T he Human Genome Project continues to produce

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1 77 kerblom, E.B. et al. (1998) Six new photolabile linkers for solidphase synthesis. 1. Methods of preparation. Mol. Divers. 3, Ordoukhanian, P. and Taylor, J-S. (1995) Design and synthesis of a versatile photocleavable DN building block. pplication of phototriggered hybridization. J. m. Chem. Soc. 117, Nakatani, K. et al. (1998) p-cyano substituted benzophenone as an excellent photophore for one-electron oxidation of DN. Tetrahedron Lett. 39, Fang, K. et al. (1998) bifunctional photoaffinity probe for ligand/ receptor interaction studies. J. m. Chem. Soc. 120, Single-nucleotide polymorphism analysis by MLDI TOF mass spectrometry Timothy J. riffin and Lloyd M. Smith Single-nucleotide polymorphisms (SNPs) have great potential for use in genetic-mapping studies, which locate and characterize genes that are important in human disease and biological function. For SNPs to realize their full potential in genetic analysis, thousands of different SNP loci must be screened in a rapid, accurate and cost-effective manner. Matrix-assisted laser desorption-ionization time-of-flight (MLDI TOF) mass spectrometry is a promising tool for the high-throughput screening of SNPs, with future prospects for use in genetic analysis. T he Human enome Project continues to produce sequence data, and it has become evident that there is substantial variation in the DN sequence between two individuals at many points throughout the genome. Most commonly, sequence variation occurs at discrete, single-nucleotide positions referred to as single-nucleotide polymorphisms (SNPs), which are estimated to occur at a frequency of approximately one per 1000 nucleotides 1 4. Therefore, for every 1000 nucleotides, the average nucleotide identity at one position will differ between any two copies of that chromosome at a substantial frequency throughout a population. SNPs are biallelic polymorphisms, that is, the nucleotide identity at these polymorphic positions is generally constrained to one of two possibilities in humans, rather than the four nucleotide possibilities that could occur, in principle 4. Single-nucleotide polymorphisms SNPs have important implications in human genetic studies. First, a subset of SNPs occurs within proteincoding sequences 3,4. The presence of a specific SNP allele can be implicated as a causative factor in human genetic disorders. Therefore, screening for such an allele in an individual might enable the detection of a genetic predisposition to disease. Second, SNPs can be used as genetic markers for use in genetic-mapping studies 2 5, which locate and identify genes of functional importance. It has been proposed that a set of 3000 biallelic SNP markers would be sufficient for wholegenome-mapping studies in humans; a map of T.J. riffin (tgriffin@u.washington.edu) is at the Department of Molecular Biotechnology, University of Washington, Box , Seattle, W, , US. L.M. Smith (smith@chem.wisc.edu) is at the Department of Chemistry, University of Wisconsin-Madison, 1101 University venue, Madison, WI, , US. or more SNPs has been proposed as an ultimate goal to enable effective genetic-mapping studies in large populations 6. Therefore, technologies capable of genotyping thousands of SNP markers from large numbers of individual DN samples in an accurate, rapid and cost-effective manner are needed to make these studies feasible. wide variety of approaches to genotyping SNPs have been developed in recent years 2,3,7,8 ; amongst the more promising technologies being developed is matrix-assisted laser desorption-ionization time-offlight (MLDI TOF) mass spectrometry (MS). This article gives a brief introduction to MLDI TOF MS, an overview of approaches previously developed for SNP analysis, as well as recent advances in the field (Table 1) and an assessment of what the future holds for MLDI TOF MS as a SNP-genotyping technology. MLDI TOF mass spectrometry MLDI was introduced in 1988 by Karas and Hillenkamp as a revolutionary method for ionizing and mass-analysing large biomolecules 9. These investigators discovered that irradiation of crystals formed by suitable small organic molecules (called the matrix) with a short laser pulse at a wavelength close to a resonant absorption band of the matrix molecules caused an energy transfer and desorption process, producing gasphase matrix ions. More importantly, they found that if a low concentration of a non-absorbing analyte, such as a protein or nucleic acid molecule, was added to the matrix in solution and embedded in the solid matrix crystals formed by drying of the mixture, the nonabsorbing, intact analyte molecules were also desorbed into the gas-phase and ionized upon irradiation with the laser, facilitating their mass analysis. Typically, predominantly singly charged molecular ions (both negative and positive) are detected by MLDI TOF TIBTECH FEBRURY 2000 (Vol. 18) /00/$ see front matter 2000 Elsevier Science Ltd. ll rights reserved. PII: S (99)

2 Table 1. SNP analysis approaches using MLDI TOF MS Method Refs Sequencing of PCR products 20, 21 Direct mass-analysis of PCR products 60 nalysis of allele-specific PCR or LCR products 33, 34 nalysis of RFLP PCR products 35, 36 Minisequencing 38 46, 61 nalysis of PN hybridization probes Direct analysis of invasive cleavage products 62 bbreviations: PCR, polymerase chain reaction; LCR, ligase chain reaction; RFLP, restriction fragment length polymorphism; PN, peptide nucleic acid. Sample plate U acc Source region Intensity MS, with these ions being created in a gas-phase protontransfer reaction with the matrix molecules 10. MLDI has been used primarily with time-of-flight mass spectrometers 11 (Fig. 1). Since its inception, MLDI TOF MS has seen widespread use in the analysis of proteins, peptides and nucleic acids nalysing nucleic acids with MLDI TOF MS has several advantages. First, it is fast; ionization, separation by size and detection of nucleic acids takes milliseconds to complete. Because signals from multiple laser pulses (~ pulses) are usually averaged to obtain a final mass spectrum, the total analysis time can take as little as ten seconds. By contrast, conventional electrophoretic methods for separating and detecting nucleic M 2 + Pulsed laser M 1 + ~ 2.0 m Field-free drift tube M 1 + M 2 + Detector Figure 1 schematic diagram of a matrix-assisted laser desorption-ionization time-of-flight mass spectrometer. Matrix and analyte ions are desorbed and ionized upon irradiance with a laser pulse in the source region; a potential (U acc ) applied to the sample plate accelerates the ions into the field-free drift tube. Ions with smaller mass-to-charge ratios () travel down the drift tube at a higher velocity than ions with a larger. The time-of-flight of each ion is measured and, with proper calibration, converted into a corresponding. This diagram shows the separation and detection of two positive, single-charged ions with different masses, M 1 and M 2. acids can take hours to complete. Second, the results are absolute, being based on the intrinsic property of the mass-to-charge ratio (). This is inherently more accurate than electrophoresis-based or hybridizationarray-based methods, which are both susceptible to complications from secondary-structure formation in nucleic acids 15,16. Furthermore, the absolute nature of detection, combined with the detection of predominantly singly charged molecular ions, makes the analysis of complex mixtures by MLDI TOF MS possible. Third, the complete automation of all steps, from sample preparation through to the acquisition and processing of the data, is feasible 17, giving MLDI TOF MS great potential for high-throughput nucleicacid analysis applications. SNP genotyping using MLDI TOF MS DN sequencing for SNP genotyping Originally, MLDI TOF MS was proposed as an alternative high-throughput technology for DN sequencing 18 to replace the conventional method of separating and detecting fluorescently labeled DN fragments produced in enzymatic sequencing reactions by gel electrophoresis 19. Enzymatic DN sequencing coupled with MLDI TOF mass spectrometric analysis has been shown to be effective at discovering previously unknown single-nucleotide substitution mutations 20,21 ; for example, PCR products from the p53 tumor suppressor gene have been sequenced and analysed by MLDI TOF MS (Ref. 20; Fig. 2). This same approach could also be used to genotype SNPs at known positions. However, it is apparent from these studies, and also from previous studies 22 24, that there is a loss of signal intensity and mass resolution with increasing DN size; this has proven to be the major current limitation to DN sequencing by MLDI TOF MS. One likely contributing factor to this phenomenon is the size-dependent tendency of the phosphodiester backbone of DN to fragment during the MLDI process 25,26, which results in a loss of signal intensity for the intact, full-length molecular ion of larger DN molecules. Other factors contributing to this size-dependent decrease in signal might include: an instrumental and/or detector bias towards smaller oligonucleotides; increased sodium and potassium cation adduction of larger oligonucleotides, resulting in division of their signal over many peaks; an ionization bias favoring the ionization of smaller oligonucleotides 27 ; and a difference in the collisional cross sections between larger and smaller oligonucleotides, making larger DN molecules more apt to collide with gas-phase molecules in the drift tube, causing them to break apart and not reach the detector. This size-dependent loss of signal has limited MLDI TOF MS DN-sequencing read lengths to, typically, 100 nucleotides per sequencing reaction 20 24, Consequently, a robust MLDI-based approach to SNP discovery, which requires sequencing of PCR products up to 300 bp in length, has not yet been demonstrated. Direct mass-analysis of PCR products This limitation in the analysis of large DN molecules has also hampered attempts at using MLDI TOF MS to analyse directly PCR amplicons containing SNPs. 78 TIBTECH FEBRURY 2000 (Vol. 18)

3 dditionally, during the MLDI process, doublestranded PCR products can dissociate into single strands of slightly different masses, which are poorly mass resolved and result in peak broadening and mass-measurement inaccuracies 32. These limitations have made the accurate differentiation between PCR products containing different SNP alleles challenging because mass differences between amplicons as small as 9 Da (the difference between d and dt nucleotides) must be resolved. One way to circumvent these limitations is to analyse amplicons that are produced in allele-specific PCR reactions, in which the allele-specific PCR primers are designed to be of sizes that are sufficiently different to enable easy mass-analysis of the PCR products 33. similar approach using the ligase chain reaction (LCR) has also been developed, wherein the presence of one SNP allele leads to the production of a singlestranded ligation product that is purified and detected by mass, but the other allele inhibits the ligation reaction, therefore no ligation product is detected in the mass spectrum 34. pproaches have also been developed for the MLDI TOF mass-spectrometric analysis of restriction fragment length polymorphism products derived from PCR amplicons 35,36. Minisequencing Minisequencing 37 for SNP analysis involves annealing a primer to a template PCR amplicon immediately downstream of an SNP position. mix of deoxynucleotide triphosphates (dntps) and dideoxynucleotide triphosphates (ddntps), or in some cases a mix of ddntps alone, are added to the PCR template and primer, along with a DN polymerase. The polymerase extends the 3 end of the primer by specifically incorporating nucleotides that are complementary to those contained in the PCR template immediately adjacent to the primer position. Extension terminates at the first position in the template where a nucleotide, complementary to one of the ddntps in the mix, occurs. MLDI TOF MS-based approaches using minisequencing have been developed 38,39 in which extended primers (generally shorter than 25 nucleotides in length) are solid-phase purified and detected by mass; the identity of the polymorphic nucleotide is determined by measuring the mass of the extended primer, which is detected at a value specific to the nucleotides added in the extension reaction. Minisequencing has become the most widely used MLDI TOF MS-based method for SNP analysis (Fig. 3), and it has been successfully applied to the genotyping of SNPs located in biologically important genes Several different variations in assay design and reaction conditions have been used, but the version of the approach used most commonly employs a thermostable DN polymerase in a temperature-cycled extension reaction, which leads to a linear amplification of the extended primer 38,41. Using this approach, successful analysis of a synthetic oligonucleotide target at a concentration as low as 400 pm has been demonstrated, although analysis of double-stranded PCR products is usually done at template concentrations approaching the micromolar range 38. Haff and Smirnov showed that multiplex SNP analysis, by minisequencing and MLDI TOF MS, is possible; they reported the successful multiplex genotyping of Primer T C C C C C T C C C C C (b) (c) (d) P P F F 40Da Heterozygous 1/2 Homozygous 1 Homozygous Figure 2 Sequencing a PCR product by MLDI TOF mass spectrometry 20,21. The sequence surrounding a single-nucleotide-polymorphism position is shown, with the polymorphic nucleotide in bold, being either a d (identified as allele 2) or a dc (identified as allele 1) nucleotide. The mass spectra shown are for the analysis of the C sequencing reactions, therefore it contains only DN fragments terminating at dc nucleotides. The heterozygous sample (b) shows a double peak for the reaction products that terminate at the dc nucleotide immediately 3 of the SNP position: one peak results from a fragment terminating with dc-3 (2 allele) and the other from a fragment, 40 Da smaller, terminating with dcc-3 (1 allele). The homozygous samples (c, d) show a single peak corresponding to the presence of only one of the two possible fragments that terminate immediately 3 of the single-nucleotide-polymorphism position. bbreviation:, mass-to-change ratio. (Reproduced, with permission, from Ref. 20.) five different SNPs occurring within the same PCR amplicon from the BRC1 exon 13 locus 44. By designing mass-tagged minisequencing primers for each SNP position that contained extra, non-complementary dt nucleotides on the 5 end of the primers, they showed that all five primers could be extended in a single reaction and that the extension products could be simultaneously mass analysed by MLDI TOF MS. Little et al. have integrated this minisequencing approach into a semi-automated system, using proprietary MLDI on a chip technology 42,45, in which nanoliter amounts of sample from minisequencing SNP-analysis reactions are piezoelectrically pipetted onto a silicon chip that is inserted directly into the mass (2) (1) TIBTECH FEBRURY 2000 (Vol. 18) 79

4 Primer + ddntps 5 3 +DN polymerase 3 * 5 PCR template Polymorphic nucleotide a (b) '' target b (b) (c) '' target (d) 'T' target c (c) d (d) (e) 'C' target (e) (f) + ddc + ddt + dd + dd Extended primer Purify extended primer 5 MLDI TOF * mass analysis 3 5 N Non-extended primer Figure 3 MLDI TOF mass spectrometric analysis of minisequencing products 38,39. Schematic diagram of a general version of minisequencing where a primer is annealed to a PCR template immediately downstream from the polymorphic position. mix containing four dideoxynucleotide triphosphates (ddntps) and a DN polymerase is added and the primer is specifically extended by one nucleotide (N), where N is complementary to the nucleotide at the polymorphic position. The extended primer is purified and mass analysed. Representative results are shown for a primer specifically extended in the presence of all four ddntps using a synthetic target containing: (b) d, (c) d, (d) dt and (e) dc, at the single-nucleotide polymorphism position. (f) negative control reaction containing a non-extended primer. ccurate mass measurement of the extended primer gives the identity of the polymorphic nucleotide. bbreviation:, mass-to-charge ratio. (Reproduced, with permission, from Ref. 38.) spectrometer, and each separate sample spot is automatically MLDI TOF MS analysed. Using this approach, they also demonstrated the ability to obtain high-quality mass spectra from 100 separate spots, each containing only eight femtomoles of an oligonucleotide control, in a total of six minutes 45. This shows the possible throughput for SNP genotyping in an automated MLDI TOF MS system, although the results were obtained from a control oligonucleotide and not from products generated from an enzymatic minisequencing reaction, which might prove to be more difficult. One drawback of the minisequencing approach is that the small mass differences between ddntps (as little as 9 Da) that are incorporated in the extension reaction require pure extension products and high instrumental mass resolution to detect heterozygous genotypes accurately. variation of the minisequencing approach has been developed that uses mass-tagged ddntps (Ref. 46), which increase the mass differences between primers extended with these ddntps to ~500 Da. Mass-tagging increases the versatility of the minisequencing approach by alleviating the need for intensive sample-preparation procedures and decreasing the necessary resolving power of the mass spectrometer. Peptide nucleic-acid hybridization probes Peptide nucleic acid (PN; Refs 47,48) is a DN analog containing the four normal nucleotides of DN attached to a neutrally charged amide backbone that retains the ability to base-pair specifically with complementary DN. The neutral backbone confers unique characteristics on the hybridization of PN with DN, including: increased thermal stability of the resulting duplex; the ability to hybridize under low ionic strength conditions; and an increased hybridization specificity for complementary DN sequences 47 49, making PN oligomers useful as allele-specific hybridization probes. dditionally, PN is easily analysed by MLDI TOF MS (Ref. 50) because, unlike DN molecules, the peptide backbone does not fragment during the MLDI process. In addition, PN oligomers do not tend to form metal cation adducts, which are detrimental to MLDI TOF mass-spectrometric analysis 22,51, because annealing of these oligomers can be done in buffers containing low salt concentrations, and also the neutral amide backbone does not have the same tendency to bind to cations that may be present to the same extent as the negatively-charged backbone of DN. iven its unique characteristics, PN is highly compatible with MLDI TOF mass-spectrometric analysis for SNP genotyping, and two similar approaches using PN have been independently developed recently 52,53. In both of these approaches, two PNhybridization probes, each corresponding to one of the two possible SNP alleles, are annealed to biotinylated, single-stranded PCR amplicons that are immobilized on streptavidin-coated magnetic beads. fter annealing the PN probes, the beads are stringently washed so that only a perfectly matched probe will remain hybridized to the PCR target. The entire bead solution is then spotted onto the MLDI probe tip and an acidic matrix solution is added to the beads, which dissociates the hybridized PN probes from the immobilized DN. The PN is then desorbed and ionized with the matrix crystals upon irradiation with the laser and detected by its mass; each uniquely mass-labeled PN probe detected corresponds to a specific SNP allele present in the PCR amplicon. 80 TIBTECH FEBRURY 2000 (Vol. 18)

5 In one approach, each allele-specific PN probe is mass-labeled by incorporating a variable number of 8-amino-3,6-dioxaoctanoic acid residues on the N- terminal end of the probes; this approach has been applied to the genotyping of four unique singlenucleotide substitution mutations contained in the human tyrosinase gene exon 4 (Ref. 52; Fig. 4). Ross et al. genotyped two SNPs located within human mitochondrial DN and also a human leukocyte antigen polymorphism using allele-specific PN probes that were uniquely mass labeled by simply adding an extra, non-complementary dt nucleobase to the 3 end of the PN oligomer 53. This approach has also been applied successfully to the genotyping of the highly polymorphic human leukocyte antigen DQ locus in 28 individuals 54. The ability to carry out multiplex analysis of SNPs using PN probes is limited by the high variability in the thermal stabilities of different probe sequences, which require the hybridization of probe pairs at each polymorphic position to be conducted in separate reaction tubes under different conditions 52. To design PN probe sequences with similar thermal stabilities, two algorithms for predicting PN:DN duplex stabilities from the sequence have been developed 55,56, which should enable single-tube, multiplex SNP genotyping to be carried out. Recent advances and future directions The recent introduction of MLDI TOF MS instruments equipped with delayed extraction has greatly improved the achievable resolution in the analysis of DN. Successful genotyping of an SNP contained in a PCR product (69 nucleotides in length) by direct MLDI TOF MS sizing of the single-stranded amplicon has been shown recently 60 using an efficient solid-phase sample purification method and delayed extraction instrumentation. The resolution achieved was sufficient to differentiate between single-stranded PCR products containing any combination of singlenucleotide differences, including d to dt transversions, which differ in mass by only 9 Da, although successful SNP analysis of a heterozygous sample was not demonstrated. Further advances in instrumentation should continue to improve the mass resolution and increase the size range of PCR products that can be analysed effectively. Perhaps the greatest promise of MLDI TOF MS for SNP analysis lies in its ability to genotype many SNPs rapidly, accurately and simultaneously. The successful multiplex genotyping of 12 different SNPs has been demonstrated recently using minisequencing 61. The 12 loci were amplified in a single reaction by multiplex PCR and primer extension reactions for all 12 SNP positions were carried out in the same tube after the PCR amplification, using minisequencing primers designed to have different masses that could be resolved easily in the resulting mass spectrum (Fig. 5). The ability to analyse SNPs in a highly parallel fashion, coupled with the proven ability to automate these types of analysis 17,45, could potentially make MLDI TOF MS the technology of choice for high-throughput SNP analysis. Recently, a more direct approach to genotyping SNPs by MLDI TOF MS has been developed that does not require a PCR-amplification step 62. Eliminating the need for target amplification is desirable PCR amplicon (b) (c) 1 2 where M 2 > M 1 + PN probes Streptavidin-coated magnetic bead WT419 WT WT419 WT Wash WT422 WT WT VR VR WT MLDI matrix MLDI TOF mass analysis Figure 4 MLDI TOF mass-spectrometric analysis of peptide nucleic-acid hybridization probes 52,53. The steps involved are detailed in the schematic diagram, where a biotinylated strand of a PCR amplicon is immobilized on streptavidin-coated magnetic beads and differentially mass-labeled PN hybridization probes are added and annealed. The beads are stringently washed so that only the perfectly matched peptide nucleic acid (PN) probe remains annealed, MLDI matrix is added to the bead mix, followed by MLDI TOF mass analysis, where the specific mass-to-change ratio () value detected divulges the identity of the single-nucleotide polymorphism. Data shown is for PCR amplicons from the human tyrosinase gene exon 4, which were analysed at four separate polymorphic positions, identified as 419, 422, 446 and 448. Each unique, labeled peak in the composite mass spectra shown corresponds to the presence of the wild-type (WT) or variant (VR) allele of a unique single-nucleotide polymorphism. (b) Sample was heterozygous for the 446 alleles and homozygous WT for the other three alleles; (c) sample was heterozygous for the 448 alleles and homozygous WT at the other three positions. (Reproduced, with permission, from Ref. 52.) TIBTECH FEBRURY 2000 (Vol. 18) 81

6 Counts T3 C CP450 C/T NFI CC6 C/ 2M TL IF LD8 ISB2 T LDLR IL1 PRS Individual 2 Counts Counts (b) (c) CP450 C/T T3 C NFI CC6 C/ 2M T3 T CP450 C/T NFI CC6 2M TL T/ IF TL T IF LD8 LD8 ISB2 T ISB2 C LDLR LDLR IL1 IL1 PRS PRS Individual 6 Individual 8 Mass () Figure 5 Mass spectra containing multiplex analysis results from minisequencing of 12 single-nucleotide polymorphisms (SNPs) for three different individuals [ individual 2, (b) individual 6, (c) individual 8 (Ref. 61)]. The labeled peaks represent each extended primer of unique mass. Unlabeled peaks are from non-extended primers that are not fully consumed in the multiplex extension reaction. Peak doublets correspond to a heterozygous genotype at a SNP position; single peaks correspond to a homozygous genotype. bbreviation:, mass-to-charge ratio. (Reproduced, with permission, from Ref. 61.) because of the considerable limitations inherent to PCR in a high-throughput system, including crossover contamination problems 63, and variability in reaction conditions and yields between different targets 64,65. This direct approach employs the Invader assay 66 (Third Wave Technologies, Madison, WI, US), a novel method for analysing SNPs that involves the sequencespecific hybridization of two oligonucleotides to form an overlapping structure at the polymorphic position. This is followed by enzymatic cleavage and amplification of an allele-specific, short oligonucleotide signal molecule, which is derived from this overlap structure. In this MLDI-based approach, the signal molecules produced in the Invader reaction contains a biotin group, enabling solid-phase sample preparation by capturing these molecules on streptavidin-coated magnetic beads. These are then washed to remove contaminants and the clean signal molecules are eluted for MLDI TOF mass-spectrometric analysis 62. This approach was used to analyse 12 separate SNPs by MLDI TOF MS directly from genomic DN (Fig. 6), demonstrating its potential for high-throughput SNP analysis at the levels necessary for comprehensive genetic studies. Outlook dvances made in recent years have shown that MLDI TOF MS clearly has the potential to become the technology of choice for high-throughput SNP genotyping. For this to become a reality, existing approaches need to be developed further and new methods discovered, with an emphasis on the development of methods that exploit, to the greatest possible 82 TIBTECH FEBRURY 2000 (Vol. 18)

7 Invader oligonucleotide 3 5 Enzymatic cleavage site 5 llele-specific probe oligonucleotide 3 * 5 enomic DN 5 3 Oligonucleotide cleavage product Polymorphic position (b) SNP 1: 5 C[T/C]TCT 3 (c) SNP 2: 5 TC[/C]CTT 3 [T] 5 TTT-biotin 1234 [] 5 TTT-biotin 1234 [C] 5 TTTT-biotin 1538 Intensity Intensity Figure 6 Single-nucleotide polymorphism (SNP) analysis using the Invader assay and MLDI TOF mass spectrometry. Schematic diagram illustrating the fundamental components of the Invader assay 66, where two oligonucleotides, the Invader oligonucleotide and the Probe oligonucleotide, are hybridized to a genomic DN target, such that an overlap structure is formed containing a flap on the 5 end of the probe oligonucleotide. This flap is enzymatically cleaved, producing a cleavage product that is amplified linearly over time. llele-specific probe oligonucleotides that produce different-sized oligonucleotide cleavage products are used for single-nucleotide-polymorphism analysis using the Invader assay and MLDI TOF mass spectometry 62. The data show representative results obtained using this approach, in which two allele-specific, biotinylated signal molecules are produced in the Invader assay for any polymorphic position analysed: dt 3 -biotin, with a deprotonated, single-charged mass [(M-H) ] of 1234 Da; dt 4 -biotin, with a (M-H) of 1538 Da. The analysis results for two separate single-nucleotide polymorphisms are presented, with the sequence context for each polymorphic position shown; the two polymorphic nucleotides are in brackets and the signal molecules from the Invader assay corresponding to each allele are also shown. (b) SNP 1 was homozygous for the T allele, as shown by the single peak at a mass-to-charge ratio () value corresponding to the T allele signal; (c) SNP 2 was heterozygous for both alleles and therefore two peaks were observed. extent, the ability of MLDI TOF MS to analyse many SNPs in a multiplex automated fashion. iven a few more advances, attaining the ultimate goal of genotyping hundreds to thousands of SNPs literally in a few pulses of a laser is within reach, an achievement that would be a significant step towards fully reaping the benefits offered by SNPs to biomedical research. cknowledgments We would like to acknowledge M. Scalf for his help in preparing this manuscript. T.J. riffin was supported (in part) by a Proctor and amble predoctoral fellowship. References 1 Wang, D.. et al. (1998) Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome. Science 280, Schafer,.J. and Hawkins, J.R. (1998) DN variation and the future of human genetics. Nat. Biotechnol. 16, Landegren, U. et al. (1998) Reading bits of genetic information: methods for single-nucleotide polymorphism analysis. enome Res. 8, Brookes,.J. (1999) The essence of SNPs. ene 234, Kruglyak, L. (1998) The use of a genetic map of biallelic markers in linkage studies. Nat. enet. 17, Collins, F.S. et al. (1998) New goals for the U.S. Human enome Project: Science 282, Ramsay,. (1998) DN chips: state-of-the-art. Nat. Biotechnol. 16, Nollau, P. and Wagener, C. (1997) Methods for detection of point mutations: performance and quality assessment. Clin. Chem. 43, Karas, M. and Hillenkamp, F. (1988) Laser desorption ionization of proteins with molecular masses exceeding daltons. nal. Chem. 60, Ehring, H. et al. (1992) Role of photoionization and photochemistry in ionization processes of organic molecules and relevance for matrix-assisted laser desorption ionization mass spectrometry. Org. Mass Spectrom. 27, TIBTECH FEBRURY 2000 (Vol. 18) 83

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