Analysis of Biosensor Chips for Identification of Nucleic Acids

Life Sciences Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee ... of the immobilized ODN, the method of labeling and detection ...
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Anal. Chem. 1997, 69, 3747-3753

Analysis of Biosensor Chips for Identification of Nucleic Acids Heinrich F. Arlinghaus* and Margaret N. Kwoka

Atom Sciences, Inc., Oak Ridge, Tennessee 37830 K. Bruce Jacobson

Life Sciences Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831

Two novel DNA-sequencing methods are described that use DNA hybridization biosensor chips. These two techniques involve either labeling the free nucleic acid with enriched stable isotopes or hybridizing DNA without labels to immobilized peptide nucleic acid (PNA) and detecting the phosphorus present in the DNA but not in the PNA. Sputter-initiated resonance ionization microprobe analysis was used to detect the presence of enriched tin isotope-labeled DNA and of phosphorus in natural DNA as a means to identify the presence of DNA after hybridization to oligodeoxynucleotides (ODNs) or PNAs, respectively, immobilized on a biosensor chip. The data clearly demonstrate that excellent discrimination between complementary and noncomplementary sequences can be obtained during hybridization of DNA to either ODNs or PNAs. The capability to detect different enriched stable isotope-labeled DNAs simultaneously allows high degrees of multiplexing which may be very advantageous for hybridization kinetics studies in complex systems, as well as significantly increasing the speed of analysis. Alternatively, by using natural DNA with PNA biosensor chips, discrimination for single-point mutation could be increased because of improved hybridization kinetics and direct analysis of genomic DNA may become possible without amplification. Both methods have the potential to provide a rapid method for DNA/RNA sequencing, diagnostics, and mapping. Rapid and automated methods of nucleic acid sequencing and diagnostics would undoubtedly lead to a deeper understanding of genetics and disease processes. However, despite the significance of sequencing genes and monitoring mutations and other variants in the genome, the present methods used in research settings are still unnecessarily slow. Hybridization of DNA fragments to oligonucleotide arrays may enable large-scale DNA analysis without electrophoresis and is particularly well suited for DNA diagnostics, sequencing of cDNAs or genomic clones, analysis of DNA sequence polymorphisms, and identification of expressed genes.1-11 (1) Drmanac, R.; Strezoska, Z.; Labat, I.; Drmanac, S.; Crkvenjakov, R. DNA Cell Biol. 1992, 9, 527-534. (2) Guo, Z.; Guilfoyle, R. A.; Thiel, A. J.; Wang, R.; Smith, L. M. Nucleic Acids Res. 1994, 22, 5456-5465. (3) Bains, W. Chem. Br. 1995, 31, 122-125. (4) Southern, E. M.; Maskos, U.; Elder, J. K. Genomics 1992, 13, 1008-1017. (5) Drmanac, R.; Crkvenjakov, R. J. Genome Res. 1992, 1, 59-79. (6) Drmanac, R.; Drmanac, S.; Labat, I.; Crkvenjakov, R.; Vicentic, A.; Gemmell, A. Electrophoresis 1992, 13, 566-573. S0003-2700(97)00267-9 CCC: $14.00

© 1997 American Chemical Society

The primary event in oligonucleotide array sequencing is the hybridization, on a solid surface, of an array of immobilized oligodeoxynucleotides (ODNs) of known sequences to a DNA/ RNA whose sequence is being sought. Hybridization between two DNA strands produces a double-stranded helix in which the strands are antiparallel and joined through hydrogen bonds. Hybridization occurs over time when the two strands are annealed at a temperature below the Tm (melting point), which is influenced primarily by the types of base pairs and the stacking interaction between bases. Any base pair combination between DNA strands other than AT and GC (Watson-Crick rules) is termed mispairing and causes destabilization of the hybrid. Sequencing with oligonucleotide arrays was originally conceived as a method for determining DNA sequences by hybridizing the DNA to a complete set of oligonucleotides that represent all possible combinations of the four nucleotides that constitute DNA; e.g., there would be 48 combinations in a set of octamers. Such a set of oligonucleotides on a surface is termed a genosensor matrix, and the entire set may occupy a ∼1 cm × 1 cm area. With such a matrix, a single DNA/RNA sample may be interrogated by all the oligonucleotides simultaneously, noting the positions in the matrix where the DNA/RNA is detected after hybridization. For medical diagnostics, abbreviated versions of the matrix should be sufficient to detect a given mutation or alteration in DNA. Such a genosensor matrix should be inexpensive and readily available when this technology matures. The target strand in sequencing with ODN arrays normally carries a label that will be identified subsequent to the hybridization reaction. Each ODN has a unique sequence, and its position on the solid surface serves as the identifier of the fixed moiety. The variables in hybridization with oligonucleotide arrays are as follows: the length and composition of the immobilized ODN, the method of labeling and detection of the hybridization, and the conditions for hybridization. Several methods of immobilizing ODNs onto surfaces have been developed. One method is to attach thiol groups to gold surfaces and then covalently bind DNA to the thiolated linker. To obtain good immobilization efficiency with this method, self(7) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767-773. (8) Lipshutz, R. J. J. Biomol. Struct. Dyn. 1993, 11, 637-653. (9) Mirzabekov, A. Trends Biotechnol. 1994, 12, 27-32. (10) Livshits, M.; Florentiev, V., Mirzabekov, A. J. Biomol. Struct. Dyn. 1994, 11, 783-795. (11) Eggers, M. D.; Hogan, M. E.; Reich, R. K.; Lamture, J. B.; Beattie, K. L.; Hollis, M. A.; Ehrlich, D. J..; Kosicki, B. B.; Shumaker, J. M.; Varma, R. S.; Burke, B. E.; Murphy, A.; Rathman, D. D. In Advances in DNA Sequencing and Technology: Keller, R. A., Katzir, A., Eds.; SPIE Proceedings 1891; SPIE: Bellingham, WA, 1993; pp 113-126.

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assembled monolayer techniques have been used.12-14 Other methods of immobilizing DNA to surfaces such as glass, pyrex, quartz, and gelatin surfaces have been reported by numerous researchers.2,15-17 Hybridization occurs directly at the surface between the immobilized ODNs and the free DNA. A surface analysis technique that is capable of detecting the hybridized nucleic acid with very high efficiency and selectivity would be optimal for sequencing and diagnostics and for studying the thermodynamics of hybridization. Currently, techniques such as 32P and fluorescent labels are used to detect hybridized DNAs/RNAs. Indirect sequencing methods such as matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) are also being studied.18,19 We have developed two new methods to detect hybridized DNA. In the first, stable isotopes are attached to the DNA and are then detected selectively.20 In the second, unlabeled DNA is hybridized to immobilized peptide nucleic acid (PNA)21,22 and is then detected by measuring the phosphorus presence in the DNA but not in the PNA.23 To detect the isotope labels or the phosphorus with high efficiency and selectivity, we used sputterinitiated resonance ionization microprobe (SIRIMP) analysis. In this paper, the first data obtained with these new methods are shown and future experimental recommendations and applications for these techniques are discussed. Peptide Nucleic Acids. The possibility of using antisense DNA to control gene expression has stimulated interest in developing methods to introduce these antisense DNA pharmaceuticals across the cell membrane and to protect them from the degradative nucleases that exist within the cell. This has led to the synthesis of DNA analogs that are not polyanions, are unable to be hydrolyzed by these enzymes, and yet retain the ability to hybridize with normal nucleic acids. In one of these analogs, PNA, both the phosphate and the deoxyribose were replaced by polyamides. The basic differences in structures between DNA and PNA are shown in Figure 1. Because the backbone of DNA contains phosphorus and PNA does not have any phosphorus, an analysis technique that identifies the presence of phosphorus on the molecular surface layer should be able to determine whether hybridization of DNA to PNA oligonucleotide analogs has taken place. Resonance Ionization Mass Spectrometry. Resonance postionization mass spectrometry is becoming recognized as an analytical technique with desirable advantages for a wide range (12) Bain, C. D.; Troughton, E. B.; Tao, Y.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-334. (13) Sabatani, E.; Rubinstein, I.; Maoz, R.; Sagiv, J. J. Electroanal. Chem. 1987, 219, 365-371. (14) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. J. Am. Chem. Soc. 1987, 109, 3559-3568. (15) Maskos, U.; Southern, E. M. Nucleic Acids Res. 1992, 20, 1679-1684. (16) Beattie, K. L.; Beattie, W. G.; Meng, L.; Turner, S. L.; Coral-Vazquez, R.; Smith, D. D.; McIntyre, P. M.; Dao, D. D. Clin. Chem. 1995, 41, 700-706. (17) Livshits, M. A.; Florentiev, V. L.; Mirzabekov, A. D. J. Biomol. Struct. Dyn. 1994, 11, 783-795. (18) Jacobson, K. B.; Arlinghaus, H. F.; Buchanan, M. V.; Chen, C. H.; Glish, G. L.; Hettich, R. L.; McLuckey, S. A. GATA 1991, 8 (8), 223-229. (19) Limbach, P. A. Mass Spectrom. Rev. 1996, 15, 297-336. (20) Arlinghaus, H. F.; Jacobson, K. B., U.S. Patent pending serial no. 08/654,181, 1996. (21) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nelsen, P. E. Nature 1993, 365, 566-568. (22) Varma, R. S. Synlett 1996, 621. (23) Arlinghaus, H. F.; Jabobson, K. B., U.S. Patent pending serial no. 08/691,614, 1996.

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Figure 1. Comparison of the backbone structures of DNA and PNA. The natural DNA backbone includes deoxyribose and phosphate groups, not present in PNA. Instead, the PNA backbone consists of repeating units of N-(2-aminoethyl)glycine linked by amide bonds. The N-terminus, shown at the top left of the figure, corresponds to the 5′ end of a DNA or RNA strand.

of applications. In resonance ionization (RI), tunable lasers are used to count neutral atoms (usually ground state atoms) of the element that is selected for analysis by sequentially exciting and then ionizing the atoms. The extremely high element specificity and sensitivity of the RI process is especially valuable for ultratrace element analysis in samples where the complexity of the matrix is a serious source of interferences. RI combined with a focused ion sputter beam and time-of-flight (TOF) mass spectrometric detection of ions is called SIRIMP and provides an exceptionally efficient analytical technique with the ability to obtain quantitative ultratrace element concentration images from surfaces with high spatial resolution and virtually no matrix effects.24-27 The surface sensitivity is due to the fact that during bombardment of a solid surface with fast primary ions, neutral atomic and molecular constituents are emitted, along with some secondary ions. These emitted particles originate almost completely from the uppermost layer of atoms and/or molecules, thus yielding direct information about its chemical composition. EXPERIMENTAL SECTION SIRIMP/SIMS. In the SIRIMP/secondary ion mass spectrometry (SIMS) analysis the sample is bombarded with a pulsed energetic ion beam to desorb particles from a solid surface (see Figure 2). The expanding cloud of sputtered particles originates primarily from the top molecular monolayer of the bombarded (24) Arlinghaus, H. F.; Spaar, M. T.; Thonnard, N.; McMahon, A. W.; Jacobson, K. B. In Optical Methods for Ultrasensitive Detection and Analysis: Techniques and Applications: Fearey, B. L., Ed.; SPIE Proceedings 1435; SPIE: Bellingham, WA, 1991; pp 26-35. (25) Arlinghaus, H. F.; Spaar, M. T.; Thonnard, N.; McMahon, A. W.;Tanigaki, T.; Shichi H.; Holloway, P. H. J. Vac. Sci. Technol. 1993, A11, 2317-2323. (26) Jacobson, K. B.; Arlinghaus, H. F. Anal. Chem. 1992, 64, 315A-327A. (27) Arlinghaus, H. F.; Joyner, C. F. J. Vac. Sci. Technol. 1996, B14, 294-300.

Figure 2. Conceptional diagram of the SIRIMP/SIMS technique.

sample and consists of mainly neutral atoms, molecular fragments, and ions. In the SIRIMP mode, the small number of ions produced in the sputtering process are removed by pulsed electric fields and electrostatic energy analysis and therefore cannot cause any interferences. The remaining neutral particles are then probed by the RI laser beams that ionize all the atoms of the selected element within the volume intersected by the laser beams. The resulting ions are then detected with a TOF mass spectrometer. In the TOF-SIMS mode, the secondary ion suppression is turned off and the RI laser beams are blocked, thus directly allowing TOF mass spectra to be taken of sputtered secondary ions. The main components of the TOF SIRIMP/SIMS instrument are a microbeam ion gun, a pulsed flood electron gun for charge compensation, a postionization laser system consisting of a pulsed Nd:YAG laser (repetition rate 30 Hz) pumping two pulsed dye lasers, a computer-controlled (x, y, z, φ) sample manipulator, a high-resolution video imaging system for sample observation, and a TOF mass spectrometer detection system. The high-precision manipulator can be positioned by stepper motors driven under computer control at a speed of 10 000 steps/s and provides submicrometer resolution. The sample holder is a carousel arrangement that can hold eight samples up to 40 mm × 40 mm in size. Spatial microcharacterization is accomplished by either changing the x and y target positions (for imaging of large areas) or scanning the ion beam (for imaging of small areas (less than 200 × 200 µm). All the important instrument functions are controlled by an IBM-compatible computer. Reagents and Materials. The probe M13(-20), having a sequence of 5′-GTA AAA CGA CGG CCA GT-3′, was obtained from Genosys Biotechnologies, Inc. This ODN was synthesized with a hexylamine group on the 5′ end to prepare it for labeling with enriched stable isotopes of tin for the oligodeoxynucleotide/DNA hybridization studies. The target ODN was then purified and reacted with the N-hydroxysuccinimide ester of triethylstannyl propionic acid (TESPA) that contained isotopically enriched tin (>90%).28 Separate preparations of the oligonucleotide were labeled with two different isotopes of tin, 118Sn and 122Sn. Two 17-mer probe ODNs, for attachment to the surfaces, were synthesized by Genosys Biotechnologies, Inc., leaving a 3′propanolamine modification for immobilization.29,30 One of the probes had a sequence that was completely complementary to the M13(-20) ODN and the sequence for the other was 5′-CTA (28) Sloop, F. V.; Brown, G. M.; Foote, R. S.; Jacobson, K. B.; Sachleben, R. A. Bioconjugate Chem. 1993, 4, 406-409.

TAG TGA GTC GTA TT-3′. PNA probe oligonucleotide analogs were obtained from PerSeptive Biosystems. These ODN analogs were 17 units long with a 5′ linker that is abbreviated as OO: 5′H2N-CH2-CH2-O-CH2-CH2-O-CH2-CO-NH-CH2-CH2-O-CH2-CH2-OCH2-CO-NH-[pna]-3′. The PNAs had the following sequences: CRD lot 4375, 5′-OO-ACT GGC CGT CGT TTT AC-3′; CRD lot 4376, 5′-OO-CTA TAG TGA GTC GTA TT-3′. One of the PNA oligonucleotides analogs (CRD lot 4375) was designed to be completely complementary to the M13(-20) target ODN. Immobilization. Pyrex, quartz, and platinum surfaces were used to immobilize probes. The oligonucleotide probes and the PNA oligonucleotide analog probes were attached to platinumcoated silicon chips supplied by W. Beattie. The probes were directly attached to the platinum surface by applying them to the chip after first cleaning it with a nitric acid solution, rinsing with water, and cleaning it with ethanol. Attaching ODNs directly to surfaces via a 3′-propanolamine linker has been shown to be faster and more reliable than previous methods.29-31 Three aliquots of 200 nL of 20 µM of each of the two probe DNA oligonucleotide solutions were immobilized to a platinum-coated silicon wafer and to quartz and glass surfaces. Aliquots of 300 nL of 20 µM solutions of the two PNA oligonucleotide analogs were immobilized to different locations on a platinum-coated silicon chip. It was expected that the efficiency of binding would be lower for PNA than the 1% that is usually achieved for DNA because the 3′-propanolamine linker has been shown by Beattie et al. to have a higher binding efficiency than the 5′-hexylamine using this immobilization method.31 Hybridization Conditions. The immobilized DNA probes were incubated with 90 µL of 1 nM M13(-20) target ODN in 6× SSC, pH 7.0, for 19 h at 5 °C and washed for 5 min in 8 mL of 6× SSC twice at 5 °C. The immobilized PNA probes were incubated with 100 µL of 50 nM M13(-20) target ODN in 3 mM Tris buffer, pH 7.4, for 2 h at 5 °C and washed for 5 min in 5 mL of purified water twice at 45 °C. RESULTS AND DISCUSSION Detection of DNA Hybridized to Immobilized ODNs. The Sn-labeled target ODNs used for hybridization to DNA probes were detected with SIRIMP. To ionize Sn, we found that a UV + visible + IR resonance ionization laser scheme was ideal (286.3 + 615.0 + 1064.1 nm). The two bound-bound transitions and the ionization step can be readily saturated, assuring unit probability of ionizing all Sn atoms. The combination of relatively low laser intensities required for the bound-bound transitions, and the use of an infrared wavelength for the ionization step, prevented nonresonant interferences from molecules, molecular fragments, or other elements. The bandwidth of the laser beams was measured at ∼8-12 GHz. This ensures that all Sn isotopes (isotope shift 90% for most of Analytical Chemistry, Vol. 69, No. 18, September 15, 1997

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them).32 In addition, greater numbers of labels are available if rare earth isotopes are employed. The rare earth elements can be contained in a caged compound known as tetraazacyclododecanetetraacetic acid (DOTA). A general synthetic route to produce the macrocyclic ring DOTA with a substituent side chain has been developed and described.33 For example, 21 enriched isotopes, 8 Sn, 7 Nd, and 6 Gd isotopes, could be used to label 21 different DNA fragments for a great improvement in speed and performance. Detection of DNA Hybridized to Immobilized PNA. To test the novel idea of detecting phosphorus in natural DNA as a means to identify its presence after hybridization to PNA, PNA oligonucleotide analogs were immobilized on platinum-coated silicon chips. These chips were then hybridized with M13(-20) and analyzed by SIRIMP for phosphorus. For most elements, a conventional laser system can be used to excite elements from the ground state to an excited electronic state using a one-photon process. However, a few elements such as phosphorus, and generally those in the upper-right-hand corner of the periodic table, require extremely high-energy photons (short wavelengths) to induce the transition. For these elements, RI is often performed using a two-photon transition; i.e., each photon has half the total energy of the transition. The selection rules are different for onephoton and two-photon transitions, and therefore, the excited state will be different in the two processes. The main advantage of a two-photon excitation is that the wavelength required is much easier to achieve and the excitation and ionization processes often require only a single wavelength. The main drawback is that twophoton excitation requires very high laser intensities which in turn requires focusing of the laser beam. This can cause interferences by nonresonant processes such as multiphoton ionization. To ionize phosphorus, a focused 305 nm laser beam was used for the two-photon resonance transition and the ionization step. The results of this three-dimensional SIRIMP phosphorus image are displayed in Figure 6. The diagram above the image shows the location of immobilized complementary and noncomplementary PNA oligonucleotide analogs. An obvious peak is observed for phosphorus in the location of the complementary analog and almost no signal is observed at the noncomplementary site, demonstrating successful binding of the PNA oligonucleotide analog to the platinum surface and successful selective hybridization of the DNA oligonucleotide to the complementary PNA oligonucleotide analog. The signal to noise ratio was ∼10:1 and could probably be increased by a factor of >1000 by using fourwave difference mixing schemes to generate and optimize vacuum ultraviolet light for the RI of phosphorus.34-37 A selective onephoton resonance scheme would also significantly increase selectivity and allow the mass spectrometer to be replaced with a simple ion detector. Other techniques such as TOF-SIMS and laser ionization mass spectrometry (LIMS) or optical techniques such as surface-enhanced Raman spectroscopy, polarization, or (32) Jacobson, K. B.; Arlinghaus, H. F.; Schmitt, H. W.; Sachleben, R. A.; Brown, G. M.; Thonnard, N.; Sloop, F. V.; Foote, R. S.; Larimer, F. W.; Woychik, R. P.; England, M. W.; Burchett, K. L.; Jacobson, D. A. Genomics 1991, 9, 51-59. (33) Garrity, M. L.; Brown, G. M.; Elbert, J. E.; Sachleben, R. A. Tetrahedron Lett. 1993, 34, 5531-5534. (34) Hilbig, R.; Wallenstein, R. Appl. Opt. 1982, 21, 933. (35) Hager, J.; Wallace, S. C. Chem. Phys. Lett. 1982, 90, 472. (36) Hilbig, R.; Wallenstein, R. IEEE J. Quant. Electr. 1983, QE-19, 194. (37) Hutchinson, M. H. R.; Thomas, K. J. IEEE J. Quant. Electr. 1983, QE-19, 1823.

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Figure 6. SIRIMP phosphorus image of a DNA oligonucleotide hybridized to a PNA oligomer. The top part of the figure shows a diagram of a biosensor chip on which two different PNA oligomers were attached in separate locations: (A) PNA oligomer that is complementary to the M13(-20) DNA oligonucleotide probe and (B) PNA oligomer that is not complementary to the M13(-20) DNA oligonucleotide probe. The PNA biosensor chip was hybridized with the M13(-20) DNA oligonucleotide. The bottom part of the figure displays a SIRIMP image of the phosphorus detected on the surface of the biosensor chip. Phosphorus is observed where the DNA oligonucleotide is located, demonstrating successful detection of hybridization of DNA to PNA.

secondary harmonic generation techniques could be used to locate DNA hybridized to PNA by detecting molecular fragments that are unique to either the DNA or the PNA.38,39 The possibilities need to be evaluated to determine which is the most efficient and economical method of analyzing DNA hybridized to a PNA biosensor chip. The use of PNA probes has several advantages. Because labeling would not be necessary, detecting DNA hybridization by analyzing for phosphorus might allow genomic DNA to be used directly for diagnostics (without the use of PCR amplification), thus significantly reducing preparation cost. Because a short sequence is likely to be repeated in a long strand of DNA, longer probes would be necessary for specific hybridization with genomic DNA. More stringent washing would also be important to reduce background signal. Another advantage of the nonlabeling technique is that DNA has as many phosphorus atoms as the number of bases present while most labeling techniques provide only one label per molecule. Thus, a 17-mer primer would have 17 phosphorus atoms, and genomic DNA has a tremendous number of phosphorus atoms. In addition, because of the binding characteristics of PNA/DNA, it is more likely that single-base mismatches can be determined with PNA/DNA hybridization on complex hybridization chips.40 Fewer bases will be necessary for the oligonucleotide analog probes due to the increased strength of PNA/DNA pairing, thus significantly reducing the size of a (38) Gillen, G.; Tarlov, M.; Herne, T.; McKenney, K. In Secondary Ion Mass Spectrometry, SIMS X; Benninghoven, A., Hagenhoff, B., Werner, H. W., Eds.; John Wiley & Sons: New York, 1997. (39) Ii, T; Okuda, S.; Hirano, T.; Tsujimoto, K.; Ohashi, M. Org. Mass Spectrom. 1993, 28, 127-131. (40) Wang, J.; Palecek, E.; Nielsen, P. E.; Rivas, G.; Cai, X.; Shiraishi, H.; Dontha, N.; Luo, D.; Farias, P. A. M. J. Am. Chem. Soc. 1996, 118, 7667-7670.

sequencing chip and, therefore, also reducing the speed and cost of the technique. CONCLUSION Two new nucleic acid detection techniques have been developed that provide rapid methods for DNA/RNA sequencing, diagnostics, and mapping. These two techniques involve either labeling the free nucleic acid with enriched stable isotopes or hybridizing DNA/RNA without labels to immobilized PNA and detecting the phosphorus, present in the DNA/RNA but not in the PNA. We have successfully shown that SIRIMP analysis can be used to detect the presence of hybridized labeled and/or unlabeled DNAs on a biosensor chip. Also, other surface-sensitive detection methods have been suggested for future investigation. The presented data clearly demonstrate that excellent discrimination between complementary and noncomplementary sequences can be obtained during hybridization of DNA to either ODNs or PNAs. Each of these two techniques has its own specific advantage over current methods. The use of enriched stable isotopes as labels offers an opportunity for multiplexing that might be very advantageous in studying hybridization kinetics in complex systems as well as to significantly increase speed of analysis. On the other hand, the use of unlabeled DNA with PNA biosensor chips could increase discrimination for single-point mutation and might allow direct analysis of genomic DNA without amplification. In future experiments we will conduct a detailed study on hybridization kinetics for both methods on more complex hybridization chips and evaluate some of the other analytical methods

to detect DNA hybridized to PNA biosensors. We will also evaluate various immobilization methods for miniaturizing biosensor chips. We conclude that the two nucleic acid-sequencing techniques have the potential to provide high-throughput methods to sequence DNA/RNA and to diagnose many sets of mutations simultaneously. Additionally, these methods can be readily applied to a variety of problems in bioanalytical chemistry (e.g., screening of combinatorial libraries, etc.). The potential cost effectiveness, accuracy, and speed of these innovative techniques could be advantageous for future applications. ACKNOWLEDGMENT This work was supported in part by the National Institutes of Health under Contract No. 1 R43 CA66525-01 to Atom Sciences, Inc. We thank T. J. Whitaker, Atom Sciences, Inc., and M. J. Doktycz, Oak Ridge National Laboratory, for helpful discussions, M. Egholm, PerSeptive Biosystems, for supplying PNA analogs, K. Beattie and W. Beattie, Houston Advanced Research Institute, for attaching ODNs and PNAs to solid surfaces, and M. Hollis, MIT Lincoln Laboratory, for preparing Si/Pt wafers. Support for K.B.J. from the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-85OR21400 with Martin Marietta Energy Systems, Inc. is gratefully acknowledged. Received for review March 10, 1997. Accepted June 30, 1997.X AC970267P X

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

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