Anal. Chem. 2001, 73, 2126-2131
MALDI-TOF Mass Spectrometric Method for Detection of Hybridized DNA Oligomers Narayana R. Isola,† Steve L. Allman, Valeri V. Golovlev, and Chung H. Chen*
Photophysics Group, Life Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6378
Two new approaches for nucleic acid hybridizations by MALDI-TOF mass spectrometry are described. Hybridization using genomic DNA without polymerase chain reaction was demonstrated. Total genomic DNA of bacteriophages bound to charge-modified nylon membranes was identified by the hybridization of species-specific oligonucleotide probes. λ-Phage DNA and M13 were used for the test with good success. Since MALDI-TOF mass spectrometry can be used to measure the molecular weights of different probes, mass spectrometry can be used for the detection of hybridizations with multiple probes. We demonstrate that multiple-probe hybridization can be resolved by mass spectrometry. Six probes with different mass tag were used for hybridization on a single spot. MALDI-TOF mass spectrometry was successfully used to measure these probes simultaneously. This provides a simple nonradioactive method for multiplex hybridization analysis. It has the potential to drastically increase the speed for microarray hybridization analysis in the future. Detection of nucleic acid sequences by hybridization with complementary sequences is the cornerstone of modern molecular biology. This is one of the most sensitive assays for research and diagnostic applications. Radioactive methods of detection of nucleic acid hybridization suffer from the disadvantages of not only handling and disposal of hazardous radioactive labels but also the limitation of the number of labels available. P32, P33, and S35 are the three most commonly used labels and with some effort the signals from each isotope can be distinguished, taking advantage of the differences in the energy of the β particles emitted.1 Fluorescent labels have become increasingly popular with the rapid advances in the detection technologies making them comparable to radioactive methods in sensitivity.2 However, the spectral resolution of fluorescence and chemiluminescence is limited due to the inherently broad emission features (typically 50-100 nm), thus limiting the number of labels that can be used simultaneously. Using sophisticated image capture and analysis methods, 27 different probes were used in fluorescence in situ † Postdoctoral Fellow, Oak Ridge Associated Universities, Oak Ridge, TN 37831. (1) Evans, M. R.; Read, C. A. Nature 1992, 358, 520-521. (2) Beck, S.; Koster, H. Anal. Chem. 1990, 62, 2258-2270. (3) Speicher, M. R.; Gwyn-Ballard, S.; Ward, D. C. Nat. Genet. 1996, 12, 368375.
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hybridization (FISH).3 Isola et al.4 demonstrated that surfaceenhanced Raman gene probes (SERGen) are a potential alternative to fluorescence probes. The SERS signals are typically narrow (10-20 nm); hence, there is a potential for simultaneous detection of more SERS probes than fluorescent probes. However, the compounds that are highly SERS active are not readily attachable to nucleic acids. All the above methods involve the addition of a label to the probe sequence. The reporter is attached either during the synthesis of DNA probes or by chemical/enzymatic manipulations after synthesis. Matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrometry is inherently very sensitive and can detect molecules in the subpicomole range. This method has found several applications in peptide/protein analysis and has recently found many applications in nucleic acid analysis as well. A detailed listing of many applications in protein and nucleic acids research can be found in Burlingame’s review.5 Both dideoxy sequencing6,7 and chemical cleavage sequencing have been successfully adapted for MALDI detection.8 Nucleic acid products generated by ligase chain reaction (LCR)9 and polymerase chain reaction (PCR) have been analyzed by mass spectrometry.10,11 This method offers a rapid nonradioactive detection system for nucleic acids with a very high resolution which could successfully complement the existing DNA analysis techniques and may even replace some of them in the future. Recently, MALDI-TOF mass spectrometry has also been used for hybridization detection.12,13 However, either synthetic DNA or PCR products were used for hybridization. Multiplexing hybridizations have not been actively pursued. (4) Isola, N. R.; Stokes, D. L.; Vo-Dinh, T. Anal. Chem. 1998, 70, 1352-1356. (5) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1998, 70, 647R716R. (6) Koster, H.; Tang, K.; Fu, D.-J.; Braun, A.; van den Boom, D.; Smith, C. L.; Cotter, R. J.; Cantor, C. R. Nat. Biotechnol. 1996, 14, 1123-1128. (7) Taranenko, N. I.; Allman, S. L.; Golovlev, V. V.; Isola, N. R.; Chen, C. H. Nucleic Acids Res. 1998, 26, 2488-2490. (8) Isola, N. R.; Allman, S. L.; Golovlev, V. V.; Chen, C. H. Anal. Chem. 1999, 13, 2266-2269. (9) Jurinke, C.; Van den Boom, D.; Jacob, A.; Tang, K.; Worl, R.; Koster, H. Anal. Biochem. 1996, 237, 174-181. (10) Tang, K.; Taranenko, N. I.; Allman, S. L.; Chang, L. Y.; Chen, C. H. Rapid Commun. Mass Spectrom. 1994, 8, 727-730. (11) Taranenko, N. I.; Potter, N. T.; Allman, S. L.; Golovlev, V. V.; Chen, C. H. Genet. Anal.: Biomol. Eng. 1999, 15, 25-31. (12) Tang, K.; Fu, D.-J.; Julien, D.; Braun, A.; Cantor, C. R.; Koster, H.; Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10016-10020. (13) O’Donnell, M. J.; Tang, K.; Koster, H.; Smith, C. L.; Cantor, C. R. Anal. Chem. 1997, 69, 2438-2443. 10.1021/ac0013711 CCC: $20.00
© 2001 American Chemical Society Published on Web 03/29/2001
Table 1. name
sequence
m/z
target oligo 1 (T1) control target (TC) probe (P) probe (P1) probe (P2) probe (P3) P1 amino (P1-A) P1 biotin (P1-B) P1 Cy5 (P1-C) P1 fluorescein(P1-F) P1 Rhodamine(P1-R)
5′-NH2-TGACCTGGAGTCTTCCAGTGT-3′ 5′-NH2-GATCGATCGATCGATCGATCG-3′ 5′-GATAACACTGGAAGACTCCAGGTCA-3′ 5′-GATGAGTTCGTGTCCGTACAACTGG-3′ 5′-GGTTATCGAAATCAGCCACAGCGCC-3′ 5′-GTTTTCCCAGTCACGACGTTGTA-3′ 5′-NH2-GATGAGTTCGTGTCCGTACAACTGG-3′ 5′-Bio-GATGAGTTCGTGTCCGTACAACTGG-3′ 5′-Cy5-GATGAGTTCGTGTCCGTACAACTGG-3′ 5′-Glu-GATGAGTTCGTGTCCGTACAACTGG-3′ 5′-Rho-GATGAGTTCGTGTCCGTACAACTGG-3′
6613 6631 7655 7684 7607 6979 7893 8119 8216 8222 8486
In this article, we present the results of experiments involving the use of different substrates to bind the analyte DNA molecules and detection of the analyte by hybridization with the synthetic oligonucleotides complementary to the analyte molecule. We also report the results on solution hybridizations and the use of mass tags for detection of multiple probes simultaneously. EXPERIMENTAL SECTION Mass Spectrometry. MALDI-TOF measurements were performed using a linear time-of-flight mass spectrometer (Voyager; PerSeptive BioSystems, Framingham, MA) equipped with a 337nm pulsed nitrogen laser for desorption/ionization. The laser fluence was 45-65 µJ/cm2 and the acceleration voltage was 26 125 V. A delayed pulsed ion extraction device was installed to increase mass resolution. Negative ion signals were collected (typically for 256 laser pulses), digitized, and averaged by a digital oscilloscope (Tektronix 520A). The matrix was a mixture of 0.5 M stock solution of 3-hydroxypicolinic acid, picolinic acid, and ammonium citrate at a 9:1:1 ratio. For MALDI-TOF mass spectrometry, it is well known that broad ion energy distribution can cause poor mass resolution. The depth distribution of samples can cause the ions to be produced at different energies. With nylon membrane as a substrate for MALDI, ions are expected to be produced at different depths due to the membrane structure. However, the thickness of the membranes used was 0.45 µm. The distance between the sample plate and the focusing grid before the entrance plate of the mass spectrometer is ∼3 mm. Thus, the limitation on the mass resolution due to the thickness of membranes is more than 3000, which is better than the resolution we normally get when using polished stainless steel as substrate. Therefore, there is no need for concern about the ion energy distribution due to the use of membranes as substrates for MALDI. Another concern is the expected shift in mass calibration due to the change of the distance between the sample plate and the focusing grid. The mass shift can be expected since the membranes used are insulators. However, the shift should be very small due to the thinness of the membrane. Nevertheless, the ion extraction field can be distorted due to the membrane. We used internal mass calibrations to determine that this effect was small enough to not be a concern. Materials. All chemicals and reagents used were molecular biology grade. Synthetic Oligonucleotides. All synthetic oligonucleotides were obtained from Oligos Etc. (Wilsonville, OR) and were used
without further purification. The list of oligonucleotide sequences used in this study is shown in Table 1. To simplify the presentation, we assigned a symbol for each oligonucleotide. (See Table 1.) Genomic DNAs. λ-Phage DNA and M13 genomic DNAs were obtained from Sigma Chemical Co. (St. Louis, MO). The membranes used in this study were nitrocellulose and Nytran (Schleicher and Schuell), Hy-bond N (Du-Pont), Quiabrene and Quiabrene plus (Quiagen), Westran PVDF membrane (Schleicher and Schuell), and Zeta-bind (Bio-Rad Laboratories). Hybridizations on Derivatized Plastic Surfaces. N-Oxysuccinimide (NOS)-derivatized polystyrene plates (DNA-BIND from Corning Co-Star, Corning, NY) were used. The target nucleic acid (T1) containing a primary amine at one end (5′) was resuspended in 100 mM carbonate buffer (pH 9.0) at a final concentration of 1 µg/µL. The target DNA was placed on the DNA-BIND plates and incubated for 30 min at 37 °C; the unbound DNA was removed by washing in 0.1 M carbonate buffer (pH 9.0). The unreacted sites on the DNA-BIND plate were blocked by adding 200 mL of 3% (w/v) bovine serum albumin (BSA) in 0.1 M carbonate buffer (pH 9.0) and incubating at 37 °C for 30 min. Hybridizations were performed in 5× saline sodium citrate (SSC) containing 0.5% sodium dodecyl sulfate (SDS), 0.1% each of Ficoll (type 400 (Pharmacia, Peapack, NJ), poly(vinylpyrrolidone), and BSA factor V (Sigma Chemical Co.) with a probe concentration of 100 ng/ mL. Hybridizations were performed at 37 °C overnight. After hybridization, the plates were washed twice in 5× SSC at room temperature and once at 37 °C. After the final stringency wash, the plates were rinsed in 75% ethanol and 100% ethanol and airdried. Matrix solution (5-10 µL) was added to the plates and was allowed to crystallize under a stream of air before being loaded into the mass spectrometer. Hybridizations on Membranes. Target nucleic acids (T1) of ∼1 µL with a concentration of 1 µg/µL were spotted onto the membranes (Qiabrene Plus) and covalently bound by cross-linking using a DNA transfer lamp (Fotodyne, Inc., Hartland, WI) for ∼1 min at a fluence of 120 W/m2. The membranes were prehybridized in 5× SSC containing 0.5% SDS, 0.1% each of Ficoll (type 400, Pharmacia), poly(vinylpyrrolidone), and BSA (factor V, Sigma) at 37 °C for 1 h. Hybridizations were performed in the same solution with the addition of probe nucleic acids (typically at a concentration of 100 ng/mL). Hybridizations were performed at 37 °C overnight. Stringency washes were performed by washing the membranes in 5× SSC at room temperature for 15 min and Analytical Chemistry, Vol. 73, No. 9, May 1, 2001
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then at 37 °C for 15 min. After stringency washes, the membranes were allowed to dry in a stream of air. The air-dried membranes were washed once in 75% ethanol and once in 100% ethanol and air-dried. The membranes were mounted onto the mass spectrometer sample plate with double-stick (Scotch 3M) tape, and the matrix solution was spotted directly onto the membrane and allowed to crystallize in a stream of warm air. Solution Hybridizations. The target DNA and probe oligonucleotides (1 µg each) were mixed in the hybridization buffer (described above) in a final volume of 10 µL. The mixture was heated at 95 °C for 5 min, allowed to cool to hybridization temperature (37 °C), and incubated at the hybridization temperature for 1 h. After hybridization, the total reaction was subjected to gel filtration using a Sephadex G-25 Spin column (Pharmacia, Peapack, NJ) equilibrated with water. Typically, two rounds of gel filtration were performed in order to remove the unbound probes completely. The high molecular weight fraction (void volume) was collected and concentrated by air-drying. The concentrated sample was passed through a ZipTip (Milllipore, Bedford, MA) and eluted in matrix directly onto a stainless steel sample plate for mass spectrometric analysis. RESULTS AND DISCUSSION Hybridizations to Derivatized Plastic Surfaces. Target oligonucleotides (T1) containing a primary amine were bound to N-oxysuccinimide-derivatized plates as described in the Experimental Section and were hybridized to probe (P) with complementary sequences. After hybridization and stringency washes, the matrix was spotted onto the plates and allowed to crystallize in a stream of air. The plates were mounted onto the mass spectrometer using double-stick tape (3M). A mass spectrometer sample plate was modified with a recessed area to accommodate the thick plastic plates and position them properly in the ion extraction region. No molecular ion species corresponding to the probe or the target oligonucleotide were observed. Narayanaswami and Levis14 reported the detection of signals corresponding to the probe using such planar surfaces. However, they used plasma-treated surfaces and synthesized the target sequences directly on the planar surface. To resolve this discrepancy, we have performed hybridizations on the DNA-BIND plates as described above, and after the stringency washes, 100 µL of ammonium hydroxide was added to each well to allow the strands to dissociate. The probe strand (in ammonium hydroxide) was collected, concentrated, and analyzed by MALDI-TOF mass spectrometry. Figure 1 shows the results of this experiment. A molecular ion species (m/z 7685) corresponding to the probe was observed in plates containing the complementary target sequence, but no such ion was observed in plates containing a noncomplementary target sequence. Molecular ion species corresponding to the target T1 (m/z 6613) and control TC (m/z 6631) sequences were not observed in either of the ammonium hydroxide extracts, indicating that the target and control sequences were covalently bound to the surface and were not extractable by ammonium hydroxide. This probably indicates that the nucleic acid hybridizations to complementary sequences did occur but the probe density might not be sufficient for detection by direct desorption. Narayanaswami and Levis14 synthesized the target oligonucleotide (14) Narayanaswami, G.; Levis, R. J. J. Am. Chem. Soc. 1997, 119, 6888-6890.
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Figure 1. Hybridizations on N-oxysuccinimide plates. Two target sequences containing a primary amino group at the 5′ end were covalently bound to DNA-BIND plates and were hybridized with the probe sequence. Direct desorption from the plate did not show the expected molecular ion species from the complementary target (A) or the control target (C). Ammonium hydroxide extraction and concentration after hybridization revealed the signal corresponding to the probe sequence only from plates containing the complementary target (B), and no such signal was observed from plates containing the control (noncomplementary) target (D).
directly on plasma-treated plates, while we have bound the target nucleic acid through the amino linker. The differences in binding chemistry might explain the differences in the results. However, the target density has been reported to be higher in direct synthesis while the density of full-length oligonucleotide is higher in postsynthesis coupling.15,16 Hybridizations on Membranes. Nitrocellulose and chargemodified Nylon membranes are being used routinely for binding nucleic acids and detection of complementary sequences by hybridizations in both radioactive and nonradioactive applications. The membranes have high binding capacity ranging from 100 to 450 µg/cm2. Use of nitrocellulose film and membranes as an additional purification step for MALDI analysis of nucleic acids was reported earlier.11 The use of MALDI for the analysis of DNA from nitrocellulose substrates was reported by Lubman and his colleagues.17,18 Several membranes were used in the initial study: namely, nitrocellulose (Schleicher and Schuell, Millipore), positively (15) Southern, E.; Mir, K.; Shchepinov, M. Nat. Genet. Suppl. 1999, 21, 5-9. (16) Lipshutz, R. J.; Fofor, S. P. A.; Gingeras, T. R.; Lockhart, D. J. Nat. Genet. Suppl. 1999, 21, 20-24. (17) Liu, Y.; Bai, J.; Zhu, Y., Liang, X.; Siemieniak, D.; Venta, P. J.; Lubman, D. Rapid Commun. Mass Spectrom. 1995, 9, 735-743. (18) Liu, Y.;, Bai, J.; Liang, X.; Lubman, D. M. Anal. Chem. 1995, 67, 34823490.
Figure 2. MALDI-TOF spectrum of 30-mer oligonucleotide: (A) Spectrum directly from the plate. (B) Oligonucleotide spotted onto Nytran membrane. (C) Spectrum after UV cross-linking. No molecular ion species corresponding to the 30 mer was observed, indicating covalent cross-linking of the DNA to the membrane.
charged nylon Nytran (Schleicher and Schuell), Hybond N+ (Amersham), and Qiabrene and Qiabrene Plus (Qiagen). One microliter of synthetic oligonucleotide (10 µg/mL) was spotted onto the membrane, air-dried, and washed in water, 2-3 mL of matrix was added, and MALDI-TOF measurements were performed. Molecular ions corresponding to the molecular weight of the oligonucleotide were observed in all of the samples, indicating that the DNA bound to the membranes is readily desorbed. When UV cross-linking was performed on the nylon membranes (Hybond N+, Qiabrene Plus, Nytran) the molecular ion peaks were not observed, indicating that the DNA was crosslinked to the membrane and could not be desorbed. Figure 2 shows a typical UV cross-linking experiment with Nytran membrane. Similar results were obtained with Hybond N+, Qiabrene Plus, and Zeta-Bind (data not shown). Oligonucleotide Hybridizations. A 21-mer target (T1) was bound by covalent cross-linking to Nytran membranes and hybridized to 25-mer probe (P) with complementary sequence to the target. After hybridizations and stringency washes, as described in the Experimental Section, the membranes were spotted with 5-10 µL of matrix solution, air-dried, and placed in the mass spectrometer using double-stick tape. Molecular ion peaks corresponding to the probe sequence (25 mer m/z 7655) were observed by direct desorption from membranes containing the target sequence while no such molecular ions were observed from membranes containing control sequence. These results indicate that oligo-oligo hybridizations could be successfully detected by in situ desorption on the membranes. The results of these experiments are shown in Figure 3. The results conclusively
Figure 3. Oligo-oligo hybridizations on membranes: (A) Spectrum of target and probe mixture. T-- and P-- represent doubly charged target and probe DNA, respectively. While T2- and P2- represent the dimer ions of target and probe oligonucleotide, respectively. (B) Hybridizations to control template. (C) Hybridizations to target. (D) Control spectrum of unhybridized membrane.
demonstrate the utility of membranes for in situ detection of nucleic acid hybridizations. Genomic Hybridizations. To further extend these results, genomic DNAs were used as targets and hybridized with complementary oligonucleotides. We chose two small genomes readily available, λ-phage (48 502 base pairs) and filamentous M13 phage (6883 base pairs). Double-stranded genomic DNA (bacteriophage λ and M13) was spotted onto the membranes at a concentration of 1 µg/µL and air-dried. The bound DNA was covalently cross-linked by UV irradiation as described earlier. The membranes were subjected to blocking (prehybridization) and hybridizations as described in the Experimental Section. After hybridizations and stringency washes, the membranes were airdried and mounted onto the mass spectrometer sample plate. One membrane containing λ and M13 phage DNAs was hybridized with a 25-nucleotide sequence complementary to λ-phage (P1). After hybridization and stringency washes as described in the Experimental Section, the membranes were analyzed by MALDI-TOF mass spectrometry. Molecular ion species corresponding to P1 were observed from the region containing the λ-phage DNA, and no signal was observed from the region containing M13 phage DNA (Figure 4). When a similar membrane was hybridized with P3 (M13 specific probe) molecular ion species corresponding to P3 were observed from the region containing M13 DNA and not from the region containing λ-phage DNA (data not shown). Unlike the planar plastic (DNA-BIND) surfaces, the membranes with their high DNA binding capacity can provide enough target densities for the detection of hybridized oligonucleotides by mass spectrometry without any further steps Analytical Chemistry, Vol. 73, No. 9, May 1, 2001
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Figure 4. Hybridizations on membrane with λ probe (P1). (A) λ Template. (B) M13 template. (C) No template.
for concentration enrichment. Since the sizes of λ-phage and M13 are known, the maximum density of λ-phage DNA molecule on nylon surface is estimated as ∼108/mm2. Since the size of the laser beam used for desorption is smaller than 1 mm2, the detection sensitivity for MALDI-TOF mass spectrometry has to be better than 1 fmol of probes. However, phage DNA was only UV cross-linked to the nylon surfaces at a few spots. Most parts of phage DNA “extruded” out into space for hybridization to take place. Thus, the true quantity of phage DNAs on the hybridization spot can be significantly higher than 108/mm2. At present, it is difficult for us to know the average cross-linked density for phage DNA molecules. Since the hybridization can still be readily observed, it can be concluded that the UV cross-linking process did not significantly reduce the efficiency of hybridization. Multiple Probes. A high order of multiplexing hybridization at a single hybridization spot would significantly increase the analysis throughput. To evaluate the feasibility of using multiple probes simultaneously, a membrane containing λ-phage and M13 phage DNA as described above was hybridized with a mixture of all three probes (P1, P2, P3). Molecular ion species corresponding to P1 (m/z 7684) and P2 (m/z 7607) were observed from the region containing λ-phage DNA, and no signal corresponding to P3 (m/z 6979) was detectable in this region. Molecular ion species corresponding to P3 (m/z 6979) was observed from the region containing M13 phage DNA, and no signal corresponding to P1 (m/z 7684) or P2 (m/z 7607) was observed in this region (Figure 5). These results clearly indicate that hybridizations were complementary based, and two probes that differ by 77 Da could be readily observed and resolved as well. Solution Hybridizations. The mass tags amino, biotin, Cy5, fluorescein, and Rhodamine were chosen since they were readily available and inexpensive. The template and the mass tagged probe mixture, probes P1, P1 amino, P1 biotin, P1 Cy5, P1 fluorescein, and P1 Rhodamine (see Table 1), were mixed and 2130 Analytical Chemistry, Vol. 73, No. 9, May 1, 2001
Figure 5. Hybridizations on membrane with mixture of probes P1, P2, and P3. (A) λ Template. (B) M13 template. (C) No template.
solution hybridizations were performed as described in the Experimental Section. After hybridization, the total mixture was subjected to two rounds of gel filtration using a Sephadex G25 spin column to remove unhybridized probes. Control experiments indicated up to 10 µg of the probe sequence could be adequately removed by one round of G25 gel filtration. We chose to perform a second round of gel filtration as an additional precaution. The void volume (containing the high molecular weight target DNAs and any hybridized probes) was concentrated and analyzed directly. The results of this experiment are shown in Figure 6. When λ-phage DNA was used as the target, five of the six probes were conclusively detected; when M13 phage was used as the target, none of the probes were detected. Since the probes were designed from λ-phage sequences, this clearly indicates that sequence-based hybridization (capture) of the probes occurred. This also demonstrates that the tagged nucleic acids could be readily resolved; thus five probes could be detected by mass spectrometry. On the basis of the fairly uniform signal intensities, it can also be concluded that the addition of the mass tag at the 5′ end of the nucleic acid did not significantly interfere with the hybridization efficiency of the sequence. Earlier reports of detection of nucleic acid hybridizations by MALDI-TOF mass spectrometry12,13,19,20 involved the covalent attachment (or synthesis) of short oligonucleotides on the substrate and detection of complementary sequence hybridizations by direct desorption. In this report, we demonstrate the use of genomic double-stranded DNA directly as templates. Jiang(19) Tang, K.; Fu, D.; Kotter, S.; Cotter, R. J.; Cantor, C. R.; Koster, H. Nucleic Acids Res. 1995, 23, 3126-3131. (20) Bleczinski, C. F.; Richert, C. Rapid Commun. Mass Spectrom. 1998, 12, 1737-1743. (21) Jiang-Beaucom, P.; Girard, J. E.; Butler, J.; Belgrader, P. Anal. Chem. 1997, 69, 4894-4898.
Figure 6. Solution hybridizations. (A) The probes complementary to the λ-phage DNA template P1, P1 amino, P1 biotin, P1 Cy5, P1 fluorescein, and P1 Rhodamine were clearly detected. Due to their small mass difference, Cy5 and fluorescein could not be well resolved. All others were clearly resolved. Rhodamine-tagged oligonucleotide gave three distinct peaks. (B) No peaks were detectable when M13 phage was used in place of λ-phage.
MALDI-TOF mass spectrometry. Since all membranes used are primarily made of nylon material, the efficiencies of UV crosslinking are expected to be similar. These results indicate that the hybridization efficiencies for DNA on these membranes are similar within the distinction capability MALDI can easily provide. We have also demonstrated the use of double-stranded genomic DNA without any modifications as targets. The use of double-stranded DNA targets offers the potential of using multiple probes simultaneously. In this article, we have demonstrated the use of two probe sequences which could be readily resolved. This offers a potential of using multiple probes that should be optimized for hybridization under similar conditions. We are presently experimenting with 25-mer probes with mass tags to evaluate the maximum number of probes usable simultaneously. We prefer the use of mass tagged probes instead of probes with different lengths because it can be more difficult to control the stringent wash conditions to distinguish perfectly complementary hybridization from single-base mismatched hybridization when probes with different lengths are used. Since the amount of target per unit area is of utmost importance, it would be interesting to evaluate this method for analysis of complex genomes such as bacteria, yeast, and humans. Since two probes could be detected simultaneously, it would be possible to detect point mutations directly from total genomic hybridizations or from PCR-amplified products. With further advances in automation of sample analysis by mass spectrometry, this method could be used for screening dot blots or ordered cDNA and genomic libraries. The advantages of using inexpensive unlabeled oligonucleotide probes together with multiplex potential will be extremely useful for gene isolations. ACKNOWLEDGMENT
Beaucom et al.21 used large double-stranded DNA sequences as targets and identified peptide nucleic acids hybridized to the targets. We also demonstrate the use of more than one probe. This method of binding double-stranded nucleic acid targets to nylon membranes and hybridization with oligonucleotides would find applications in mutation detection and other types of dot blots such as screening ordered arrays (libraries, amplification reactions, etc.). SUMMARY We demonstrated that probes hybridized to complementary sequences bound to nylon membranes could be detected by
This research is sponsored by the Office of Biological and Environmental Research, U. S. Department of Energy, and DOE/ EMSP program under Contract DE-AC05-00OR22725 with UTs Battelle, LLC. We also express our thanks to PerSeptive Biosystems (a subsidiary of Perkin-Elmer, Inc.) for providing the mass spectrometer for this work.
Received for review November 21, 2000. Accepted February 19, 2001. AC0013711
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