Discrimination of Single-Nucleotide Polymorphisms in Human DNA

Elmer, Applied Biosystems Division, Foster City, CA), and 500. nM forward and ... J.; Nielsen, P. E.; Tsui, L-C.; Zielinski, J. Nature (London) 1996, ...
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Anal. Chem. 1997, 69, 4197-4202

Discrimination of Single-Nucleotide Polymorphisms in Human DNA Using Peptide Nucleic Acid Probes Detected by MALDI-TOF Mass Spectrometry Philip L. Ross,* Katherine Lee,† and Phillip Belgrader

DNA Technology Development Branch, Center for Molecular and Medical Genetics, Armed Forces Institute of Pathology, Rockville, Maryland 20850

Human genomic and mitochondrial DNA contain large numbers of single-nucleotide polymorphisms (SNPs), many of which are linked to known diseases. Rapid and accurate genetic screening for important SNPs requires a general methodology which is easily implemented. We present here an approach to SNP discrimination based on high-specificity hybridization of peptide nucleic acid (PNA) probes to PCR-amplified DNA. The assay is directly applied to polymorphisms located within hypervariable region 1 of the human mitochondrial genome and type 1 suballeles of the human leukocyte antigen DQr gene. Captured, single-stranded DNA molecules prepared by PCR amplification are hybridized with PNA probes in an allele-specific fashion. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is then used for rapid, precise, and unambiguous detection and identification of the hybridized PNA probes. Since PNA oligomers bind strongly to complementary DNA under minimal salt conditions, the use of PNA probes is compatible with MALDI-TOFMS. The unparalleled ability of MALDI-TOFMS analysis in terms of molecular weight resolution and accuracy, in conjunction with the highly specific PNA hybridization afforded by this method, offers promise for development into a multiplexed, high-throughput screening technique.

an assay should be expandable to a multiplex format, whereby a large number of possible mutations of a single or several genes can be surveyed. Current PCR-based methods to characterize SNPs include allele-specific amplification,5 allele-specific oligonucleotide hybridization,6,7 genetic bit analysis,8 ligation assays,9-13 and restriction fragment-length polymorphism.14 These techniques utilize slab gel electrophoresis, plate readers, or reverse dot-blot hybridization strips for analysis. A strategy for screening an entire panel of known SNPs requires a technology that is much more rapid than those conventionally used. Mass spectrometry, specifically matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOFMS),15,16 is a technique that possesses the versatility and rapid analysis capability desirable for high throughput genotyping. MALDI-TOFMS has evolved into an accurate, sensitive, and rapid method for molecular weight and sequence determination of peptides, proteins and small DNA fragments.17 Problems have been encountered in size analysis and/or sequence determination of DNA oligomers larger than 30 bases. This difficulty stems primarily from the extensive fragmentation18 that occurs during the MALDI process or the formation of cation adducts that severely degrade resolution and sensitivity. Typically, a series of time- and labor-intensive steps are required to preconcentrate and purify DNA for successful analysis by MALDI-TOFMS.19-21

The extensive effort currently devoted to genome sequencing has revealed a strong correlation between sequence polymorphism, particularly base deletions and substitutions, and the occurrence of numerous genetic diseases.1 It has been estimated that single-nucleotide polymorphisms (SNPs) occur at ∼1 in every 400 nucleotides,2 a frequency that is continually updated as the human genome is unraveled. SNPs therefore provide a valuable source of genetic markers for determining identity, establishing genetic linkages, and predicting/diagnosing diseases.3,4 To take maximum advantage of such genetic information for clinical or forensic purposes, it is necessary to develop practical and inexpensive yet reliable assays for SNP detection. Ultimately, such

(5) Wu, D. Y.; Ugozzoli, L.; Pal, B. K.; Wallace, R. B. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 2757-2760. (6) Saiki, R. K.; Bugawan, T. L.; Horn, G. T.; Mullis, K. B.; Erlich, H. A. Nature 1986, 324, 163-166. (7) Saiki, R. K.; Walsh, P. S.; Levenson, C. H.; Erlich, H. A. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 6230-6234. (8) Nikiforov, T. T.; Rendle, R. B.; Goelet, P.; Rogers, Y.-H.; Kotewicz, M. L.; Anderson, S.; Trainer, G. L.; Knapp, M. R. Nucleic Acids Res. 1994, 22, 4167-4175. (9) Baron, H.; Fung, S.; Aydin, A.; Bahring, S.; Luft, F. C.; Schuster, H. Nature Biotechnol. 1996, 14, 1279-1282. (10) Barany, F. Proc. Natl. Acad. Sci. US.A. 1991, 88, 189-193. (11) Nickerson, D. A.; Kaiser, R.; Lappin, S.; Stewart, J.; Hood, L.; and Landegren U. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 8923-8927. (12) Belgrader, P.; Marino, M. A.; Lubin, M.; Barany, F. Genome Science and Technol. 1996, 1, 77-87. (13) Delahunty, C.; Ankener, W.; Deng, Q.; Eng, J.; Nickerson, D. A. Am. J. Hum. Genet. 1996, 58, 1239-1246. (14) Boehm, C. D. Clin. Chem. 1989, 35, 1843-1848. (15) Hillenkamp, F.;, Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A-1202A. (16) Cotter, R. J. Anal. Chem. 1992, 64, 1027A-1039A. (17) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1996, 68, 599R651R. (18) Tang, W.; Zhu, L.; Smith, L. M. Anal. Chem. 1997, 69, 302-312. (19) Ch’ang, L. Y.; Tang, K.; Schell, M.; Ringelberg, C.; Matteson, K. J.; Allman, S. L.; Chen, C. H. Rapid Commun. Mass Spectrom. 1995, 9, 772-774.

* Corresponding author (e-mail: [email protected]). † University of MarylandsBaltimore County, 5401 Wilkens Ave., Baltimore, MD 21228. (1) Guyer, M. S.; Collins, F. S. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 1084110848. (2) Cooper, D. N.; Smith, B. A.; Cooke, H. J.; Niemann, S.; Schmidke, D. Hum. Genet. 1995, 69, 201-205. (3) Landegren, U.; Kaiser, R.; Caskey, C. T.; Hood, L. Science 1988, 242, 229237. (4) Antonarakis, S. E. N. Engl. J. Med. 1989, 320, 153-163. S0003-2700(97)00396-X CCC: $14.00

© 1997 American Chemical Society

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Table 1. DNA Sequences and Corresponding PNA Probes for Loci Possessing Single-Nucleotide Polymorphisms location

local DNA sequencea

PNA probe sequenceb

probe mol wtc

mt 16220A mt 16220G mt 16293A mt 16293G HLA DQR 1.1 HLA DQR 1.2, 4

CAGCAATCAACCCTCAA CAGCAATCAGCCCTCAA ACCTACCCACCCTTAACA ACCTACCCGCCCTTAACA GAGATGAGGAGTTCTAC GAGATGAGCAGTTCTAC

GCAATCAACCCTC GCAATCAGCCCTCT CTACCCACCCTTA CTACCCGCCCTTAT TAGAACTCCTCATCTCC TAGAACTGCTCATCTCCT

3449.9 3732.2 3401.2 3684.2 4500.1 4806.6

a Forward DNA sequence is listed. For mtDNA fragments, biotin label is attached to the reverse PCR primer, while for HLA DQR fragments, forward primer is biotinylated. b HLA DQR PNA probes are complementary to the reverse DNA strand. Polymorphic sites are indicated in boldface. The 16220‘G’, 16293‘G’, and DQR 1.2, 4 probes have an extra T nucleotide tail to aid in visualization of molecular weight differences. c PNA oligomer molecular weights are based on monomer values (A, 275.3; C, 251.3; G, 291.3, T, 266.3) plus 17.0 for the amino terminus.

Peptide nucleic acids (PNAs)22 are a recently developed DNA analog in which the sugar phosphate backbone has been replaced by a poly[N-(2-aminoethyl)glycine] structure carrying normal purine and pyrimidine nucleobase side chains. PNA mimics DNA in the ability to hybridize to complementary strands of DNA,22 and it has been demonstrated that a single base mismatch located at a central position lowers the melting temperature by 8-20 °C. The use of PNAs in DNA typing, however, has largely remained limited to PCR clamping,23 whereby PNA binds tightly to a target site and prevents primer annealing during PCR. Application of PNA probes toward SNP recognition utilizing free solution capillary electrophoresis (CE) has also been reported.24,25 In CEbased experiments, PNA-DNA hybrids are prepared and analyzed at elevated temperature in the presence of denaturants to control hybridization stringency. Single base discrimination of a PCR product was demonstrated by Perry-O’Keefe et al.25 However, since mutant and wild-type probes cannot be simultaneously discriminated under the conditions described, the analysis requires separate individual CE runs to distinguish a correct genotyping result from background. The successful application of MALDI-TOFMS to rapid characterization of PNA oligomers with varying base compositions has been demonstrated.26 The strong peptide-like backbone of the PNA molecules is resistant to fragmentation during the MALDI process. Since single-base substitutions in PCR products cannot be directly addressed by MALDI-TOFMS, a probe-based strategy for SNP determination is required. Use of PNA probes with mass spectrometric detection is a particularly attractive approach. Since PNA-DNA hybridization is favored under low-salt conditions,22 MALDI-TOFMS should be directly compatible with in situ detection of hybridized PNA probes. We report here a method for SNP detection that couples the sensitivity and accuracy of MALDI-TOFMS with the specificity and stability of PNA-DNA hybridization. Following PCR ampli(20) Jurinke, C.; van den Boom, D.; Jacob, A.; Tang, K.; Wo¨rl, R.; Ko ¨ster, H. Anal. Biochem. 1996, 237, 174-181. (21) Doktycz, M. J.; Hurst, G. B.; Habibi-Goudarzi, S.; McLuckey, S. A.; Tang, K.; Chen, C. H.; Uziel, M.; Jacobson, K. B.; Woychik, R. P.; Buchanan, M. V. Anal. Biochem. 1995, 230, 205-214. (22) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. Nature (London) 1993, 365, 566-568. (23) Theide, C.; Bayerdorffer, E.; Blasczyk, R.; Wittig, B.; Neubauer, A. Nucleic Acids Res. 1996, 24, 983-984. (24) Carlsson, C.; Jonsson, M.; Norde´n, B.; Dulay, M. T.; Zare, R. N.; Noolandi, J.; Nielsen, P. E.; Tsui, L-C.; Zielinski, J. Nature (London) 1996, 380, 207. (25) Perry-O’Keefe, H.; Yao, X-W.; Coull, J. M.; Fuchs, M.; Egholm, M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14670-14675. (26) Butler, J. M.; Jiang-Baucom, P.; Huang, M.; Belgrader, P.; Girard, J. Anal. Chem. 1996, 68, 3283-3287.

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fication, the method is applied directly to polymorphisms located within human mitochondrial DNA (mtDNA) and in genomic DNA using human leukocyte antigen (HLA) DQR polymorphisms. PNA probes complementary to each of two known alleles were designed for each of the aforementioned loci. The probes were hybridized to single-stranded PCR-amplified DNA immobilized onto magnetic particles.27 Following washing, the immobilized PNA-DNA complex is then directly analyzed by MALDI-TOFMS. The PNA molecules denature from the DNA during the MALDI process, providing accurate mass spectral data. The ability to distinguish SNPs in amplified DNA consistently with high specificity is demonstrated. EXPERIMENTAL SECTION Synthesis and Purification of PNA Probes. The PNA probes (Table 1) were synthesized on a PerSeptive Biosystems Expedite nucleic acid synthesizer modified for peptide nucleic acid chemistry. The crude PNA product was removed from the universal PNA synthesis column by treatment with a 4:1 TFA/ Cresol mixture. Following precipitation and washing with cold ether, the pure PNA molecules were isolated using reversed-phase HPLC on a Zorbax C18 analytical column held at a temperature of 55 °C. A linear gradient of 50:50 acetonitrile/water with 0.1% TFA was used to purify PNA synthesis mixtures. PNA purity in collected fractions was determined by MALDI-TOF analysis. Impurities were detected at low abundance but were in molecular weight ranges that did not overlap with any of the pure PNA probes used. PNA oligomers for hybridization experiments were quantitated by 260 nm absorbance measurements. DNA Amplification. DNA was isolated from whole blood from individuals with known mitochondrial sequences using the Puregene (Minneapolis, MN) DNA extraction kit. Amplification was performed using buffer containing 2.5 mM MgCl2, 2 mM each deoxynucleotide triphosphate, Perkin-Elmer 10× PCR buffer, 1 ng/µL DNA, 1.25 units of Amplitaq DNA polymerase (Perkin Elmer, Applied Biosystems Division, Foster City, CA), and 500 nM forward and reverse primers and typically 25 ng of DNA. The primers were synthesized using standard phosphoramidite chemistry on an Applied Biosystems 392 DNA synthesizer. For mtDNA amplification, the primer pair (forward primer 5′-TTG ACC AAC TGT AGT ACA TA, reverse primer 5′-biotin-GAG GAT GGT GGT CAA GGG AC) was designed to amplify a 268-bp product between position 16143 and 16410 of the human mitochondrial genome. Appropriate primers were 5′ labeled with biotin using Biotin-On (27) Tang, K.; Fu, D.; Ko ¨tter, S.; Cotter, R. J.; Cantor, C. R.; Ko ¨ster, H. Nucleic Acids Res. 1995, 23, 3126-3131.

Phosphoramidite (Clontech Labs, Palo Alto, CA). The purity of the primers was assessed by MALDI-TOFMS analysis to be close to 100% and they were therefore used without purification. PCR was performed on a Perkin Elmer Model 9600 thermal cycler using a program starting with a 94 °C soak for 1 min, followed by 32 cycles of 94 °C denature for 30 s, 56 °C anneal for 10 s, and 70 °C extension for 30 s, followed by a 5 min soak at 70 °C. Amplification products were stored at 4 °C prior to use. For amplification of HLA DQR products (forward primer 5′-biotin-GCC AGT ACA CCC ATG AAT TTG, reverse primer 5′-ACA GCC ATG TTT CTC AGT GCA), reaction conditions were similar to those described above with the following exceptions: 4 mM final concentrations of MgCl2 were used, and a 60 °C, 30 s anneal step was used in the thermal cycling program. Mass Spectrometry. MALDI-TOF mass spectrometry was performed on a PerSeptive Biosystems Voyager RP workstation operating in linear, positive ion mode with a static accelerating voltage of 25 kV. All analyses used a matrix solution containing 10 mg/mL sinapinic acid (SA) in a 2:1 water/acetonitrile solution. Matrix solutions were regularly analyzed for the presence of PNA or other contamination. All spectra were collected by averaging between 30 and 50 individual laser shots. Instrument calibration was performed using either cytochrome c or PNA standards. PNA Hybridization Experiment. Streptavidin-coated magnetic beads (PerSeptive Biosystems) in wash buffer (0.5 M NaCl, 0.005 M EDTA) were added to 50 µL of PCR reaction mixture and incubated for 15 min. The captured double-stranded product was then washed and denatured by treatment with 0.1 N NaOH. Following removal of the free strands, the beads were resuspended in hybridization buffer and combined with an equimolar mixture of PNA probes in quantities ranging from 2 to 25 pmol. A variety of hybridization buffer compositions, hybridization temperatures, and posthybridization wash protocols were examined to optimize specificity. Hybridization solutions consisting of 0.1 M NaCl and 0.005 M EDTA, 1× SSPE, or 1× TBE were used at temperatures ranging from 45 to 72 °C. Hybridization experiments were also conducted at 70 °C using 10 mM phosphate buffer (pH 7.0). Trials using hybridization solutions containing 20-50% formamide (Boehringer-Mannheim, Indianapolis, IN, prepared according to manufacturers instructions and diluted accordingly for final formamide concentration) were carried out at temperatures ranging from 37 to 50 °C. After initial experiments, a prehybridization step using bovine serum albumin (BSA, Life Technologies, Gaithersburg, MD) at concentrations ranging from 0.05 to 0.4% was included to eliminate background hybridization. The hybridization mixture was incubated for time periods ranging from 10 to 30 min, following which the beads were washed with several aliquots of deionized water. The captured beadDNA-PNA complex was combined with 5 µL of matrix solution, and 1 µL of the resultant mixture was deposited onto the mass spectrometer sample probe.

Figure 1. Schematic outline of PNA hybridization MALDI-TOFMS experiment. Biotin-labeled DNA is captured onto streptavidin-coated magnetic particles. The remaining PCR solution components are washed away and then the DNA is denatured with NaOH. The free strands and NaOH are washed away, and the captured single strands are hybridized with PNA mixture. Hybridization buffer and unbound PNA are washed away, and the remaining captured DNA:PNA hybrids are mixed with matrix solution and analyzed by MALDI-TOFMS. During the MALDI process, the hybridized PNA probe desorbs from the immobilized ssDNA, giving a direct readout of the PNA molecular weight.

RESULTS The principle components of the procedure are outlined in Figure 1. Probe pairs were designed to recognize polymorphisms at position 16 220 and 16 293 in hypervariable region 128-31 of the human mitochondrial genome and type 1 HLA DQR suballeles.

Extracted DNA was selected from individuals known to possess target polymorphisms through analysis by cycle sequencing (mtDNA) or reverse dot-blot hybridization kits (HLA DQR typing kit, Perkin Elmer Corp.). Pertinent sequence data of the PCR products and of the corresponding PNA probes are listed in Table 1. Both mitochondrial pairs match A f G polymorphisms, while the HLA DQR probe pair is a G f C polymorphism. The additional T bases (Table 1) are included to aid in visualization of molecular weight differences between probe pairs. Since these T bases are at the 3′ ends of the PNA probe and are not complementary, they do not significantly influence the effective melting temperature of the respective PNA-DNA duplex. Typical results observed from application of the assay are illustrated in Figure 2. Figure 2A shows the MALDI-TOF mass spectrum obtained from direct analysis of an equimolar mixture (0.5 pmol each) of PNA probes 16293‘A’ and 16293‘G’. This spectrum establishes that equivalent concentrations of each probe have close to equal detection sensitivity. Panels B and C show direct analysis of captured PNA-DNA duplex following hybridization with an equimolar mixture of 16293A and 16293G PNA probes with amplified DNA. Typical results obtained for individuals with ‘A’ and ‘G’ alleles are shown. The experiment was performed using a 15 min prehybridization step at 45 °C, followed by a 30 min hybridization period. Both prehybridization and hybridization

(28) Stoneking, M.; Hedgecock, D.; Higuchi, R. G.; Vigilant, L.; Erlich, H. A. Am. J. Hum. Genet. 1991, 48, 370-382. (29) Vigilant, L.; Pennington, R.; Harpending, H.; Kocher, T. D.; Wilson, A. C. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 9350-9354.

(30) Horai, S.; Hayasaka, K. Am. J. Hum. Genet. 1990, 46, 828-842. (31) Anderson, S.; Bankier, A. T.; Barrell, B. G.; de Bruijn, M. H. L.; Coulson, A. R.; Drouin, J.; Eperon, I. C.; Nierlich, D. P.; Roe, B. A.; Sanger, F.; Schreier, P. H.; Smith, A. J. H.; Staden, R.; Young, I. G. Nature 1981, 290, 457-465.

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Figure 2. Mass spectra obtained (A) after mixing equimolar quantities (1.0 pmol) of purified mt16293 probe pair with SA matrix and (B) and (C) following 30 min hybridization of mt16293 PNA probe pair with denatured PCR product containing 16293A and 16293G sequences, respectively. Prehybridization and hybridization were carried out using buffer containing 37.5% formamide and 0.1% BSA.

were carried out using a buffer containing 37.5% formamide and 0.1% BSA. A mixture containing 3 pmol of each probe was added to mark the beginning of the hybridization step. The spectra (Figure 2B,C) demonstrate specificity between alleles differing by a single nucleotide with minimum nonspecific background. The experimentally observed signal intensities are shown with each spectrum. These values reveal a substantial decrease in the overall signal intensity between analysis of the probe mixture (Figure 2A) and analysis of the captured PNADNA duplex (Figure 2B,C). Such a drop is expected since, under stringent hybridization conditions, a considerable number of perfectly matched PNA probes will remain unbound. Additionally, the presence of the magnetic particles and the bound DNA will interfere with the cocrystallization of the PNA with the matrix. The presence of the interfering magnetic particles also necessitates an increase in laser power of ∼20% over pure PNA before signals appear. Use of automatic peak labeling produced molecular weight assignments that differed by less than 0.2% between experiments, without the need for instrument recalibration with each experiment. The results shown in Figure 3 verifies that high specificity can be achieved consistently between individuals with similar genotypes. Both 16220A type individuals show essentially 100% specificity between the 16220 A and G probe pair. The data for individual 3 (Figure 3B) were obtained using 15 min prehybridization and 30 min hybridization, while for individual 4 (Figure 3C), results with equal specificity were obtained using 5 min 4200 Analytical Chemistry, Vol. 69, No. 20, October 15, 1997

Figure 3. Mass spectra obtained (A) following mixing equimolar quantities (0.5 pmol) of purified mt16220 probe pair with SA matrix, (B) following 15 min prehybridization and 30 min hybridization, and (C) following 5 min prehybridization and 10 min hybridization of mt16220 PNA probe pair with denatured PCR product from different individuals having 16220A alleles.

prehybridization and 10 min hybridization steps. There is a resultant 2-fold decrease in signal intensity which may be correlated with the shorter hybridization time; however, there is ample signal for accurate data interpretation. To demonstrate that SNPs in amplified genomic DNA can be discriminated with equivalent specificity, we selected human DNA samples with known HLA DQR type 1 suballeles. The 1.1 allele contains a unique sequence motif and is distinguished by the 1.1 PNA probe, whereas the 1.2 and 4 alleles share a common sequence region recognized by the 1.2, 4 PNA probe. The 1.1 and 1.2, 4 probes differ by a single G f C polymorphism (Table 1). Figure 4 shows results from hybridization of HLA DQR PNA probes using 10 min prehybridization and 15 min hybridization at 45 °C with buffer containing 37.5% formamide and 0.1% BSA. Clear single-base discrimination between 1.2 and 1.1 genotypes is demonstrated (Figure 4B), as is identification of a heterozygous 1.1, 4 individual (Figure 4C). DISCUSSION The ability to discriminate between SNPs with specificity approaching 100% via MALDI-TOFMS analysis of allele-specific PNA probes is demonstrated here. This level of specificity is achieved under identical experimental conditions for PCR-amplified human mitochondrial and genomic DNA. The main difference with respect to detection sensitivity and difficulty in achieving high allele specificity between mitochondrial and genomic DNA lies in the larger template copy number of mtDNA. Although this

Figure 4. MALDI-TOFMS spectra obtained from (A) an equimolar mixture of HLA DQR 1.1 and 1.2,4 PNA probes, (B) hybridization of equimolar mixture of 1.1 and 1.2, 4 probes with PCR-amplified DNA having 1.2, 2 and (C) 1.1, 4 genotypes. Prehybridization and hybridization (formamide/BSA) times were 10 and 20 min, respectively.

circumstance may increase somewhat the final copy number obtained following PCR amplification, it is shown here that SNP discrimination in DNA from either source is accomplished under identical conditions. By elimination of nonspecific hybridization, the unambiguous detection of heterozygosity is also demonstrated. The incorporation of MALDI-TOFMS with solid-phase capture of DNA has a number of distinct and important advantages over recently described approaches utilizing PNA oligomers and free solution capillary electrophoresis.24,25 In the CE-based procedures, hybridization stringency is controlled via buffer composition and elevated column temperatures, and the resultant PNA-DNA duplexes are identified by a shift in mobility from the unhybridized oligonucleotide. Using mass spectrometry, however, an accurate, numerical molecular weight readout can be obtained reproducibly within seconds, as illustrated above, without the requirement of regular instrument recalibration or standardization as experimental conditions change. The results presented here (Figures 2-4) were all obtained using an external calibration file. The calibration file used for Figure 4 was generated during a different loading of the sample plate, yet mass accuracy within 2 Da (0.05%) was observed. In general, mass accuracies determined using calibration files generated in some cases on a different day varied by up to 0.2%, which does not significantly compromise discrimination with the PNA probes used here. The molecular weight stability indicated by these findings greatly simplifies the analysis for users with minimal familiarity with the instrumentation.

Another important aspect of this technique lies in the ability to acquire MALDI-TOFMS information by direct interrogation of the immobilized PNA-DNA hybrids. The purification of PCR product and subsequent hybridization of PNA can therefore be performed in a single reaction tube. This compatibility of MALDITOFMS analysis with solid-phase capture also facilitates drastic alteration of chemical conditions, particularly hybridization and washing stringency, with virtually no change on the effective PNA probe molecular weight obtained. As a result, hybridization stringency close to 100% for single-nucleotide discrimination has been achieved. Complete elimination of nonspecific background in this fashion is an absolute necessity to reduce ambiguity in assigning heterozygosity. The incorporation of fluorescent chemistry as is encountered in oligonucleotide hybridization assays is not necessary since SNP identification is based on an intrinsic property of the corresponding PNA probe, its exact molecular weight. A final, unanticipated advantage imparted by the solid-phase nature of this approach is that samples, once co-deposited with matrix solution and allowed to dry onto the sample plate, were observed to remain stable in crystallized form for several hours. Spectra were obtained from samples kept covered but at ambient temperature and pressure up to 18 h with no loss in genotyping accuracy or specificity and only marginal reduction in signal intensity. Further investigation into the stability of deposited PNA-DNA samples was not pursued. A variety of hybridization conditions were investigated, some of which yielded an undesirable level of nonspecific background. Relative background levels of noncomplementary PNA probe approaching 20% were observed in experiments conducted with both mt16220 and mt16293 probe pairs under low-salt (no formamide) conditions at 70 °C. This background persisted even with the inclusion of prehybridization with BSA. Although the nominal melting temperatures for PNA-DNA hybrids with a single base mismatch have been determined to be at least 8 °C lower than a perfect complement,22 the actual melting curve is perhaps a more significant characteristic with respect to maximizing absolute specificity and eliminating background signal. Overlap in melting curves can occur such that 50% melting of a perfect PNA-DNA complement is observed at the temperature where only 90% of single-base mismatched PNA-DNA duplexes have melted. In our case, such an overlap explains the consistently present nonspecific PNA probe signal in 70 °C, low-salt experiments. Since background PNA hybridization to the magnetic particles was eliminated by prehybridization, we hypothesize that there is a sharper melting profile of the PNA-DNA hybrid at lower temperatures in 37.5% formamide. This hypothesis is in agreement with the findings of Perry-O’Keefe et al.,25 who reported an apparent 13 °C temperature interval in 30% formamide with no effective hybridization overlap between matched and single-base mismatched PNA probes. It is observed with the experiments completed to date that there is no substantial loss in peak resolution between MALDITOF analysis of the purified PNA mixtures and analysis of the immobilized, hybridized PNA probe. The high molecular weight resolution is perhaps the most powerful aspect of this technique. Probes with similar lengths and only minor variations in nucleotide composition can be readily distinguished and unambiguously identified on the basis of accurate mass assignment alone. Furthermore, we have demonstrated that PNA probes ranging in Analytical Chemistry, Vol. 69, No. 20, October 15, 1997

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PNA and DNA concentrations. These preliminary results show that resolution between PNA oligomers of similar length but differing base composition is straightforward. This may have particularly significant implications in the context of simultaneous probing of multiple mutation sites using PNA probes varying in length by one or two bases with similar hybridization characteristics. Enhancement of data resolution through implementation of currently available signal enhancement capabilities32 remains unexplored in MALDI-TOFMS analysis of PNA oligomers. An increase in resolution of probes with similar molecular weight translates to a greater effective data space from which to construct appropriate multiplex PNA probe libraries. We aim to explore further the development of multiplex analysis using PNA probes incorporating improved MALDI-TOFMS instrumentation.

Figure 5. MALDI-TOFMS spectra obtained (A) before and (B) following hybridization of equimolar quantities of mt16220 and mt16293 PNA probes with PCR product from individual having 16220A and 16293A polymorphisms.

length from 13 to 18 bases are hybridized in a highly specific manner under identical conditions. The ability to analytically resolve a large number of PNA probes with a considerable range of available sequence combinations is suggested by these findings and represents a definitive step toward general-purpose multiplex assays based on PNA hybridization and MALDI-TOFMS analysis. As an initial exploration toward the ultimate goal of multiplex SNP determination, several preliminary experiments using simultaneous hybridization of multiple probe sets were conducted. As shown in Figure 5 for an individual with 16220‘A’ and 16293‘A’ polymorphisms, encouraging results have been obtained, demonstrating the feasibility of expansion to multiplex analysis. For simultaneous detection of multiple SNPs located within a single PCR fragment, it will be necessary to establish optimum relative (32) Roskey, M. T.; Juhasz, P.; Smirnov, I. P.; Takach, E.; Martin, S. A.; Haff, L. A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4724-4729.

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CONCLUSIONS We have demonstrated that mass spectrometric detection of allele-specific PNA probes is a straightforward, accurate, and highly specific means of detecting SNPs in amplified DNA. The incorporation of solid-phase capture of allele-specific PNA-DNA duplexes permits the entire procedure to be carried out in a single reaction tube using simple operations that can be implemented into an automated format. The most powerful feature of the approach is the use of MALDI-TOFMS analysis, which permits direct analysis in seconds. Through MALDI-TOFMS analysis, positive and unambiguous probe detection with minimal background is accomplished for normal, mutant, and heterozygous polymorphisms. The rapid and stringent PNA hybridization observed suggests that the procedure as described here can be simplified dramatically, perhaps to within 30 min following PCR. Finally, encouraging preliminary results indicating the ability to expand the assay to simultaneous detection of multiple SNPs are also presented. ACKNOWLEDGMENT The authors wish to thank Kristal Allen-Weaver for technical assistance. The views stated here are the opinions of the authors and in no way reflect the position of the U.S. Army, the U.S. Air Force, or the Department of Defense. Received for review April 15, 1997. Accepted August 5, 1997.X AC9703966 X

Abstract published in Advance ACS Abstracts, September 15, 1997.