Forensic Science International 154 (2005) 111–121

www.elsevier.com/locate/forsciint

A DNA microarray system for forensic SNP analysis

Anna-Maria Divne, Marie Allen*
Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory 75 185 Uppsala, Sweden

Received 13 April 2004; received in revised form 27 September 2004; accepted 28 September 2004
Available online 2 December 2004

Abstract

Forensic DNA analysis is routinely performed using polymorphic short tandem repeat (STR) markers. However, for
degraded or minute DNA samples, analysis of autosomal single nucleotide polymorphisms (SNPs) in short fragments might be
more successful. Furthermore, sequencing of mitochondrial DNA (mtDNA) is often performed on highly degraded or scarce
samples due to the high copy number of mtDNA in each cell. Due to the increasing number of complete mtDNA genome
sequences available, the limited discrimination power of an mtDNA analysis, may be increased by analysis of coding region
polymorphisms in addition to the non-coding variation. Since sequence analysis of the coding region would require more
material than generally present in forensic samples, an alternative SNP analysis approach is required. We have developed a one-
colour microarray-based SNP detection system for limited forensic materials. The method is based on minisequencing in
solution prior to hybridisation to universal tag-arrays. In a first outline of a forensic chip, a combination of 12 nuclear and 21
mitochondrial SNP markers are analysed simultaneously. The mitochondrial markers on the chip are polymorphisms within the
hypervariable region as well as in the coding region. Even though the number of markers in the current system is limited, it can
easily be extended to yield a greater power of discrimination. When fully developed, microarray analysis provides a promising
system for efficient sensitive SNP analysis of forensic samples in the future.
# 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Microarrays; Universal tag-arrays; Forensic; mtDNA; SNPs

1. Introduction discriminating and can be used for small amounts of DNA

DNA-based methods have greatly enhanced the ability to
match the genetic profiles of evidence and reference materials,
and are widely used in criminal investigations. Forensic DNA
analysis is traditionally performed using short tandem repeat
(STR) markers, due to the high number of length-variants at
each locus [1]. Routine analysis of STR markers is highly
Abbreviations: mtDNA, mitochondrial DNA; STR, short tan-
dem repeat; SNP, single nucleotide polymorphism; S/N, signal-to-
noise ratio; HVI, hypervariable region I; HVII, hypervariable
region II
* Corresponding author. Tel.: +46 18 471 48 03;
fax: +46 18 471 48 08.
E-mail address: marie.allen@genpat.uu.se (M. Allen).
using the LCN strategy [2]. However, the amplified fragments
of 100–400 base pairs can prove too large for successful
amplification on degraded DNA. For this type of samples,
analysis of single nucleotide polymorphisms (SNPs) utilising
very short amplicons can provide a more sensitive analysis.
Characterization of the human genome has revealed a large
number of nuclear SNPs, which might be useful as genetic
markers for forensic DNA analysis. In order to obtain highly
discriminating information, however, the biallelic nature of
these markers must be compensated for with a larger set of
markers. Theoretical calculations have shown that even a
relatively small array of about 50 biallelic SNP markers would
be as informative as 12 STRs [3].
Nevertheless, severely degraded or minute forensic DNA
samples may only be feasible using mitochondrial markers

0379-0738/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.forsciint.2004.09.134
112 A.-M. Divne, M. Allen / Forensic Science International 154 (2005) 111–121
[4–6]. The specific features of the mitochondrial genome,
such as high copy number per cell, high substitution rate and
uniparental inheritance make mitochondrial DNA (mtDNA)
suitable for forensic analysis and ancient DNA studies [7]. A
major drawback of using mtDNA for individual identifica-
tion is the low discrimination power of the analysis, which
generates a random match probability of about 1/200 for the
hypervariable regions HVI and HVII of the mitochondrial
genome [8–10]. However, discrimination may be increased
by analysis of highly polymorphic SNPs in the coding
region, in addition to HVI and HVII [11,12]. Sequencing
of mtDNA is, nevertheless, time consuming and labour
intensive, and complete coding region sequencing is not
suited for analysis of limited forensic samples. Thus, a rapid
SNP detection method with high throughput capability
would be more appropriate for coding region analysis of
forensic samples.
Methods that have been developed for analysis of nuclear
or mitochondrial SNPs include Pyrosequencing [11], melt-
ing curve analysis using DASH [13] or the LightCycler
platform [14], electrophoresis-based detection of primer
extension products such as SNaPshot [15–17] and the Taq-
Man genotyping system [18]. Moreover, microarray-based
DNA analysis has advanced rapidly in recent years, provid-
ing a powerful tool for fast and cost-effective detection of a
large number of SNPs simultaneously. Ongoing develop-
ment and evaluation of the microarray technology is there-
fore in progress in the forensic community [19,20]. SNPs
can be analysed either by direct target detection using primer
extension [21,22] or ligation [23] directly on the chip, or by
indirect detection using hybridisation of a ligated or
extended target sequence to a probe on the array [24–27].
In this study, we used a one-colour microarray-based
system, allowing detection of C and T polymorphisms. The
assay relies on minisequencing in solution prior to hybridi-
sation onto universal tag-arrays. To ensure high sensitivity,
the PCR system was designed to amplify very short frag-
ments. Some of the technical issues related to microarray
analysis of forensic samples were explored on a limited set
of SNP markers. The evaluated forensic chip contains 12
autosomal SNP markers and 21 mitochondrial polymorph-
isms in the hypervariable and coding regions. These markers
have been analysed in control samples, as well as non-
probative forensic casework samples. The post-PCR hand-
ling takes about 3.5 h and a large number of arrays can be
processed simultaneously.
2. Materials and methods
2.1. Extraction of DNA
DNA from 19 unrelated Swedish blood donors was
extracted using a salting out procedure. DNA from non-
probative forensic samples, collected on swabs was
extracted using the following components of the Wizard1
Genomic Purification System (Promega, Madison, WI)
according to manufacturer’s protocol. DNA from hair was
extracted in a total volume of 212 ml with a final concentra-
tion of 1
PCR Buffer II (Applied Biosystems), 33 mM
DTT and 0,24 mg/ml Proteinase K (SIGMA, St. Louis, MO).
Chelex extraction was used when blood samples were used
as reference material [28].
2.2. Markers and controls
In order to design a chip containing a set of highly
discriminating markers, 10 SNP markers with a frequency
ranging from 0.23 to 0.47 of the less common allele in
European populations were chosen (Table 1). The selection
was made from the HGVbase (http://hgvbase.cgb.ki.se) in
which the marker is represented as a nine digit SNP number
(i.e. marker 24 = SNP000000024). Marker 2629, corre-
sponding to SNP PD-1.5, was obtained from a separate
in-house study [29] and the sex-determining marker was
obtained from the literature [30]. Mitochondrial markers
were selected from the literature [8,10,31,32]. Genotypes for
the markers XY and 2629, as well as HVI/HVII sequence
data were available for most of the 19 control samples.
Genotypes for the autosomal markers selected from
HGVbase, and the mtDNA coding markers were available
from sequencing data for four of the samples. Sequence data
for the mtDNA HVI and HVII regions were available for all
forensic samples and coding region sequence data were
available for one casework sample.
2.3. Primer design
Six PCR primer pairs covering polymorphic sites in the
mtDNA coding region, and 12 primer pairs specific for
nuclear SNP containing regions were designed to yield
74–130 bp fragments, using Primer Express 3.1 (Applied
Biosystems, Foster City, CA) (Table 1). Each primer was
tagged with 50 -GCGTATAGCGTACCACGTG-30 at the 50 -
end. The tag sequence has been shown to stabilise the
primers and facilitate multiplex amplification [33]. Reverse
primers carry phosphorothioate (PTO) in the first five bases
at the 50 -end to protect the strand from degradation during
exonuclease treatment [34]. Primer pairs for amplification of
two longer fragments of the d-loop were untagged and
without PTO.
2.4. Oligonucleotides
Oligonucleotides were obtained as custom synthesis
products from ThermoHybaid, Germany. The 39-mer zip
codes contain 15 T residues as a spacer coupled to an
aminolinker C7 at the 30 -end. The 24-mer zip codes were
designed as per Gerry et al. [25]. In short, the zip code
sequences consist of six tetramers, each of which differs
from all others by at least two bases. All zip codes were
designed to have similar Tm-values. Each minisequencing
Table 1
PCR-primers, zip code sequences and minisequencing primers used for detection of the 33 mitochondrial and nuclear markers

A.-M. Divne, M. Allen / Forensic Science International 154 (2005) 111–121

Each PCR-primer pair except HVI and HVII carries the tag-sequence 50 -GCGTACTAGCGTACCACGTG-30 in the 50 -end. The HVI and HVII regions were amplified using one primer
pair for each region. The zip codes were designed according to Gerry et al. [25]. Each zip code contains a spacer of 15 T residues coupled to a C7 aminolinker in the 30 -end. The
minisequencing primer carries a zip-address complementary to the corresponding zip code in the 50 -end not shown in the table. The base I: Inosine in minisequencing primer mt16189.

113
114 A.-M. Divne, M. Allen / Forensic Science International 154 (2005) 111–121
primer covers 20 bases upstream of the SNP and contains a
tag-sequence (zip-address) complementary to a specific zip
code sequence at the 50 -end (Table 1).
2.5. PCR amplification
PCR amplifications of mitochondrial and nuclear DNA
were performed in 50 ml single or duplex reactions contain-
ing 0.8 mM dNTPs, 2.5 mM MgCl2, 1
PCR Taq Gold
buffer (Applied Biosystems), 1.25 U Taq polymerase
(Applied Biosystems), 0.3 mM forward and reverse primers
and 10–50 ng of DNA (1 ng of the forensic materials).
Thermal cycling was performed with an initial hot start at
95 8C for 10 min, followed by 35 cycles of denaturation at
94 8C for 30 s, annealing at 55 8C for 30 s, and extension at
72 8C for 30 s. Duplex reactions were run in the following
pairs: HVI and HVII, 4216 and 7028; 10463 and 16519;
12705 and 14766; 3412 and 2629; 8093 and 8075; 2397 and
24; 3288 and XY; 8101 and 5175; 155 and 8102. Amplified
products were treated with 0.27 U/ml T7 Gene 6 Exonu-
clease (USB Corporation, Cleveland, OH) in 1
T7 Gene 6
Exonuclease reaction buffer at 37 8C for 15 min, followed by
inactivation of the enzyme at 80 8C for 15 min. Single
stranded PCR reactions were pooled two and two, and
purified using the QIAquickTM PCR purification kit (Qiagen,
GmbH, Germany). All purified PCR fragments were pooled
into a mitochondrial and a separate nuclear reaction. The
reactions were precipitated and the residual pellets were
resuspended in elution buffer to yield 2.25 times concen-
trated products.
2.6. Multiplex minisequencing
Separate C and T minisequencing reactions were carried
out in 15 ml volumes containing 5 ml pooled, concentrated
single stranded PCR products, 0.25 mM ddATP, ddGTP,
0.25 mM of either ddCTP or ddTTP and 1.7 mM of the
fluorescently labelled TAMRA-ddCTP (NEL473, NEN Life
Science, Boston, MA) or TAMRA-ddUTP (NEL472, NEN
Life Science, Boston, MA), 0.5 mM of each minisequencing
primers, 1
Thermo Sequenase reaction buffer and 0.32 U/
ml Thermo Sequenase enzyme (Amersham Bioscience, Pis-
cataway, NJ). The cycling procedure was performed by
preheating at 95 8C for 1 min followed by 10 cycles at
94 8C for 15 s, 50 8C for 30 s, 68 8C for 1 min, 10 cycles
at 94 8C for 15 s, 45 8C for 30 s, 68 8C for 1 min, and 10
cycles at 94 8C for 15 s, 40 8C for 30 s, 68 8C for 1 min in a
9700 thermal cycler (Applied Biosystems).
2.7. Array preparation and blocking procedure
Two sets of 4
6 oligonucleotide arrays, with two
vertical replicates were printed on Silylated Slides
(ArraIt/Telechem, Sunnyvale, CA) with reactive aldehyde
groups from Arrayit Telechem using a GMS 417 Arrayer
(Affymetrix, Santa Clara, CA). The spot solution consisted
of 60 mM zip code oligonucleotide, 200 nM NaHCO3 pH 9.0
and 0.07% SDS and printed arrays were left for 12 h at room
temperature. Uncoupled oligonucleotides were removed and
unbound aldehyde residues were blocked by sequential
washing twice in 0.2% SDS for 2 min, ddH2O for 2 min,
0.5 g NaBH4 resuspended in 150 ml 1
PBS and 50 ml
99.5% ethanol for 5 min, 0.2% SDS for 2 min, followed
by ddH2O for 2 min. The slides were stored at 4 8C until
used. All arrays were spotted with 3 mM of a control
oligonucleotide with sequence 50 -aminolinkC6-Poly
(dT)35-GACT-TAMRA-30 at the upper left corner and the
lower right corner of the array. The TAMRA control was
used to indicate successful binding to the support.
2.8. Hybridisation of minisequencing products to
zip code arrays
Arrays were pre-incubated in 4
saline sodium citrate
(SSC) for 10 min. Two separate reaction chambers (15
15
or 9
9 Frame Seal Incubation Chambers, MJ Research,
Watertown, MA) were placed on the slides. The mitochon-
drial and nuclear minisequencing reactions were analysed
either on separate arrays or pooled on the same array. For
separate reactions, 10 ml of the C- or T-minisequencing
reaction were hybridised at 50 8C for 1–2 h in 4
SSC in
a rotisserie. Final volume was 50 ml or 30 ml depending on
the size of the incubation chamber. Slides were then washed
in 6
SSC/0.05% N-lauroyl sarcosine for 2
5 min, and in
0.2
SSC/0.1%SDS for 3
15 min at 50 8C on a shaker.
For pooled reactions, 9 ml of the nuclear and 15 ml of the
mitochondrial reaction were added followed by hybridisa-
tion and washing under the same conditions as above.
2.9. Image analysis
Positive signals on the arrays were detected using the
confocal four-colour laser scanner ScanArray1 5000 at
543.8 nm (Green HeNe), laser 80–96% and 80–90% photo-
multiplier tube (Packard Bioscience, Wellesly, MA). The
resulting images were analysed and quantified using the
QuantArray1 software 2.11. An adaptive quantitation method
with a p-value of 0.0001 was used and, in general, total
intensities rather than mean intensities were read. Genotype
determinations were based on a signal-to-noise ratio (S/N) of
2 for a true positive signal. In addition to the S/N value for
triplicate spots, spots of reagent blanks (spot solution and
water) and non-printed areas were quantified as controls for
background signal. Signals that showed S/N = 2, but were
lower than the control spots, were considered as failed calls.
3. Results and discussion
An overview of the microarray-based system used in this
study is shown in Fig. 1. The regions of interest are first
amplified by PCR in 20 singleplex or ten duplex reactions,
 A.-M. Divne, M. Allen / Forensic Science International 154 (2005) 111–121 115

Fig. 1. (A) The region of interest is amplified and used as template in the minisequencing reaction. Fluorescent-labelled ddNTPs are
incorporated at the SNP site in the 30 -end of the minisequencing primer. The zip-addresses are then hybridised to their complementary zip code
on the array. (B) In each reaction chamber, two sets of zip code-arrays (mt and nuclear) are spotted in triplicates below each other. A TAMRA-
labelled control oligonucleotide was placed in the upper left and lower right corner (black spots). Grey spots show where zip codes are placed
while white spots contain reagent and water controls.

and the 33 nuclear and mitochondrial SNPs are detected by
separate C and T minisequencing reactions performed in
one 12-plex reaction (nuclear markers) and one 21-plex
reaction (mitochondrial markers). In the minisequencing
reaction, primers are extended with TAMRA-labelled
dideoxynucleotides at the polymorphic site. The minise-
quencing primer has a region complementary to the
sequence located 20 base pairs adjacent to the SNP, and
one tag-sequence (zip-address) complementary to the oli-
gonucleotide (zip code) printed on the chip. The zip-address
oligonucleotides are hybridised to their complementary zip
code on the array, and positive signals are detected by a
confocal laser-scanner.
3.1. Developmental testing
Initial comparison of on-chip extension and hybridisation
to zip code arrays using a small subset of mt-markers in a
two-colour system (ddCTP-TAMRA, ddUTP-Cy5) was per-
formed. Although the on-chip extension strategy would save
time, it was not as robust as the hybridisation strategy in our
experiments. An evaluation of ddUTP coupled to Cy5,
Fluorescein, FAM and TAMRA was made as unequal signal
intensities were observed between incorporation of ddCTP
and ddUTP. Experiments showed that a one-colour system
using TAMRA in separate C and T reactions revealed the
most sensitive and reliable detection, and was therefore used
in subsequent development of the system. To further
enhance the signal intensities on the array, the binding
capacity of the solid support was evaluated. Oligonucleotide
in concentrations of 20–120 mM (20 mM increments) for
three nuclear markers was analysed. A 1.5–2-fold increase in
signal intensity for each increment up to 60 mM was
detected, with only minor increases thereafter. Therefore,
60 mM was used as standard concentration.
3.2. mtDNA basecalling and nuclear genotype
determination
The signal intensities varied up to 20-fold between
different markers on the same chip. Variations were also
observed between the two TAMRA-labelled reactions. The
signal intensities from incorporation of the TAMRA-
labelled C nucleotide were generally higher, and the back-
ground signal lower than from the TAMRA-T nucleotide.
Basecalling of the mitochondrial markers was in general
straightforward due to their haploid state. In evaluation of
the nuclear markers, the homozygous genotypes CC and TT
were in most cases unambiguously interpreted, with low
signals from the background channel. Signal-to-noise ratios
for heterozygous genotypes in general displayed a one to
three-fold (1:1–1:3) difference between the C and T channel,
whereas homozygous genotypes showed at least a four-fold
difference. Fig. 2 shows the S/N obtained in a combined
detection of nuclear and mt-markers in two individuals.
Occasionally the S/N for the background channel exceeded
the threshold of 2 for known homozygous genotypes, while a
four- to five-fold difference in S/N between the two channels
were seen (e.g. marker 24 in Fig. 2). A signal above the
threshold from the background channel was also observed
for some of the mt-markers e.g. marker 195 in individual 1
and marker mt12705 in individual 2 (Fig. 2). In these cases,
116 A.-M. Divne, M. Allen / Forensic Science International 154 (2005) 111–121

Fig. 2. Combined detection of mitochondrial and nuclear markers in two individuals. (A) Superimposed scan images from the C (red) and T
(green) reactions. In each picture, the mt-markers are located on the left side (array 1) and the nuclear markers on the right (array 2). Signals from
spots that are located on the same place in the superimposed picture appear in yellow. The yellow spots in the upper left and lower right corner of
each array represent a TAMRA-labelled control oligonucleotide (spots 1:1:1, 1:4:6, 2:1:1 and 2:3:6). (B) Signal-to-noise ratios (S/N) of
background subtracted signal intensities for each marker and channel, and the ratio between the C and T-channels. S/N of 2 was generally
accepted as a threshold for a true positive signal. For the nuclear markers, ratios between channels of 1:1–1:3 indicate heterozygous genotypes.
The value zero resulting from negative intensities is treated as 1 for the S/N ratio between channels. Due to its location on the X-chromosome, the
marker 24 is haploid for male sexes. Base callings and genotypes that differ between the two samples are shown in bold.

the channel showing the highest intensity agreed with
sequencing data, where available.
3.3. Nuclear markers
A total of 19 individuals were analysed for the 12 nuclear
markers. Of these, nine samples were performed in dupli-
cate, as well as combined mtDNA and nDNA experiments.
Of all nuclear genotype calls, 320 of 336 (95%) were
successfully determined (duplicate samples included). Most
of the failed calls were attributed to the markers 2397, 8101
and 8102, which generally generated low signal intensities.
Genotypes for the 10 HGVbase selected markers were
known for four individuals (three included in the duplicate
 A.-M. Divne, M. Allen / Forensic Science International 154 (2005) 111–121 117

Table 2
Duplicate analysis of a sample showing the variation in signal intensities between markers on the array and variation between repeated
experiments
Marker C bkg corr T bkg corr S/N C S/N T C bkg corr T bkg corr S/N C S/N T Genotype
24 944829 106067 15 1 2571173 3529 11 2 CC
155 483057 502242 7 4 856250 621053 7 5 CT
2397 172989 240478 3 2 231526 114734 3 3 CT
2629 326131 70911 8 0 964335 126424 5 0 CC
3288 472346 340572 6 3 693971 312111 7 4 CT
3412 17665 239301 0 2 81375 286349 1 3 TT
5175 392495 93309 4 0 422391 186981 6 0 CC
8075 708306 396843 9 3 1090550 445394 9 4 CT
8093 332760 330653 4 2 367949 198382 5 4 CT
8101 204727 83267 3 0 254179 177919 4 0 CC
8102 206397 100607 3 0 208521 141922 4 0 CC
XY 430220 113229 8 0 1214832 35289 6 0 CC
Background subtracted signal intensities (C bkg corr and T bkg corr) and S/N ratios for each marker and channel, are shown for repeated
experiments of the same sample. S/N = 2 is generally accepted as a threshold for a true positive signal. The value zero results from negative
intensities. A linear regression of the S/N from the experiments resulted in R2 = 0.84.

experiment) and the status of marker XY and 2629 were
known for 19 and 17 individuals, respectively. Thus, a total
of 124 genotypes were known. Of the successful calls for
which the genotypes were known, 117 of 119 (98%) showed
correct genotypes. The two incorrect calls were observed for
marker XY in the first duplicate experiment and for marker
2629 in the second.
Duplicate experiments of nine control samples showed
concordant genotypes in 93% of the 216 calls. Four calls
showed disagreeing genotypes, and 12 calls failed in the first
or second duplicate experiment. Table 2 shows an example
of the variation in signal intensities between repeated experi-
ments for one individual. Of the 10 autosomal markers
selected from HGVbase, six showed allele frequencies
between 0.37 and 0.5 for the less abundant variant (155,
2397, 2629, 3288, 8093 and 8102) among the 19 individuals
in this study. The other four markers displayed allele fre-
quencies 0.25 for the less common allele.
3.4. mtDNA markers
Polymorphisms are often clustered in the mitochon-
drial non-coding region, which can destabilise the priming
event resulting in reduction or complete loss of signal [21].
The marker mt16189 failed in most (93%) experiments.
Moreover, incorrect basecalling or signals from both chan-
nels were seen in 7% of the basecallings for marker mt195
when a T was expected. The minisequencing primer for
these two markers covers a region in which several poly-
morphisms are located. Signals from both channels were
observed occasionally for markers mt7028 and mt12705.
Although the difference in the signals from both channels
was at least four-fold, contamination, multi-contributor
samples and heteroplasmy should be considered. However,
sequence analysis could not confirm heteroplasmy or
sample mixtures in these samples. The marker mt146
had a generally low signal resulting in a poor typing success
rate (25%).
A total of 19 individuals were analysed for the 21
mitochondrial markers, with nine samples analysed in dupli-
cates. Fig. 3 shows an example of mtDNA analysis in four
individuals. Additional discrimination between the indivi-
duals is seen for the markers mt4216, mt10463 and mt16519
outside the HVI–HVII region. Of the total number of base-
callings (duplicate experiment included), 129 of 588 (22%)
could not be determined. Of these, 54 failed calls were due to
template and primer binding site mismatches, based on
previous sequence data, and the remaining 71 calls failed
due to low PCR yield or unknown causes. Excluding the
failed calls due to mismatches in the primer binding site and
calls for the mt146 and mt16189 markers, the estimated
failure rate was 4% (19 of 478).
A total of 532 calls had known SNP status from HVI/
HVII sequence data for a majority of the controls, and
coding sequence data for a few of the controls. Of the
successfully called SNPs to which controls were available,
402 of 411 (98%) showed concordant results. Of the incor-
rect calls, marker mt195 accounted for four of 411 (1%),
while the other five showed equally high signals from both
channels (markers mt195 and mt12705). The reproducibility
in the duplicate experiment was 94% (342 calls), excluding
markers mt146 and mt16189. Three calls were disagreeing,
three calls showed signals from both channels (markers
mt195, mt7028 and mt12705), and 14 calls failed in the
first or second duplicate experiment.
Of the coding markers on the array, the marker mt4216 is
the only polymorphism listed in the MITOMAP [35] data-
base that might be associated with disease. The variant
4216 C is considered a secondary LHON (Leber’s hereditary
optic neuropathy) mutation that, in combination with pri-
mary LHON mutations, may contribute to LHON expres-
sion. Primary LHON mutations are preferentially clustered
118 A.-M. Divne, M. Allen / Forensic Science International 154 (2005) 111–121

Fig. 3. The analysis of mtDNA markers in four different individuals illustrates additional discrimination from markers outside the control
region. (A) Superimposed scan images. (B) Base callings made from the four samples. The yellow spots in the upper left and lower right corner of
each array represent a TAMRA-labelled control oligonucleotide (spots 1:1 and 4:6 in each array). The polymorphisms 16186T and 16189C
(previous sequence data) in individual 1 cause a mismatch at the minisequencing primer-binding site resulting in signal loss. CRS: Cambridge
reference sequence. nd: not determined, S/N < 2; nd*: not determined, S/N < 2 due to mismatch at the primer-binding site.

in mtDNA haplogroup J, which would act as a susceptibility
background for LHON expression [36]. The mtDNA mar-
kers on this chip however cannot be used for identification of
individuals belonging to haplogroup J, and neither are any of
the primary LHON mutations detectable on the array.
3.5. Casework examples
The one-colour system was tested on non-probative
samples in a maternity investigation, and two previously
analysed forensic cases. The evidence materials, consisting
of hair and skin debris samples, were analysed using the
mitochondrial part of the microarray as mtDNA sequence
data are available.
3.5.1. Case 1
In a robbery of a post office in Sweden, a pair of textile
gloves was found as evidence during the crime scene
investigation. DNA from the inside of the glove and a hair
sample from a suspect were analysed using 19 mtDNA
markers (mt195 and mt16189 were excluded). One marker
failed in the analysis of the evidence sample and four
markers failed for the reference sample. Comparison of
the successfully scored markers revealed five mtDNA dif-
ferences between the samples of which three were observed
in the coding region (mt7028, mt12705, mt14766). Sequen-
cing data for the HVI and HVII regions was concordant with
the microarray results.
3.5.2. Case 2
A woman claimed to be a previously unknown daughter
of the famous Swedish actress Greta Garbo, who died in the
early 1990s. According to the woman, Garbo had given her
up for adoption soon after her birth. While most such claims
can be easily dismissed, the woman’s birth did coincide with
an eight-month stay by the actress in Sweden. As reference
material hair from the adoptive mother, and from a known
biological daughter of the adoptive mother were used. These
samples were analysed and compared with a blood sample
from the woman claiming to be Garbo’s daughter (coding
markers were not analysed). The two reference hairs and the
blood sample showed identical mtDNA results for the d-loop
markers indicating that the three women are maternally
related (mtDNA type: 152C, 195C, 16126C, 16186T and
 A.-M. Divne, M. Allen / Forensic Science International 154 (2005) 111–121 119

Table 3
Results for the mtDNA markers obtained from the evidence material and the four suspects in case 3
SNP name Evidence Suspect 1 Suspect 2 Suspect 3 Suspect 4 CRS
mt146 nd T C T T T
mt150 C T C C C C
mt152 T nd* C T T T
mt295 C C C C C C
mt4216 C T T T C T
mt7028 T T T C T C
mt10463 C T T T C C
mt12705 C C C C C C
mt14766 T T T C T T
mt16126 C T T T C T
mt16186 nd* C C C nd* C
mt16192 nd* nd* C C nd* C
mt16223 C C C nd C C
mt16224 T T C T T T
mt16256 C C C C C C
mt16270 C T nd nd C C
mt16294 T C C C T C
mt16311 T T C T T T
mt16519 C T C C C T
No. of diff. 7 7 6 0
The Cambridge reference sequence (CRS) is shown in the outer right column. Marker mt16189 and mt195 were excluded from the experiment
due to previous experience of low success rate and ambiguous typing results. No. of diff. denotes the number of nucleotide differences between a
suspect and the evidence. nd: not determined base call due to S/N < 2; nd*: not determined base call due to S/N < 2 due to mismatches at the
primer-binding site based on previous sequence data.

16311T). Although impossible to prove without a sample
from the actress, the results are arguing against adoption and
the claimant being Garbo’s daughter. The results were in
agreement with previous mtDNA sequence analysis.
3.5.3. Case 3
Cell material in a pair of gloves found in an escape car
was one of several evidence materials in a bank robbery
investigation. Four suspects were investigated for possible
involvement in the crime. DNA from blood samples from the
suspects was compared with DNA from skin debris found
inside the gloves using the mtDNA markers on the chip.
The analysis showed a match between suspect 4 and the
evidence material, while the other three suspects were
excluded as donors by multiple SNPs (Table 3). Previous
mtDNA sequence analysis of the samples revealed identical
results.
3.6. Addressing accuracy and ambiguity
The accuracy was 98% for both the mt-markers and the
nuclear markers, and reproducibility was 94% for the mt-
markers (excluding 146 and 16189) and 93% for the nuclear
markers. Although these rates are comparable with a similar
study of 76 SNPs [26], they are unacceptable in forensic
analysis and replacement or redesign of several of the
markers is therefore necessary. Twenty-two of the 33 mar-
kers could be unambiguously determined in all analysed
samples, consequently approximately one third of the mar-
kers require further development. Markers mt195, 2629 and
XY need to be replaced due to inaccurate results, while
markers mt146 and mt16189 failed in most experiments.
Markers 8102, 8101 and 2397 displayed low signal inten-
sities, while markers 24, mt7028 and mt12705 displayed
very high signal intensities, hindering unambiguous inter-
pretation. In addition, the markers 8101 and 8102 are closely
located. When this study was initiated, the exact location of
these markers was unknown, but recent data shows that they
are separated by approximately 20 kb.
A major advantage of using universal tag-arrays is that
the system is flexible and easy to reorganize, since marker
redesign or replacement requires only a new minisequencing
primer. This system was developed to detect SNPs of C and
T variants alone. While less complex, a disadvantage is a
reduction in the number of informative SNPs that can be
used, especially in the mitochondrial genome. Moreover, the
minisequencing primer cannot be redesigned for detection
on the opposite strand, a strategy which might reduce the
interpretation difficulties for a marker [26]. Upon redesign of
the nuclear typing system, other informative markers can be
selected. However, for the mitochondrial markers, especially
those in the hypervariable region where primer-template
mismatches can affect the results, alternative strategies
are desired. To reduce signal loss for the marker mt16189
due to mismatches at 16186, Inosine was synthesized in this
position in the minisequencing primer. Despite this, detec-
tion of the mt16189 marker failed in most experiments. A
similar approach, in which the synthesis of the mini-sequen-
120 A.-M. Divne, M. Allen / Forensic Science International 154 (2005) 111–121
cing primers includes degenerate positions where poly-
morphisms are expected to occur, will be evaluated as an
alternative.
3.7. SNP markers in forensic analysis
When the autosomal markers were selected for this study,
only a limited number of SNP markers were available in
public databases, whereas today this number exceeds
1,000,000. This resource is now attracting interest as a
source of polymorphic markers for the forensic community,
particularly for analysis of degraded samples. An overview
of autosomal, Y-chromosome and mitochondrial SNP
makers identified and used in forensic analysis will be
available at the STRbase website (http://www.cstl.nist.gov/
biotech/strbase) (personal communication, J.M. Butler).
For selection of coding mitochondrial polymorphisms,
close to 1000 complete genomes are now sequenced
[32,37,38]. In a collection of over 700 genomes from
different populations, more than 250 SNPs in the coding
region have a frequency higher than 0.01. Many of these sites
may be useful in forensic mtDNA identification, especially
for resolving the most common HVI/HVII types (http://
www.genpat.uu.se/mtDB). In a recent study, 59 mtDNA
SNPs were identified as useful markers for additional dis-
crimination in forensic analysis, illustrating the demand for
SNP typing technologies with high parallel analysis cap-
ability [37]. In fact, several assays for coding region analysis
based on Pyrosequencing or SNaPshot have already been
developed [11,15–17].
3.8. Final remarks
This is the first outline of a one-colour microarray system
using 12-plex nDNA and 21-plex mtDNA minisequencing-
based analysis of SNPs for forensic identification. Due to its
limited number of markers, the system does not represent a
fully developed array with an optimal set of markers for
forensic analysis, and future expansion is required to obtain a
higher discriminating power. The system allows detection of
both mitochondrial and nuclear markers on the same micro-
array, but the mtDNA and nuclear analysis should be sepa-
rated during extraction and amplification procedures to
minimise contamination. In this study, all tests were per-
formed on at least 1 ng of input DNA, and further tests on
samples with lower amounts are necessary for both the
nuclear and mitochondrial part of the chip to fully evaluate
the sensitivity and robustness of the system.
A crucial factor to minimise the consumption of evidence
material, but also to save time and labour is the multiplexing
capacity of the PCR reactions. Even if recent improvements
have reduced the number of amplifications in this system to
six in total, a single nDNA and a single mtDNA multiplex
needs to be developed when the final collection of markers is
set. Despite the technical difficulties, successful amplifica-
tions combining up to 28 different fragments in a single
reaction have been described [27]. Furthermore, interpreta-
tion of DNA mixtures and occurrence of allelic dropout in
the nuclear SNP analysis has to be evaluated [3]. Finally,
although a major redesign of this 33-marker array is
required, addition and replacement of markers can offer a
promising system for efficient sensitive SNP analysis of
forensic samples in the future.
Acknowledgement
This work was supported by grants from Kjell and Märta
Beijers Foundation and the Swedish Research Council for
Medical Sciences (VR Grants K2002-31P-12577-05C and
K2002-31X-13095-04B).
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