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DNA PROFILING: STATE OF THE ART

Since its development in the mid-1980s (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 (100–400 bp) and their amenability to multiplex analysis have rendered STRs the most important forensic DNA markers. The establishment of a laboratory-independent 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 inter-national 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 discrimi-nation 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 discrimi-nation 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 infor-mation than just the fragment length. This has been shown for selected STR alleles by direct sequencing analysis (6–9) and the identification of small haplotype blocks in which SNPs are tightly linked to the STRs (10–12). Based on sequencing experiments,

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

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

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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 polymor-phisms (SNPs) is another niche in forensic genetics. The short fragment

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 high-multiplex format that would be required to achieve the necessary discrimi-nation 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 appre-ciated, 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 afore-mentioned 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 infor-mation per se to investigate forensic samples by direct sequencing and SBE typing (15,16).

LIQUID CHROMATOGRAPHY-ELECTROSPRAY IONIZATION MASS SPECTROMETRY

General Principle

The first attempts to apply the analytical method originated from the online hyphenation of ion-pair reversed-phase liquid chromatog-raphy to electrospray ionization mass spectrometry (ICEMS) for nucleic acids research date back to the 1990s (17–21). Applications focused on the quality check of synthetic oligonucle-otides (22,23), the identification of metabolites of therapeutic oligonucle-otides (18,24–26), and the character-ization of covalently modified DNA molecules (27–30). The first attempts to use ICEMS for genotyping were based on the analysis of short single-stranded oligonucleotide sequences (8-mers) derived from amplified genomic DNA via enzymatic digestion (31). The first two articles demon-strating the usability of ICEMS for the characterization of sequence varia-tions 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

Figure 1. Analytical system suitable for nucleic acid analysis by ion-pair reversed-phase liquid chromatography to electrospray ionization mass spec-trometry (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 spec-trometer; 13, electrospray ionization (ESI) source; 14, gas inlet; 15, ion optics; 16; vacuum system; 17, collision cell; 18, pusher; 19, reflectron; 20, detector.

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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 condi-tions, the molecular mass is an intrinsic property that is independent from the physical environment.

The success of the mass spectro-metric 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, double-stranded PCR products from solution into the gas phase⎯is 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 charac-terization is advantageous because of the division in half of the masses of the detected species and the possi-bility to identify base substitutions by

measuring mass differences ranging in size between 9.01 and 40.02 amu. If required, denaturation can be easily accomplished during the chromato-graphic run by using elevated column temperatures (65°–75°C).

Another reason for using chroma-tography as a sample preparation method is its ability to fractionate nucleic acid mixtures enabling the thorough mass spectrometric character-ization of samples that would have been too complex for direct analysis. One parameter that influences the chromato-graphic efficiency is the applied column material. Highest performance is obtained by using rods filled either with octadecylated silica particles (36,37), octadecylated polystyrene/divinyl-benzene particles (38), or monolithic polystyrene/divinylbenzene (30,39). The second important chromato-graphic parameter that has an impact on separation efficiency is the solvent composition. Several different amines were suggested as solvent additives (40–42). The use of butyldimethyl-amine 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 10–25 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 post-column 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 demon-strated 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 recom-mended.

Proper tuning of the mass spectrometer can have an influence on the detectability of nucleic acids (47–49). 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, interpre-table mass spectra can be obtained from oligonucleotide solutions with concen-trations in the low amol/ μL 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 (34,50–52). 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 spectro-metric 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 60–70 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 fabri-cation. 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 multi-plexed PCR assay represents an easy to accomplish task. Recently, guide-lines for selecting ICEMS-compatible PCR reagents were published (54).

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An aliquot of the crude PCR solution (0.5–1.0 μL) 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 double-stranded PCR products are completely denatured into the corresponding single strands by setting the column temper-ature to 65°–75°C. The molecular masses of the single-stranded species are determined and compared with the theoretical molecular masses calcu-lated 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 measure-ments. 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 (56–61).

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,

which have a retarding effect on new developments.

Application of ICEMS for SNP Typing

ICEMS has been successfully applied for the genotyping of forensi-cally relevant SNPs. One of the first attempts focused on the character-ization 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 62-bp-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 previ-ously 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 charac-teristics 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

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

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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 C-stretch regions. This is a well-known phenomenon, which can make data interpretation difficult in some cases. Here, ICEMS offered the consid-erable advantage that length variants were clearly separated. Deconvoluted mass spectra resulted in easily distin-guishable 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 intro-duced 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

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 HVS-II regions, which are usually charac-terized in routine forensic casework by Sanger sequencing. The amplicon lengths were in the range of 55–131 bp, which made the assay highly appro-priate 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 nucle-otide position. The impact of a detected variation on the phenotypic expression remained uncertain. Thus, some additional amount of genetic infor-mation 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 repre-senting 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 variations—13 base substi-tutions and one deletion—were detected by ICEMS and confirmed by

sequencing. The genetic information obtained by the 23-plex PCR-ICEMS assay was combined with HVS-I/HVS-II 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 fragment-length 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 fragment-length 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 fluorescence-based 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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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

REFERENCES

1. Gill, P., A.J. Jeffreys, and D.J. Werrett. 1985. Forensic application of DNA ‘finger-prints’. Nature 318:577-579.

2. Grubwieser, P., R. Mühlmann, B. Berger, H. Niederstätter, M. Pavlic, and W. Parson. 2006. A new “miniSTR-multiplex” displaying reduced amplicon lengths for the analysis of degraded DNA. Int. J. Legal Med. 120:115-120.

3. Butler, J.M. 2006. Genetics and genomics of core short tandem repeat loci used in human identity testing. J. Forensic Sci. 51:253-265.

4. Bär, W., B. Brinkmann, B. Budowle, A. Carracedo, P. Gill, P. Lincoln, W. Mayr, and B. Olaisen. 1994. DNA recommenda-tions—1994 report concerning further recom-mendations of the DNA Commission of the ISFH regarding PCR-based polymorphisms in STR (short tandem repeat) systems. Int. J. Legal Med. 107:159-160.

5. Gill, P., L. Fereday, N. Morling, and P.M. Schneider. 2006. New multiplexes for Europe-amendments and clarification of stra-tegic development. Forensic Sci. Int. 163:155-157.

6. Urquhart, A., C.P. Kimpton, T.J. Downes, and P. Gill. 1994. Variation in short tandem repeat sequences—a survey of twelve micro-satellite loci for use as forensic identification markers. Int. J. Legal Med. 107:13-20.

7. Rolf, B., M. Schurenkamp, A. Junge, and B. Brinkmann. 1997. Sequence polymor-phism at the tetranucleotide repeat of the human beta-actin related pseudogene H-beta-Ac-psi-2 (ACTBP2) locus. Int. J. Legal Med. 110:69-72.

8. Grubwieser, P., R. Mühlmann, H. Niederstätter, M. Pavlic, and W. Parson. 2005. Unusual variant alleles in commonly used short tandem repeat loci. Int. J. Legal Med. 119:164-166.

9. Grubwieser, P., B. Zimmermann, H. Niederstätter, M. Pavlic, M. Steinlechner, and W. Parson. 2007. Evaluation of an ex-tended set of 15 candidate STR loci for pa-ternity and kinship analysis in an Austrian population sample. Int. J. Legal Med. 121:85-89.

10. Mountain, J.L., A. Knight, M. Jobin, C. Gignoux, A. Miller, A.A. Lin, and P.A. Underhill. 2002. SNPSTRs: empirically de-rived, rapidly typed, autosomal haplotypes for

inference of population history and mutation-al processes. Genome Res. 12:1766-1772.

11. Ramakrishnan, U. and J.L. Mountain. 2004. Precision and accuracy of divergence time estimates from STR and SNPSTR varia-tion. Mol. Biol. Evol. 21:1960-1971.

12. Agrafioti, I. and M.P. Stumpf. 2007. SNPSTR: a database of compound microsat-ellite-SNP markers. Nucleic Acids Res. 35:D71-D75.

13. Jobling, M.A. and C. Tyler-Smith. 2003. The human Y chromosome: an evolutionary marker comes of age. Nat. Rev. Genet. 4:598-612.

14. Brion, M., J.J. Sanchez, K. Balogh, C. Thacker, A. Blanco-Verea, C. Borsting, B. Stradmann-Bellinghausen, M. Bogus, et al. 2005. Introduction of a single nucleodite polymorphism-based “Major Y-chromosome haplogroup typing kit” suitable for predict-ing the geographical origin of male lineages. Electrophoresis 26:4411-4420.

15. Brandstatter, A., T.J. Parsons, and W. Parson. 2003. Rapid screening of mtDNA coding region SNPs for the identification of west European Caucasian haplogroups. Int. J. Legal Med. 117:291-298.

16. Parson, W., L. Fendt, D. Ballar, C. Borsting, B. Brinkmann, A. Carracedo, M. Carvalho, M.D. Coble, et al. Identification of West Eurasian mitochondrial haplogroups by mtDNA SNP screening: results of the 2006-2007 EDNAP collaborative exercise. Forensic Sci. Int.: Genetics. (In press).

17. Bleicher, K. and E. Bayer. 1994. Analysis of oligonucleotides using coupled high perfor-mance liquid chromatography-electrospray mass spectrometry. Chromatographia 39:405-408.

18. Griffey, R.H., M.J. Greig, H.J. Gaus, K. Liu, D. Monteith, M. Winniman, and L.L. Cummins. 1997. Characterization of oli-gonucleotide metabolism in vivo via liquid chromatography/electrospray tandem mass spectrometry with a quadrupole ion trap mass spectrometer. J. Mass Spectrom. 32:305-313.

19. Apffel, A., J.A. Chakel, S. Fischer, K. Lichtenwalter, and W.S. Hancock. 1997. Analysis of oligonucleotides by HPLC-elec-trospray ionization mass spectrometry. Anal. Chem. 69:1320-1325.

20. Huber, C.G. and A. Krajete. 1999. Analysis of nucleic acids by capillary ion-pair re-versed-phase HPLC coupled to negative ion-electrospray ionization mass spectrometry. Anal. Chem. 71:3730-3739.

21. Huber, C.G. and H. Oberacher. 2001. Analysis of nucleic acids by on-line liquid chromatography-mass spectrometry. Mass Spectrom. Rev. 20:310-343.

22. Gilar, M. 2001. Analysis and purification of synthetic oligonucleotides by reversed-phase high-performance liquid chromatography with photodiode array and mass spectrometry detection. Anal. Biochem. 298:196-206.

23. Fountain, K.J., M. Gilar, and J.C. Gebler. 2003. Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ioniza-tion mass spectrometry. Rapid Commun. Mass Spectrom. 17:646-653.

24. Gaus, H.J., S.R. Owens, M. Winniman, S. Cooper, and L.L. Cummins. 1997. On-line HPLC electrospray mass spectrometry of phosphorothioate oligonucleotide metabo-lites. Anal. Chem. 69:313-319.

25. Zhang, G., J. Lin, K. Srinivasan, O. Kavetskaia, and J.N. Duncan. 2007. Strategies for bioanalysis of an oligonucle-otide class macromolecule from rat plasma using liquid chromatography-tandem mass spectrometry. Anal. Chem. 79:3416-3424.

26. Lin, Z.J., W. Li, and G. Dai. 2007. Application of LC-MS for quantitative analy-sis and metabolite identification of therapeu-tic oligonucleotides. J. Pharm. Biomed. Anal. 44:330-341.

27. Apruzzese, W.A. and P. Vouros. 1998. Analysis of DNA adducts by capillary meth-ods coupled to mass spectrometry: a perspec-tive. J. Chromatogr. A. 794:97-108.

28. Andrews, C.L., P. Vouros, and A. Harsch. 1999. Analysis of DNA adducts using high-performance separation techniques coupled to electrospray ionization mass spectrometry. J. Chromatogr. A. 856:515-526.

29. Chen, H., W. Zhang, R. Song, H. Ma, Y. Dong, G. Sheng, Z. Zhou, and J. Fu. 2004. Analysis of DNA methylation by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ioniza-tion mass spectrometry. Rapid Commun. Mass Spectrom. 18:2773-2778.

30. Xiong, W., J. Glick, Y. Lin, and P. Vouros. 2007. Separation and sequencing of isomeric oligonucleotide adducts using monolithic col-umns by ion-pair reversed-phase nano-HPLC coupled to ion trap mass spectrometry. Anal. Chem. 79:5312-5321.

31. Laken, S.J., P.E. Jackson, K.W. Kinzler, B. Vogelstein, P.T. Strickland, J.D. Groopman, and M.D. Friesen. 1998. Genotyping by mass spectrometric analysis of short DNA fragments. Nat. Biotechnol. 16:1352-1356.

32. Oberacher, H., W. Parson, R. Mühlmann, and C.G. Huber. 2001. Analysis of poly-merase chain reaction products by on-line liquid chromatography-mass spectrometry for genotyping of polymorphic short tandem repeat loci. Anal. Chem. 73:5109-5115.

33. Oberacher, H., P.J. Oefner, W. Parson, and C.G. Huber. 2001. On-line liquid chromatog-raphy mass spectrometry: a useful tool for the detection of DNA sequence variation. Angew. Chem. Int. Ed. Engl. 40:3828-3830.

34. Oberacher, H., C.G. Huber, and P.J. Oefner. 2003. Mutation scanning by ion-pair reversed-phase high-performance liq-uid chromatography-electrospray ionization mass spectrometry (ICEMS). Hum. Mutat. 21:86-95.

35. Oberacher, H., W. Parson, G. Holzl, P.J. Oefner, and C.G. Huber. 2004. Optimized suppression of adducts in polymerase chain reaction products for semi-quantitative SNP genotyping by liquid chromatography-mass spectrometry. J. Am. Soc. Mass Spectrom. 15:1897-1906.

36. Willems, A.V., D.L. Deforce, W.E. Lambert, C.H.V. Peteghem, and J.F. Van Bocxlaer. 2004. Rapid characterization of oligonucleotides by capillary liquid chroma-tography-nano electrospray quadrupole time-

Review

Supplement to Vol. 43 ı No. 4 | 2007 www.biotechniques.com ı BioTechniques ı xiii

of-flight mass spectrometry. J. Chromatogr. A. 1052:93-101.

37. Fountain, K.J., M. Gilar, and J.C. Gebler. 2004. Electrospray ionization mass spectro-metric analysis of nucleic acids using high-throughput on-line desalting. Rapid Commun. Mass Spectrom. 18:1295-1302.

38. Oberacher, H., A. Krajete, W. Parson, and C.G. Huber. 2000. Preparation and evalu-ation of packed capillary columns for the separation of nucleic acids by ion-pair re-versed-phase high-performance liquid chro-matography. J. Chromatogr. A. 893:23-35.

39. Premstaller, A., H. Oberacher, and C.G. Huber. 2000. High-performance liquid chro-matography-electrospray ionization mass spectrometry of single- and double stranded nucleic acids using monolithic capillary col-umns. Anal. Chem. 72:4386-4393.

40. Potier, N., A. Van Dorsselaer, Y. Cordier, O. Roch, and R. Bischoff. 1994. Negative electrospray ionization mass spectrometry of synthetic and chemically modified oligonu-cleotides. Nucleic Acids Res. 22:3895-3903.

41. Cheng, X., D. Gale, H.R. Udseth, and R.D. Smith. 1995. Charge state reduction of oli-gonucleotide negative ions from electrospray ionization. Anal. Chem. 67:586-593.

42. Muddiman, D.C., X. Cheng, H.R. Udseth, and R.D. Smith. 1996. Charge-state reduc-tion with improved signal intensity of oligo-nucleotides in electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 7:697-706.

43. Oberacher, H., H. Niederstätter, F. Pitterl, and W. Parson. 2006. Profiling 627 mito-chondrial nucleotides via the analysis of a 23-plex polymerase chain reaction by liquid chromatography-electrospray ionization time-of-flight mass spectrometry. Anal. Chem. 78:7816-7827.

44. Griffey, R.H., H. Sasmor, and M.J. Greig. 1997. Oligonucleotides charge states in nega-tive ionization electrospray-mass spectrom-etry are a function of solution ammonium ion concentration. J. Am. Soc. Mass Spectrom. 8:155-160.

45. Apffel, A., J.A. Chakel, S. Fischer, K. Lichtenwalter, and W.S. Hancock. 1997. New procedure for the use of high-perfor-mance liquid chromatography-electrospray ionization mass spectrometry for the analy-sis of nucleotides and oligonucleotides. J. Chromatogr. A. 777:3-21.

46. Huber, C.G. and A. Krajete. 2000. Sheath liquid effects in capillary high-perfor-mance liquid chromatography-electrospray mass spectrometry of oligonucleotides. J. Chromatogr. A. 870:413-424.

47. Oberacher, H., H. Niederstätter, and W. Parson. 2005. Characterization of synthetic nucleic acids by electrospray ionization quad-rupole time-of-flight mass spectrometry. J. Mass Spectrom. 40:932-945.

48. Oberacher, H., W. Walcher, and C.G. Huber. 2003. Effect of instrument tuning on the detectability of biopolymers in electro-spray ionization mass spectrometry. J. Mass Spectrom. 38:108-116.

49. Hofstadler, S.A., J.J. Drader, and A. Schink. 2006. Selective ion filtering by digi-tal thresholding: a method to unwind complex

ESI-mass spectra and eliminate signals from low molecular weight chemical noise. Anal. Chem. 78:372-378.

50. Van Ert, M.N., S.A. Hofstadler, Y. Jiang, J.D. Busch, D.M. Wagner, J.J. Drader, D.J. Ecker, J.C. Hannis, et al. 2004. Mass spectrometry provides accurate characteriza-tion of two genetic marker types in Bacillus anthracis. BioTechniques 37:642-648.

51. Oberacher, H., H. Niederstätter, B. Casetta, and W. Parson. 2005. Detection of DNA se-quence variations in homo- and heterozygous samples via molecular mass measurements by electrospray ionization time-of-flight mass spectrometry. Anal. Chem. 77:4999-5008.

52. Gross, E., G. Hölzl, N. Arnold, E. Hauenstein, A. Jacobsen, K. Schulze, J. Ramser, A. Meindl, et al. 2007. Allelic loss analysis by denaturing high-performance liquid chromatography and electrospray ionization mass spectrometry. Hum. Mutat. 28:303-311.

53. Syvanen, A.C. 2001. Accessing genetic vari-ation: genotyping single nucleotide polymor-phisms. Nat. Rev. Genet. 2:930-942.

54. Oberacher, H., H. Niederstätter, B. Casetta, and W. Parson. 2006. Some guidelines for the analysis of genomic DNA by PCR-LC-ESI-MS. J. Am. Soc. Mass Spectrom. 17:124-129.

55. Hölzl, G., H. Oberacher, S. Pitsch, A. Stutz, and C.G. Huber. 2005. Analysis of biologi-cal and synthetic ribonucleic acids by liquid chromatography-mass spectrometry using monolithic capillary columns. Anal. Chem. 77:673-680.

56. Ni, J., S.C. Pomerantz, J. Rozenski, Y. Zhang, and J.A. McCloskey. 1996. Interpretation of oligonucleotide mass spectra for determination of sequence using electro-spray ionization and tandem mass spectrom-etry. Anal. Chem. 68:1989-1999.

57. Rozenski, J. and J.A. McCloskey. 2002. SOS: a simple interactive program for ab inition oligonucleotide sequencing by mass spectrometry. J. Am. Soc. Mass Spectrom. 13:200-203.

58. Oberacher, H., B. Wellenzohn, and C.G. Huber. 2001. Comparative sequencing of nu-cleic acids by liquid chromatography-tandem mass spectrometry. Anal. Chem. 74:211-218.

59. Oberacher, H., P.J. Oefner, G. Hölzl, A. Premstaller, and C.G. Huber. 2002. Re-sequencing of multiple single nucleotide polymorphisms by liquid chromatography-electrospray ionization mass spectrometry. Nucleic Acids Res. 30:e67.

60. Oberacher, H., W. Parson, P.J. Oefner, B.M. Mayr, and C.G. Huber. 2004. Applicability of tandem mass spectrometry to the auto-mated comparative sequencing of long-chain oligonucleotides. J. Am. Soc. Mass Spectrom. 15:510-522.

61. Oberacher, H., B.M. Mayr, and C.G. Huber. 2004. Automated de novo sequencing of nucleic acids by liquid chromatography-tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 15:32-42.

62. Oberacher, H., F. Pitterl, G. Huber, H. Niederstätter, M. Steinlechener, and W. Paron. Increased forensic efficiency of DNA fingerprints through simultaneous resolution

of length and nucleotide variability by high-performance mass spectrometry. Hum. Mutat. (In press).

63. Berger, B., G. Hölzl, H. Oberacher, H. Niederstätter, C.G. Huber, and W. Parson. 2002. Single nucleotide polymorphism ge-notyping by on-line liquid chromatography-mass spectrometry in forensic science with the Y-chromosomal locus M9. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 782:89-97.

64. Brion, M., B.M. Dupuy, M. Heinrich, C. Hohoff, B. Hoste, B. Ludes, B. Mevag, N. Morling, et al. 2005. A collaborative study of the EDNAP group regarding Y-chromosome binary polymorphism analysis. Forensic Sci. Int. 153:103-108.

65. Niederstatter, H., B. Berger, H. Oberacher, A. Brandstätter, C.G. Huber, and W. Parson. 2005. Separate analysis of DYS385a and b versus conventional DYS385 typing: is there forensic relevance? Int. J. Legal Med. 119:1-9.

66. Sanchez, J.J., M. Brion, W. Parson, A.J. Blanco-Verea, C. Borsting, M. Lareu, H. Niederstatter, H. Oberacher, et al. 2004. Duplications of the Y-chromosome specific loci P25 and 92R7 and forensic implications. Forensic Sci. Int. 140:241-250.

67. Oberacher, H., H. Niederstätter, C.G. Huber, and W. Parson. 2006. Accurate determination of allelic frequencies in mi-tochondrial DNA mixtures by electrospray ionization time-of-flight mass spectrometry. Anal. Bioanal. Chem. 384:1155-1163.

68. Oberacher, H., H. Niederstätter, and W. Parson. 2007. Liquid chromatography-elec-trospray ionization mass spectrometry for simultaneous detection of mtDNA length and nucleotide polymorphisms. Int. J. Legal Med. 121:57-67.

69. Gabriel, M.N., C.D. Calloway, R.L. Reynolds, S. Andelinovic, and D. Primorac. 2001. Population variation of human mito-chondrial DNA hypervariable regions I and II in 105 Croatian individuals demonstrated by immobilized sequence-specific oligonucle-otide probe analysis. Croat. Med. J. 42:328-335.

Address correspondence to Walther Parson, Institute of Legal Medicine, Innsbruck Medical University, Müllerstrasse 44, 6020 Innsbruck, Austria. e-mail: walther.parson@i-med.ac.at

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