Review

Forensic DNA fingerprinting by

liquid chromatography-electrospray
ionization mass spectrometry
Herbert Oberacher and Walther Parson
BioTechniques 43:Svii-Sxiii (October 2007)
doi 10.2144/000112581

DNA PROFILING: STATE OF

THE ART
Since its development in the mid1980s (1), advancements in forensic
DNA analysis were characterized by a
continuous increase of sensitivity and
discrimination power. This process
was paralleled by a size reduction of
the investigated DNA fragments in
order to comply with the requirement
to type severely degraded DNA in
challenging forensic samples (2). The
most powerful method that combines
the aforementioned requirements
is the analysis of autosomal short
tandem repeats (STRs). Forensic
relevant STRs are DNA segments
that are typically found in noncoding
regions of the human genome and are
composed of repeating units of tri- to
pentanucleotide sequence motifs. The
elevated mutation rate of STRs has
led to a high degree of polymorphism
in humans, which renders STR typing
powerful for identity testing (3). Their
relative small amplicon size (100400
bp) and their amenability to multiplex
analysis have rendered STRs the most
important forensic DNA markers.
The establishment of a laboratoryindependent nomenclature (4) allowed
for the direct comparison of typing
results across borders and set the basis
for the foundation of national DNA
databases that are successfully used as
intelligence tools by executive forces.
Nowadays, national DNA databases

contain millions of STR fingerprints

that effectively help to link an unknown
stain to the perpetrator. The STR
fingerprint that contains the evidential
information consists of the combined
genotyping information obtained from
a selected number of well-characterized
and validated STR loci. Harmonization
of technology and of STR markers
has thoroughly been pursued by international forensic scientific working
groups such as the European DNA
Profiling Group (EDNAP; www.
isfg.org/ednap/ednap.htm) and the
European Network of Forensic
Science Institutes (ENFSI) DNA
Working Group (www.enfsi.org/ewg/
dnawg). This has led to the selection
of core loci that were adapted by the
forensic community and constitute
the basic configuration of the national
DNA databases. The International
Standard Set of Loci (ISSOL) that is
recommended by the Interpol DNA
Monitoring Expert Group (www.
interpol.int/Public/Forensic/DNA/
DNAMEG.asp) involves the STR loci
vWA, TH01, D21S11, FGA, D8S1179,
D18S51, and D3S1358. Depending on
the typing chemistry that is used by the
laboratory, the following STR loci add
to the standard set: D2S1338, D19S433,
D16S539, D7S820, D13S317, D5S818,
CSF1PO, Penta D, Penta E, TPOX,
and SE33 (www.interpol.int/Public/
Forensic/dna/HandbookPublic.pdf). In
a recent attempt to extend the discrimination power of STR profiles for

samples containing heavily degraded

DNA, so-called miniSTRs have been
evaluated and suggested as additional
loci (D2S441, D10S124, D22S1045)
(5).
STR typing is usually accomplished
via selective amplification using PCR
and consecutive electrophoretic sizing
of the amplified fragments. Their size
is determined via the comparison of
observed migration times to those of
size standards. The individual alleles are
denoted by comparing their migration
times to those of the allelic ladder, a
selection of sequenced allele variants
that need to be co-analyzed with the
samples in question. So far, capillary
electrophoresis (CE) with multicolor
fluorescence detection represents the
method of choice for STR typing, as it
offers 1-bp resolution for the discrimination of all allelic length variants
within an STR fingerprint. Length
variants have been rigorously studied in
all world populations. This serves as the
basis for the calculation of combined
allele frequencies that are used to
determine statistical values to support
the weight of evidence (e.g., www.
strbase.org). STR amplicons, however,
may contain more discriminative information than just the fragment length.
This has been shown for selected STR
alleles by direct sequencing analysis
(69) and the identification of small
haplotype blocks in which SNPs are
tightly linked to the STRs (1012).
Based on sequencing experiments,

Institute of Legal Medicine, Innsbruck Medical University, Innsbruck, Austria

Supplement to Vol. 43 No. 4 | 2007

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Review

Figure 1. Analytical system suitable for nucleic acid analysis by ion-pair reversed-phase liquid chromatography to electrospray ionization mass spectrometry (ICEMS). 1, Low pressure binary gradient micropump; 2, solvent reservoirs; 3, degasser unit; 4, splitting tee-piece; 5, restriction capillary; 6,
autosampler; 7, injector; 8, needle; 9, dispensor; 10, column thermostat; 11, monolithic capillary column; 12, quadrupole/quadrupole/time-of-flight mass spectrometer; 13, electrospray ionization (ESI) source; 14, gas inlet; 15, ion optics; 16; vacuum system; 17, collision cell; 18, pusher; 19, reflectron; 20, detector.

STRs were classified as simple (repeats

that contain only units of identical
length and sequence), compound
(repeats that comprise two or more
adjacent simple repeats), and complex
(repeats that contain several repeat
blocks of variable unit lengths along
with more or less variable intervening
sequences), which indicates that there is
additional sequence variability in STRs
that would allow for discrimination of
fragments with identical length. There
is no doubt that sequencing of STRs
would not substitute the established
fragment-length analysis for multiple
reasons, including hands-on time and
cost. However, a method that is capable
of discriminating sequence differences
in STR amplicons would be beneficial
for a number of forensic applications.
While autosomal STR analysis
remains the gold standard in human
identification, other technologies and
markers have been developed to further
assist forensic investigations. This
involves the analysis of Y-chromosomal
STRs that are technically identical to
autosomal STR analysis but beneficial
in cases in which the male lineage
of a sample is relevant or where the
background of female DNA (victim)
is present in excessive amounts. The
analysis of single nucleotide polymorphisms (SNPs) is another niche in
forensic genetics. The short fragment
viii BioTechniques www.biotechniques.com

lengths involved in SNP analyses seem

promising, however SNPs are unlikely
to replace autosomal STRs as core DNA
markers, as there are technological
difficulties for the analysis in highmultiplex format that would be required
to achieve the necessary discrimination power. Further, their analysis
is currently not as sensitive compared
with STR typing, they are difficult to
interpret in mixture samples (that are
often found in casework analysis),
and finally, the established national
DNA intelligence databases that are
composed of STRs have a retarding
effect on the development of new
markers. In individual cases however,
the advantages of SNPs are well appreciated, such as in identification cases
where bone and teeth samples are to be
analyzed. Also, the haploid genomes
that play a role in forensic genetics are
investigated by SNPs (i.e., the aforementioned Y-chromosome and the
mitochondrial DNA or mtDNA). While
Y-SNP analysis is complementing
Y-STR typing and currently most often
performed with single base extension
(SBE) technology (13,14), the analysis
of mtDNA is using the SNP information per se to investigate forensic
samples by direct sequencing and SBE
typing (15,16).

LIQUID CHROMATOGRAPHYELECTROSPRAY IONIZATION

MASS SPECTROMETRY
General Principle
The first attempts to apply the
analytical method originated from
the online hyphenation of ion-pair
reversed-phase liquid chromatography to electrospray ionization mass
spectrometry (ICEMS) for nucleic
acids research date back to the 1990s
(1721). Applications focused on the
quality check of synthetic oligonucleotides (22,23), the identification of
metabolites of therapeutic oligonucleotides (18,2426), and the characterization of covalently modified DNA
molecules (2730). The first attempts
to use ICEMS for genotyping were
based on the analysis of short singlestranded oligonucleotide sequences
(8-mers) derived from amplified
genomic DNA via enzymatic digestion
(31). The first two articles demonstrating the usability of ICEMS for
the characterization of sequence variations directly from amplified genomic
DNA samples were published in 2001
(32,33).
The instrumental setup required for
the characterization of nucleic acids
by ICEMS is depicted in Figure 1.
The analytical system consists of two
Supplement to Vol. 43 No. 4 | 2007

Review
major parts: (i) the chromatographic
part and (ii) the mass spectrometric
part. Electrospray ionization (ESI) in
negative ion mode is used to hyphenate
the two methods and to transfer the
charged nucleic acids from solution
into the gas phase. The analytically
useful information is solely gathered
from the mass spectrometric part.
Either the molecular mass of an intact
molecule or the masses of fragment
ions obtained from a tandem mass
spectrometric experiment give a deep
insight into the composition and/or
the sequence of a nucleic acid species.
In general, sequence variations are
detected with high sensitivity by
ICEMS (34). While other indicators
like retention or migration times are
influenced by experimental conditions, the molecular mass is an intrinsic
property that is independent from the
physical environment.
The success of the mass spectrometric analysis largely depends on the
purity of the nucleic acid molecules
introduced into the mass spectrometer.
Due to partial substitution of protons
in the sugar-phosphate backbone by
variable numbers of cations, such as
sodium, potassium, or magnesium,
the multiply deprotonated molecules
are dispersed among several different
species, resulting in highly complex
spectra and poor detection limits.
Chromatography represents one of the
most efficient methods to purify and
desalt nucleic acids prior to their mass
spectrometric characterization (35).
The advantageous feature of ESI
the transfer of intact, doublestranded PCR products from solution
into the gas phaseis disadvantageous
in the context of genotyping. Base
substitutions are difficult to identify in
double-helical DNA by molecular mass
measurements, because A/T and G/C
base pairs have very similar average
masses of 615.4 atomic mass units
(amu) and 616.4 amu, respectively, and
A/T or G/C substitutions do not cause
any mass shift at all. Denaturation of
double-stranded PCR products into
the corresponding single strands prior
to their mass spectrometric characterization is advantageous because
of the division in half of the masses
of the detected species and the possibility to identify base substitutions by
Supplement to Vol. 43 No. 4 | 2007

measuring mass differences ranging

in size between 9.01 and 40.02 amu.
If required, denaturation can be easily
accomplished during the chromatographic run by using elevated column
temperatures (6575C).
Another reason for using chromatography as a sample preparation
method is its ability to fractionate
nucleic acid mixtures enabling the
thorough mass spectrometric characterization of samples that would have been
too complex for direct analysis. One
parameter that influences the chromatographic efficiency is the applied column
material. Highest performance is
obtained by using rods filled either with
octadecylated silica particles (36,37),
octadecylated polystyrene/divinylbenzene particles (38), or monolithic
polystyrene/divinylbenzene (30,39).
The second important chromatographic parameter that has an impact
on separation efficiency is the solvent
composition. Several different amines
were suggested as solvent additives
(4042). The use of butyldimethylamine or cyclohexyldimethylamine
instead of the conventionally applied
triethylamine has been proposed as
a consequence of the favorable mass
spectrometric performance obtained
with both amines (33,43). Solvents
containing 100 mM of the ion-pair
reagent are best suited for efficient
chromatographic separation of nucleic
acids. Such solvents, however, are
inappropriate for ESI-MS, as ionization
of nucleic acids is impaired by the high
ionic strength of the eluent (44,45).
Thus, the concentration of the ion-pair
reagent is usually reduced to 1025
mM, which results in a moderate loss of
chromatographic resolving power, but
improves mass spectrometric detection
limits considerably (20). Besides the
ionic strength, the amount of organic
solvent within the eluent was also
claimed to have some impact on mass
spectrometric performance. The postcolumn addition of sheath liquid (e.g.,
acetonitrile) was suggested to increase
the detection efficiency. In an early
study, a greater than 7-fold increase
in signal intensity was reported due to
the use of sheath liquid (46). In a more
recent study, however, it was demonstrated that the addition of sheath
liquid had only a moderate impact on

mass spectrometric performance (47).

Thus, on modern instruments, the use
of sheath liquid is no longer recommended.
Proper tuning of the mass
spectrometer can have an influence
on the detectability of nucleic acids
(4749). Depending on the length of
the analyzed molecule, the settings of
certain instrumental parameters, such as
sprayer position or lens voltages, need
to be carefully adopted to reach lowest
limits of detection. On an optimized
mass spectrometric system, interpretable mass spectra can be obtained from
oligonucleotide solutions with concentrations in the low amol/ L range.
The mass analyzer represents the
core part of any mass spectrometer.
Its performance in regard to mass
accuracy and resolution restricts the
maximum allowable molecular mass
and therefore the length of nucleic
acids for the unequivocal detection of
base exchanges (34,5052). Ion trap
(IT) and time-of-flight (TOF) mass
analyzers are the two most commonly
applied types of instrument. Typically,
a high-priced TOF instrument offers
a considerable better mass spectrometric performance than a more
inexpensive IT instrument. Whereas
on a TOF instrument any single base
exchange can be detected in nucleic
acids with lengths up to approximately
250 nucleotides, molecules must not
be larger than 6070 nucleotides for
unequivocally genotyping with an IT.
Thus, the TOF analyzer represents the
more appropriate tool for ICEMS than
the IT.
Workflow
In Figure 2, the principle workflow
used for genotyping genomic DNA
samples by ICEMS is shown. The
procedure starts with the sample fabrication. PCR is used to amplify one or
more genomic regions of interest. In
contrast to many other genotyping
methods (53), sample preparation is
limited to a single molecular biological
reaction that needs to be optimized.
Thus, even the development of a multiplexed PCR assay represents an easy
to accomplish task. Recently, guidelines for selecting ICEMS-compatible
PCR reagents were published (54).
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Review
which have a retarding effect on new
developments.
Application of ICEMS for SNP
Typing

Figure 2. The steps involved in the analysis of nucleic acids by ion-pair reversed-phase liquid chromatography to electrospray ionization mass spectrometry (ICEMS).

An aliquot of the crude PCR solution

(0.51.0 L) is analyzed by ICEMS.
Optionally, prior to analysis, reagents
that bind to small inorganic ions (e.g.,
EDTA) can be added to the sample
solution to improve the desalting
efficiency (35,55). For the detection
of nucleotide variability, the doublestranded PCR products are completely
denatured into the corresponding single
strands by setting the column temperature to 6575C. The molecular
masses of the single-stranded species
are determined and compared with the
theoretical molecular masses calculated from the reference sequence
(the putative sequence of the PCR
product). A mass difference larger
than the typically observed error of
measurement indicates the presence
of a sequence variation. The type of
sequence variability can be deduced
from the magnitude of the detected
mass difference. It is important to
note here that the exact location of the
sequence alteration cannot be defined
via intact molecular mass measurements. Sanger sequencing is usually
applied to unequivocally identify the
varying position. Alternatively, tandem
mass spectrometric approaches that
enable the automated reconstruction
of nucleic acid sequences from highly
specific fragment ion mass spectra may
be applied for this purpose (5661).
x BioTechniques www.biotechniques.com

Application of ICEMS for STR

Typing
STR typing was among the first
applications of ICEMS (32). By
typing the TH01 locus in a number of
homo- and heterozygous samples, it
was shown that ICEMS can efficiently
distinguish length variants. Even those
alleles that differed in length by a single
nucleotide were correctly identified.
More recently, we proved that ICEMS
is highly suitable to simultaneously
detect length and nucleotide variability
within forensically important STR
markers (62). Probably, ICEMS is the
only method available that can be used
for rapid and efficient characterization
of SNPSTR markers (12). Overall, 21
STRs were screened in an Austrian
population sample for the occurrence
of nucleotide variability within or
close to the repeat region. Eleven of
the investigated loci (SE33, D2S1338,
vWA, D21S11, D3S1358, D16S539,
D8S1179, D7S820, D13S317, D5S818,
D2S441) brought additional allele
(sequence) variants. Forensic efficiency
as determined by statistical parameters
was increased by 20%30%. The
beauty of ICEMS-STR analysis is the
fact that it represents one of the few
technological advancements that allow
direct comparison of newly generated
data with existing data, such as data
stored in DNA intelligence databases,

ICEMS has been successfully

applied for the genotyping of forensically relevant SNPs. One of the first
attempts focused on the characterization of the Y-chromosomal marker
M9 (63). The marker M9 defines an
ancestral lineage that is found in all
geographic regions except Africa.
Its apparent absence in Africa and
extensive distribution and frequency
in Europe and other parts of the world
suggest that it occurred initially outside
of Africa. As a consequence of genetic
drift during a bottleneck, this mutation
dispersed widely, differentiating into
several lineages. For genotyping, the
marker was amplified within a 62bp-long PCR product. To evaluate
the performance of ICEMS for the
characterization of M9, 90 different
DNA samples from unrelated males
were analyzed using both ICEMS and
an enzymatic method. As expected, the
genotypes determined by both methods
correlated perfectly. In the following
years, the number of Y-chromosomal
markers analyzable by ICEMS was
extended (64,65). A procedure for
genotyping the SNP 92R7 is among
these recently developed assays. Based
on genotyping results obtained with
ICEMS and other methods, it was
recognized that 92R7, which had previously been classified a simple SNP
locus with binary polymorphism, is not
a unique locus, but exhibits at least one
paralogous sequence variant (66).
Genotyping of mtDNA markers is
another field of application in which
ICEMS has found broad applicability.
The locus 16519T/C was extensively
used to gather the performance characteristics of ICEMS (51). Furthermore,
this locus served as a model to evaluate
the benefits of ICEMS in allelic
frequency determination (67). First,
artificially prepared mtDNA mixtures
served as samples. For 13 different
allelic mixtures with C contents in the
range of 1.0% to 99.0%, an average
error of 1.2% and a maximum error of
2.2% were observed. Next, ICEMS was
applied to the quantitative genotyping
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Review
of eight selected individuals. Among
these samples, four were heteroplasmic
with C contents in the range of 1.9%
to 34.1%. To check the reliability of
these results, allelic proportions were
additionally determined by a cloning
assay. The results of the two assays
correlated well. In all cases, deviations
were obtained that were smaller than
5.4%. The observed assay performance
suggested that ICEMS represents one
of the most powerful assays for the
determination of allelic frequencies.
Another ICEMS-based assay was
developed for the characterization of
certain regions of the first and second
hypervariable segments (HVS-I and
HVS-II, respectively) of the mtDNA
control region (68). Both segments
contain polycytosine (C) tracts, which
display length heteroplasmy at a
substantial rate in the population. The
two sections were simultaneously
amplified and analyzed by ICEMS in 90
maternally unrelated mother-offspring
pairs. The findings were compared
with the results obtained by Sanger
sequencing of the PCR products.
Sequence electropherograms showed
a characteristic out-of-phase pattern
downstream of the heteroplasmic Cstretch regions. This is a well-known
phenomenon, which can make data
interpretation difficult in some cases.
Here, ICEMS offered the considerable advantage that length variants
were clearly separated. Deconvoluted
mass spectra resulted in easily distinguishable peak patterns directly related
to observed heteroplasmic fragment
lengths. Hence, treatment of mass
spectrometric data was a rather simple
task, which enabled the unequivocal
identification of the length variants.
Moreover, the quantitative information
inherently present in the obtained mass
spectra in the form of peak intensities
was used for the determination of
relative contents of heteroplasmic
length variants.
Recently, a rapid and informative
mtDNA profiling system has been introduced that is based on the analysis of a
23-plex PCR by ICEMS. The developed
assay can be utilized as a prescreening
technology complementing standard
control region sequencing to eliminate
multiple suspects from an inquiry or
to discriminate between stains in high
Supplement to Vol. 43 No. 4 | 2007

volume casework. Target SNPs were

selected on their ability to increase
forensic discrimination within West
Eurasian populations. All of them are
located outside of the HVS-I and HVSII regions, which are usually characterized in routine forensic casework
by Sanger sequencing. The amplicon
lengths were in the range of 55131
bp, which made the assay highly appropriate for the analysis of degraded DNA
samples. For all except two targeted
sites, the designed primer pairs were
flanking more nucleotide positions than
just the target SNP. Thus, an overall
number of 627 nucleotide positions
were screened for variability. Rare or
even private polymorphisms became
detectable, which clearly improved
the forensic efficiency. Due to the
inability of ICEMS to locate the site of
the sequence variability, the obtained
sequence information was just linked to
an amplicon and not to a certain nucleotide position. The impact of a detected
variation on the phenotypic expression
remained uncertain. Thus, some
additional amount of genetic information was obtained with a minimum
loss of medical genetic privacy. Within
a single run, molecular masses of
more than 60 different single-stranded
species were determined. As far as we
know, no other mass spectrometric
assay has ever been able to characterize
such a large number of different nucleic
acids present within a single sample.
The measured molecular masses
were compared with the molecular
masses calculated for amplicons representing the reference sequence. Due
to the performance of the TOF mass
analyzer, an average molecular mass
measurement error of approximately 10
parts per million (ppm) was observed.
Significantly higher molecular mass
deviations indicated the presence and
enabled the identification of changes
within the nucleotide compositions
of the amplicons. The vast majority
of observed sequence variations were
explainable by alterations of the allelic
states of the target SNPs. Nevertheless,
within an Austrian population sample
comprised of 90 unrelated men, 14
different, nontarget SNP-related
sequence variations13 base substitutions and one deletionwere
detected by ICEMS and confirmed by

sequencing. The genetic information

obtained by the 23-plex PCR-ICEMS
assay was combined with HVS-I/HVSII sequencing results to create one
highly discriminating mtDNA profile,
which covered approximately 7.5% of
the total mtDNA genome. With the aid
of these highly discriminating mtDNA
profiles, the Austrian population sample
was resolved into 80 different lineages,
with 72 of them appearing only once in
the data set. Concerning rapid mtDNA
screening methods, the observed
power of discrimination (98.6%)
clearly surpasses those obtained with
alternative approaches (15,69). The
observed robustness and sensitivity
underlined the practical applicability
of the assay in forensic science, which
was proven by typing representative
casework samples.
SUMMARY
The determination of the molecular
mass of a DNA sequence has several
benefits over conventional fragmentlength analysis that are advantageous to
the forensic field: (i) sequence variation
is captured that increases the power
of discrimination compared with that
obtained by conventional fragmentlength analysis. First experiments
showed that this increase makes up to
20%30% for STR analysis. The new
technical approach does not invalidate
established developments and data, but
adds to this information with additional
discriminative categories. (ii) ICEMS is
faster and cheaper than electrophoresis,
does not require internal size standards,
allelic ladders, or spectral calibration,
which are necessary for fluorescencebased electrophoresis. (iii) ICEMS
can unequivocally detect any single
sequence variation in DNA molecules
with lengths up to 250 nucleotides. This
allows for maximum discrimination of
forensically relevant DNA fragments,
covering all sorts of STRs, SNPs, and
also the analysis of the hypervariable
segments of mtDNA. More effort,
however, needs to be put into software
development that escorts the analysis
and data interpretation processes to
make this technology manageable for
the practical user.

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Review
ACKNOWLEDGMENTS

The authors thank Applied

Biosystems and the Austrian Research
Promotion Agency (FFG, Project
810998) for their financial support.
COMPETING INTERESTS
STATEMENT

The authors declare no competing

interests
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Address correspondence to Walther Parson,

Institute of Legal Medicine, Innsbruck
Medical University, Mllerstrasse
44, 6020 Innsbruck, Austria. e-mail:
walther.parson@i-med.ac.at
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