs were left at room temperature
for at least 6 weeks, slightly more results were obtained with the STR-kits
when ATL-buffer was used. However, we cannot exclude the possibility that this
difference was due to differences in the amount of fingerprints present on the
glass slide. (3)The amount of DNA recovered when the swabs were left at room
temperature for at least 1 week until 8 weeks was slightly lower than when DNA
extraction was done immediately. However, there was no decrease in the amount
of DNA recovered between the different time periods neither in the possibility
to type the STRs suggesting that degradation and loss of DNA is a slow process
after sampling fingerprints when swabs remain at room temperature. (4)From the
6 donors, only one person was a good “shedder”. This was reproducible and the
amount of DNA recovered was sufficient for STR analysis and SNP amplification.
For the other donors, either no DNA or low quantity DNA was obtained and the
STR-profiles showed evidence for allele-drop-out, locus-drop-out and the
presence of additional alleles. The presence of mixed profiles indicates the
presence of additional DNA on the glass slide. We cannot exclude the
possibility of secondary transfer. Further experiments should clarify this.
(5)No significant difference was seen between the different methods used for DNA
extraction. (6)We were able to obtain DNA after enhancement with different
methods of the glass slides. The amount of DNA recovered was slightly less than
without enhancement and no inhibition was observed in the PCR-reactions.
* Presenting and corresponding
author: ronny.decorte@med.kuleuven.be
O-12
Characterization of parameters
influencing autosomal STR mutations
Hohoff C1, Fimmers R2,
Baur, MP2 und Brinkmann B1
1 Institut für Rechtsmedizin,
Universitätsklinikum Münster, Röntgenstr. 23, 48149 Münster
2 Institut für
Medizinische Biometrie, Informatik und Epidemiologie, Medizinische Fakultät der
Rheinischen
Friedrich-Wilhelms-Universität Bonn
Friedrich-Wilhelms-Universität Bonn
In
our routine parentage testing and additional meioses studies in Caucasoid
populations we have observed 189 de novo mutations in more 100,000
meiotic transfers at autosomal STR loci.
The
following criteria were chosen to call a Non-Mendelian transfer a mutation:
isolated mismatch(es) (1 - 2), sequencing of all involved alleles and inclusion
of the mutation in the biostatistical calculation with a resulting paternity
value W > 99,97%.
By
application of ’maximum likelihood’ estimates we have been able to evaluate
system-specific parameters such as gender, gain or loss of repeat units,
parental age at conception and the sequence structure of the particular repeat.
This
work is an important step to increase our understanding of the basic principles
of STR mutations to estimate allele-related mutation rates in the future.
Address for correspondence
Prof. Dr. med. Bernd Brinkmann, Institut für
Rechtsmedizin, Universitätsklinikum Münster, Röntgenstrasse 23, D-48149
Münster, Germany, Fax: 00 49 (0) 251 8355158,
eMail: remed@uni-muenster.de
O-13
Highly efficient
semi-quantitative genotyping of single nucleotide polymorphisms in
mitochondrial DNA mixtures by liquid chromatography electrospray ionization
time-of-flight mass spectrometry
Niederstätter H, Oberacher H,
Parson W
Institute of Legal Medicine, Innsbruck Medical University,
Innsbruck, Austria
Sanger sequencing represents
the “golden standard” for the typing of mtDNA. Accordingly, sequencing is
commonly used for the detection and the semi-quantification of heteroplasmic
mixtures. Typically, a minimum contribution of 20% of the minority allele is
required to unequivocally reveal the presence of point-heteroplasmy, which
clearly restricts the use of this method for the quantification of allelic
contents. A number of different technologies have been introduced for the
determination of allelic frequencies in DNA mixtures including allele-specific
oligonucleotide hybridization, minisequencing, denaturing gradient gel
electrophoresis, real-time PCR, and denaturing high-performance liquid
chromatography.
Here, the combination of
ion-pair reversed phase high-performance liquid chromatography and electrospray
ionization quadrupole time-of-flight mass spectrometry is presented as an
efficient method for the semi-quantitative genotyping of single nucleotide
polymorphisms (SNPs). Artificially prepared and naturally occurring
mitochondrial DNA mixtures showing different levels of heteroplasmy at
nucleotide position 16519 served as reference samples. Allelic frequency determinations
were based on the comparisons of allele-specific peak intensities in the
obtained deconvoluted mass spectra. Deviations between measured and observed
allelic frequencies were caused by differential PCR amplification and
ionization of single alleles. Biased estimates were corrected by measuring the
allele-specific signal intensities of equimolar allelic mixtures. Afterwards,
measured and expected allelic frequencies correlated well (R² = 0.9983). An
average error of 1.2% and a maximum error of 2.2% demonstrated the accuracy of
the method. An average standard deviation of 2.45% and a maximum deviation of
5.35% proved the reproducibility of the assay. Due to the sensitivity of the
applied mass spectrometric detection system alleles occurring at a frequency of
1.0% were unequivocally detected. The limit of quantification was found in the
range of 5% minority component. The observed assay performance suggests that
the described mass spectrometric technique represents one of the most powerful
semi-quantitative genotyping assays available today.
O-14
A
Problem in Paradise: the Development and Forensic
Interpretation of the Y Chromosome in New Zealand
Harbison S1, Brash
K1, Fris B2, McGovern C1
1Forensic Biology,
Institute of Environmental Science and Research Ltd, Auckland, New Zealand
2Forensic Science
Programme, Department of Chemistry, University
of Auckland, New Zealand
The
Y chromosome offers much for the analysis of forensic samples in both a
traditional crime solving capacity and also in matters of mass disaster such as
experienced recently in Asia. At ESR we are interested in developing Y
chromosome systems for use in forensic casework to complement the SGM Plus™
system currently in place. A difficulty
we face is the unique, multicultural nature of New Zealand, comprised as the
population is of individuals of European, Asian and Polynesian descent and
mixtures thereof. This complexity was
demonstrated by the discovery of significant amounts of linkage disequilibrium
in our STR population data and consequent adoption of relatively high Fst (θ)
values of up to 5% in calculations of match probabilities.
In
this paper we have evaluated both Y chromosome STRs and Y chromosome SNPs as
potential systems for development. We
have used both the Y plex 12™ system from Reliagene and the Powerplex-Y system
from Promega to investigate the distribution of Y chromosome haplotypes in our
population. We have found differences
between population groups, including haplotypes common to some population
groups and not others. We offer suggestions as to how these differences could
be utilized in a forensic investigation.
We
also describe the development of Y-chromosome based SNP marker systems,
designed with the requirements of a forensic laboratory in mind. These
multiplex systems comprise 5 or 6 SNP loci and have been built using
mini-sequencing technology from Applied Biosystems, the SNAPSHOTTM SNP
system. Loci were specifically selected
for typing Y-chromosome lineages within Polynesian populations. These systems
stand as a stepping-stone to the development of larger broad-based Y-chromosome
and autosomal SNP marker systems that could complement or replace the STR
systems currently in use.
We
illustrate our work with case examples that demonstrate the usefulness of these
techniques.
O-15
Relative Y-STR mutation rates
estimated from the variance inside SNP defined lineages
Soares P1, Pereira F1,2, Brion M3,
Alves C1, Carracedo A3, Amorim A1,2, Gusmão L1
1IPATIMUP, Instituto de Patologia e Imunologia da
Universidade do Porto, Portugal; 2Faculdade de Ciências da Universidade
do Porto, Portugal; 3Unidad de Genética Forense, Inst. Medicina Legal, Univ. Santiago de Compostela, Spain.
Apart from the important role
in general population genetics and in discerning male counterpart of
demographic history, Y-chromosome has major forensic applications. Y specific
microsatellites (STRs) have been widely used in forensic and population
genetics in age estimates of human male lineages. Previously estimates of
mutation rates from father-son pairs have shown quite variable results in
different studies, essentially due to the rare nature of the mutational
phenomenon. We propose an indirect approach for the determination of relative
mutation rate of Y-chromosome microsatellites based on STR allele size
intra-lineages variance. Indeed, the present distribution of STR alleles offers
us an insight into the mechanisms that have generated that diversity, not a
direct observation of a mutation occurrence but the observation of past
mutations. We performed simulations using a Stepwise Mutation Model which
showed that the variance of allele size distribution in Y-chromosome presented
a linear relation with elapsed time that could be expressed as V=d×t
(where V is the variance of the allele size distribution, d is
the slope of the curve and t is the number of generations). From this
equation: t=V/d. Therefore, if we compare the variances inside the same
SNP defined lineage for two microsatellites (e.g. A and B), as time lapse is
the same for both, we will have VA/dA=VB/dB
and therefore dA= (VA/VB)×dB.
Since the slope of the curve and mutation rates also presented a linear
relation in the simulations, the equation can be changed to µA=
(VA/VB) ×µB (or µA= RAB×µB
if we considered R as the relative mutation rate of A to B). Using the
calculated relative mutation rates, a linear relation shows up between the
variance of each microsatellite inside a lineage (V) and 1/R. For
new STRs, or to those where few data concerning mutation studies have been
accumulated, it would be thus possible to establish a relative mutation rate
using lineage’s microsatellite variance, according to the equation V=m×(1/R) (where m is the lineage specific
slope). The relative mutation rate (R) between two microsatellites will
be the same in the distinct SNP defined lineages (p and q) so we will have Vp/mp=Vq/mq
and, since V=d×t and dp=dq for the same
microsatellite, we could modify the equation to tp×mq=tq×mp,
and therefore, tp=(mp/mq) ×tq.
This means that the relative age of the lineage could be obtained through the
relation of the lineages’ specific slopes (m). In a sample of 950
unrelated Iberian individuals belonging to different Y-lineages, we selected
those which presented n>30 to avoid too large confidence intervals and
spurious results. Among those we selected those with well defined unimodal
distributions and not too dispersed allele distributions in order to decrease
inaccuracies due to random demographic factors (genetic drift, bottlenecks and
founder effects) and to avoid significant departures from the theoretical model
due to different mutational behaviour of the extreme size alleles in a STR
allele distribution. Applying these criteria, we obtained three lineages
(defined by SNPs M172, M201 and M269) and we calculated the relative mutation
rates for all pairs of seven commonly used Y-microsatellites (DYS19, DYS389I,
DYS389II, DYS390, DYS391, DYS392 and DYS393). Contact: pedros@ipatimup.pt
O-16
Relaunch of the Y-STR
haplotype frequency surveying method based on metapopulations
Willuweit S1,
Krawczak M2, Roewer L1
1Inst. of Legal
Medicine, Charité-University Medicine
Berlin, Germany;
2Institute of Medical Informatics and Statistics,
Christian-Albrechts-University, Kiel,
Germany
The successful
implementation of Y-STR analysis in forensic practice led to the establishment
of large web-based population databases which facilitate the assessment of
match probabilities for haplotypic profiles. Thanks to international
collaboration the current release 15 of the Y-STR Haplotype Reference Database
(YHRD) consists of more than 22,000 different haplotypes from 249 population
samples. YHRD provides frequencies for haplotypes found in geographically or
linguistically defined metapopulations. Metapopulations are here defined as
pools of population samples with an (assumed) high degree of relatedness. To
obtain the observed haplotype frequency, it is sufficient to search a profile against
the database or metapopulation and count the number of matches. The frequency
surveying approach instead [1] takes the genetic similarities of a searched
haplotype profile to its closely related “neighbours” into account and thus
allows the frequency estimation even of those haplotypes which are rare and not
observed in the database. In order to ensure that the extrapolated frequencies
retain their high evidential power, it is important to perform the analysis for
specified homogeneous data sets which can be identified by population genetic
analysis. Using AMOVA and MDS analysis such pools with a high degree of genetic
relatedness of Y-STR haplotypes have been identified for Europe
based on a representative dataset 12.700 haplotypes from 91 populations [2].
Starting with release 16 all European haplotypes of the YHRD were assigned to
these genetically defined metapopulations. Now we present the re-programmed
web-based frequency surveying method adapted to metapopulation pools. To define
such data pools we introduce a test for the assessment of homogeneity of
population pools used for the frequency extrapolations.
[1] Roewer L, Kayser M, de
Knijff P, Anslinger K, Betz A, Caglia A, Corach D, Furedi S, Henke L, Hidding
M, Kärgel HJ, Lessig R, Nagy M, Pascali VL, Parson W, Rolf B, Schmitt C, Szibor
R, Teifel-Greding J, Krawczak M (2000) A new method for the evaluation of
matches in non-recombining genomes: application to Y-chromosomal short tandem
repeat (STR) haplotypes in European males.' Forensic Sci Int. 114 (1):
31-43. [2] Roewer L, Croucher PJ, Willuweit S, Lu TT, Kayser M, Lessig R, de
Knijff P, Jobling MA, Tyler-Smith C, Krawczak M (2005) Signature of recent
historical events in the European Y-chromosomal STR haplotype distribution. Hum
Genet. 116 (4): 279-91.
O-17
Mitochondrial
DNA pseudogenes in the nuclear genome as possible sources of contamination
Goios A1,2, Amorim A1,2, Pereira
L1
1IPATIMUP (Instituto de Patologia e Imunologia
Molecular da Universidade do Porto), Porto, Portugal; 2Faculdade de
Ciências Univ. Porto, Porto, Portugal
Since shortly after the
discovery of the mitochondrial genome, mitochondrial DNA-like sequences have
been identified in the nuclear genome. The presence of these nuclear
mitochondrial insertions (NUMTs) may lead to accidental amplifications of
nuclear fragments with primers specifically designed for mitochondrial DNA (mtDNA).
Depending on the homology of each NUMT to the mtDNA, this problem may be more
or less relevant. In this work, we focused on the NUMTs that may be a cause of
contamination in forensic analyses. Following the report of the complete human
genome sequence, various studies have been published describing and listing all
mitochondrial pseudogenes that exist in the different chromosomes. Of the 247
NUMTs reported in one of these studies (Mishmar et al, 2004), we analysed 19
that encompass the fragments of the D-loop that are usually used for forensic
purposes, and identified the homologies to the primer annealing zones. We
observed that none of the primers used for amplifying the Hypervariable Regions
(HVRs) I and II in forensic studies (Wilson et al, 1995) anneals completely in
any NUMT. The highest homology was observed in three NUMTs (chromosomes 4 and
17), where the annealing sites of both forward and reverse HVRI primers present
one point substitution. Therefore, we concluded that an accidental amplification
of one of these NUMTs with the HVRI and HVRII primers is very unlikely to
occur. However, forensic and anthoropological studies have been focusing more
and more on the coding region, and much information is now obtained by using
SNaPshot multiplexes or sequencing of various fragments outside the D-loop. The
longest and most similar NUMT from Mishmar’s study, with 97% homology to the
region between 3914-9755np of the Cambridge Reference Sequence (CRS),
encompasses a target region for several analyses performed by forensic
researchers. This high homology enables 11 primers used in a SNaPshot multiplex
for mtDNA typing (Quintáns et al, 2004) to anneal perfectly to this NUMT. In
order to establish whether this is an issue that forensic investigators must take
into account when studying mtDNA, we will perform PCR with primers specifically
designed for the NUMT sequences that may be a source of accidental
amplifications and compare the results with what is obtained for mtDNA-targeted
primers. We will present results of this analysis made on samples with
different mtDNA content, such as blood, hair and buccal swabs.
Mishmar D, Ruíz-Pesini E, Brandon M, Wallace, DC (2004) Hum Mutat.
23:125-133
Quintáns B, Álvarez-Iglesias V, Salas A, Phillips C, Lareu MV, Carracedo
A (2004) Forensic Sci Int. 140:251-257
Wilson MR, DiZinno JA,
Polanskey D, Replogle J, Budowle B. (1995) Int J Legal Med. 108:68-74.
O-18
Genotyping
coding region mtDNA SNPs for Asian and Native American haplogroup assignation
Álvarez-Iglesias V, Salas A, Cerezo M, Ramos-Luis E,
Jaime JC, Lareu MV, Carracedo A
Unidad de
Genética Forense, Instituto de Medicina Legal, Universidade de Santiago de
Compostela, Galicia, Spain
Based on
phylogenetic criteria we selected 34 mitochondrial DNA (mtDNA) coding region
SNPs that allow to distinguish Asian and Native American mtDNA haplogroups. SNP
genotyping is carried out in a single multiplex reaction that involves a
20-amplicons PCR amplification, followed by a single minisequencing reaction
using SNaPshot (Applied Biosystems, Foster City, CA, USA). The
polymorphisms selected increase the discrimination power of the mtDNA
hypervariable regions (HVS-I/II) in populations. Consequently, these combined
SNPs are of particular interest in forensic casework, clinical and population
genetics research. Here we show preliminary results using a sample from
south-east Asia (Taiwan) and Native Americans from Argentina.
Contact: apimlase@usc.es
O-19
Haplogroup-level
coding region SNP analysis and subhaplogroup-level control region sequence
analysis for East Asian mtDNA haplogroup determination in Koreans
Hwan Young Lee1,
Ji-Eun Yoo1, Myung Jin Park1, Ukhee Chung1,
Kyoung-Jin Shin1,2, Chong-Youl Kim1,2
1Department of
Forensic Medicine, College of Medicine, Yonsei University, Seoul, Korea; 2Human
Identification Research Institute, Yonsei University, Seoul, Korea
We have established a high quality mtDNA control region
sequence database for 593 Koreans. Based on previously reported patterns of
shared haplogroup-specific or haplogroup-associated polymorphisms in control
region sequence, we also classified 592 Korean mtDNAs (99.8%) into various East
Asian haplogroups or subhaplogroups using the program mtDNAmanager (K-J Shin,
Yonsei University, unpublished). These haplogroup-directed database comparisons
and posteriori phylogenetic analysis confirmed the absence of major systematic
errors in our data. We collated the basic informative control region SNPs and
suggested the important mutation motifs for the assignment of East Asian
haplogroups. However, quite a few haplogroup-diagnostic SNPs are located in
mtDNA coding region, and in some haplogroups, scoring of coding region SNPs is
required for exact haplogroup determination due to the lack of informativity in
their control region sequences. Accordingly, we have selected 21 coding region
SNP markers and designed the 3 multiplex systems applying single base extension
methods. Using 2 multiplex systems, we allocated all 593 Korean mtDNAs into 15
haplogroups: M, D, G, D4, D5, M7, M8, M9, M10, M11, R, F, B, A and N9. Using
the other multiplex, we further determined D4 subhaplogroups; D4a, D4b D4e, D4g
and D4j, since D4 haplotypes occurred most frequently in Koreans. In this way,
we could complement coding region information to control region mutation motifs
and also confirm our control region mutagenic motifs for the assignment of East
Asian haplogroups. Moreover, these 3 multiplex systems are expected to work
well in degraded samples, since they have been designed to contain small PCR
products (101~163 bp) for SNaPshot reactions. Therefore, we performed SNP
scoring in 98 old skeletal remains using 3 multiplex and proved the utility of
these multiplex in degraded samples. The targeting and preferential
amplification of mtDNA control region using small amplicons and the selective
scoring of highly informative SNPs in coding region using the 3 multiplex
systems in this study is expected to represent a promising means for most
application involving East Asian mtDNA haplogroup determination and
haplogroup-directed stringent quality control even in degraded samples.
O-20
Dissection of mitochondrial
haplogroup H using coding region SNPs
Brandstätter A1,
Salas A2, Gassner C3, Carracedo A2, Parson W1
1Institute of Legal
Medicine, Innsbruck
Medical University,
6020 Innsbruck, Austria
2Instituto de Medicina
Legal, Facultad de Medicina, 15705 Santiago de Compostela, Spain
3Central Institute
for Blood Transfusion, General
Hospital and University
Clinics, Innsbruck, Austria
Analysis of single nucleotide
polymorphisms (SNPs) is a promising application in forensic human
identification. We selected 45 SNPs from the coding region of the human
mitochondrial DNA in order to ascribe samples belonging to mitochondrial
haplogroup H (hg-H) to one of the previously described sub-lineages of hg-H.
SNP selection was carried out using the available literature on population and
forensic genetics and extended by means of phylogenetic analysis of complete
genomes (>400) and control region profiles. The selected SNPs are amplified
in two PCR-multiplex reactions and subsequently targeted in three
multiplex-systems via the application of the SNaPshotTM kit. Samples
belonging to haplogroup H (approximately 40% of West-Eurasians) can in most
cases not be distinguished from each other based on control region
polymorphisms. By screening the selected coding region SNPs after sequencing of
the control region however, we would be able to rapidly differentiate between
stains or hairs in high volume case work or to eliminate multiple suspects from
an inquiry. The presented hg-H screening strategy was conceived as a
high-throughput method and the distribution of the selected SNPs and targeted
haplogroups was inferred from a huge population sample.
contact: Walther.Parson@uibk.ac.at
O-21
Analysis
of mtDNA mixtures from different fluids: an inter-laboratory study
Montesino M1, Salas A2,
Crespillo M3, Albarrán C4, Alonso A4,
Alvarez-Iglesias V2, Cano JA5, Carvalho M6,
Corach D7, Cruz C8, Di Lonardo9, Espinheira R8,
Farfán MJ10, Filippini S9, Garcia-Hirchfeld J4
, Hernández A11, Lima G12, López-Cubría CM5,
López-Soto M10, Pagano S13, Paredes M3 ,
Pinheiro MF12, Sala A7, Sóñora S13, Sumita DR14, Vide MC6, Whittle MR14, Zurita
A11, Prieto L1
1Comisaría General de Policía Científica, Spain 2Inst.
Medicina Legal Santiago de Compostela, Spain 3Instituto de
Toxicología y Ciencias Forenses de Barcelona, Spain 4Instituto de
Toxicología y Ciencias Forenses de Madrid, Spain. 5Dirección General
de la Guardia Civil, Spain. 6Delegação de Coimbra do Inst. Nacional
de Medicina Legal, Portugal 7Servicio de Huellas Digitales
Genéticas, U. Buenos Aires, Argentina 8Delegação de Lisboa do
Instituto Nacional de Medicina Legal, Portugal 9Banco Nacional de
Datos Genéticos, Buenos Aires, Argentina 10Inst. de Toxicología y
Ciencias Forenses de Sevilla, Spain 11Inst. Toxicología
y Ciencias Forenses de Canarias, Spain 12Delegação do Porto do
Instituto Nacional de Medicina Legal, Portugal. 13Dirección Nacional
de Policía Técnica, Uruguay 14Genomic Engenharia Molecular Ltda, São
Paulo, Brasil.
The analysis of mixed stains is a routine practice in
forensic casework, mainly related to sexual assault cases. These analyses are
commonly performed using differential lysis that allows the separation of
epithelial cells DNA from that at of spermatozoa, followed by nuclear STR
typing. In a number of cases, however, it could be interesting to know the
mitochondrial DNA (mtDNA) haplotypes that contributed to the mixture (e.g.
degraded or low-copy number reference samples, exclusion of a maternal
relationship between victim and suspect in rape cases, etc). In the last GEP-ISFG mtDNA proficiency
exercise (2003’04), the mtDNA analysis of a mixture stain (saliva from a female
plus 1:20 diluted semen)
yielded an unexpected consensus result: only the mtDNA hypervariable I and II saliva
haplotype was detected, in contrast to the predominant presence of the male autosomal STR profile. Hence, the use of only mtDNA
typing for this mixture sample could in this case lead to a false exclusion.
Several additional experiments carried out by some laboratories pointed to the
existence of different relative amounts of nuclear and mtDNA in saliva and
semen (Crespillo et al. 2005, in press). In order to disentangle this puzzle, the mtDNA GEP-ISFG
working group decided to carry out an inter-laboratory study.
We have studied mixtures from
three semen donors and three saliva/blood female donors. Three semen dilutions (pure, 1:10 and 1:20)
from each donor were mixed with saliva or alternatively, blood taken from each
female donor (see Table 1). No a priori
information was provided to the participating laboratories concerning either
the mitochondrial haplotypes of contributors or the dilutions of semen. Each
laboratory used their routine methodologies in order to carry out differential
lysis, cell count, nuclear or mtDNA quantification, PCR and sequencing. There
was a high consensus between labs for the epithelial fractions. In contrast,
results concerning the seminal fractions were more ambiguous. In addition, some
laboratories reported contamination problems in the male fraction. The most
plausible explanation to this finding is that, after differential lysis, female
and male mitochondria remain in the epithelial fraction and, theoretically, no
mtDNA should be found in the male fraction (assuming effective differential
lysis). Nevertheless, the first lysis is not always completely effective, so
that mtDNA is also detected in the seminal fraction. The detection level of the
male component decreased in accordance with the degree of semen dilutions,
although the loss of signal was not uniform throughout all the nucleotide
positions. There were clear differences between the mixtures prepared from
different donors and body fluids. In some cases
the male component was not detected. This may indicate that there are
differences in the number of mitochondria (or cellular content) contributed by
different donors and body fluids.
In conclusion, we
can tentatively say that special care should be taken when analysing mtDNA in
mixtures. There are several variables that we should bear in mind: the types of
body fluids involved in the mixture, the possibility of contamination mainly in
male fractions, the loss of signal in some nucleotide positions (but not in
others), and the fact that differences in cellular content between donors are
also possible. In addition, unlike the autosomal STR mixtures, the
interpretation of mtDNA mixtures can be supported by using a phylogenetic
approach.
contact: lourditasmt@ya.com
|
Female/male pair
number
|
Haplogroups
|
Female saliva / semen mixtures |
Female blood /
semen mixtures
|
|
1
|
Female T2
|
50 ml of saliva + 50 ml of pure semen
50 ml of saliva + 50 ml of semen 1:10
50 ml of saliva + 50 ml of semen 1:20
|
50 ml of blood + 50 ml of pure semen
50 ml of blood + 50 ml of semen 1:10
50 ml of blood + 50 ml of semen 1:20
|
|
Male H
|
|||
|
2
|
Female K
|
50 ml of saliva + 50 ml of pure semen
50 ml of saliva + 50 ml of semen 1:10
50 ml of saliva + 50 ml of semen 1:20
|
50 ml of blood + 50 ml of pure semen
50 ml of blood + 50 ml of semen 1:10
50 ml of blood + 50 ml of semen 1:20
|
|
Male H
|
|||
|
3
|
Female H
|
50 ml of saliva + 50 ml of pure semen
50 ml of saliva + 50 ml of semen 1:10
50 ml of saliva + 50 ml of semen 1:20
|
50 ml of blood + 50 ml of pure semen
50 ml of blood + 50 ml of semen 1:10
50 ml of blood + 50 ml of semen 1:20
|
|
Male J2
|
Table 1.
Composition of the mixture stains analysed in the inter-laboratory study.
O-22
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