Multiplexed DNA Sequencing and Diagnostics by Hybridization with

A new DNA diagnostic and sequencing system has been developed that uses time-of-flight resonance ionization mass spectrometry (TOF-RIMS) to provide a ...
0 downloads 0 Views 252KB Size
Download PDF
Anal. Chem. 1997, 69, 1510-1517

Multiplexed DNA Sequencing and Diagnostics by Hybridization with Enriched Stable Isotope Labels Heinrich F. Arlinghaus,* Margaret N. Kwoka, and Xiao-Qin Guo

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

Health Science Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831

A new DNA diagnostic and sequencing system has been developed that uses time-of-flight resonance ionization mass spectrometry (TOF-RIMS) to provide a rapid method of analyzing stable isotope-labeled oligonucleotides in form 1 sequencing by hybridization (SBH). With form 1, the DNA is immobilized on a nylon membrane and enriched isotope-labeled individual oligonucleotide probes are free to seek out complementary DNAs during hybridization. The major advantage of this new approach is that multiple oligonucleotides can be labeled with different enriched isotopes and can all be simultaneously hybridized to the genosensor matrix. The probes can then be simultaneously detected with TOF-RIMS with high selectivity, sensitivity, and efficiency. By using isotopically enriched tin labels, up to 10 labeled oligonucleotides could be examined in a single hybridization to the DNA matrix. Greater numbers of labels are available if rare earth isotopes are employed. In the present study, matrices containing three different DNAs were prepared and simultaneously hybridized with two different probes under a variety of conditions. The results show that DNAs, immobilized on nylon surfaces, can be specifically hybridized to probes labeled with different enriched tin isotopes. Discrimination between complementary and noncomplementary sites of better than 100 was obtained in multiplexed samples. This new SBH method, which employs stable isotopic labels to locate target DNAs and TOF-RIMS to detect the labels, will be a very versatile and extensive multiplexing method.

labeled individual oligonucleotide probes are free to seek out the DNAs during hybridization.1-3 With form 2, individual oligonucleotide probes are attached to the surface and labeled DNAs that have been sheared are free to seek out the oligonucleotides.4-8 Form 1 is useful for assessing a large number of DNAs for the presence of a number of short defined sequences. Form 2 is useful for sequencing a smaller number of DNA fragments. This paper reports a new approach to label and detect oligonucleotides in SBH form 1.9 Typically, large DNAs are attached to nylon membranes and hybridization/detection is performed by using radioactive phosphorus (32P) for labeling oligonucleotides and autoradiography for detection.2,3 However, the use of radioactive materials has certain limitations, poses health hazards, and increases disposal costs. For example, autoradiography is not very quantitative, has low spatial resolution, and requires long exposure time. Fluorescence labeling is another technique often employed in DNA studies. However, difficulties are encountered using fluorescence labels in SBH form 1 because of nonspecific binding and substrate fluorescence background. Employing nonradioactive enriched stable isotopes as labels and time-of-flight resonance ionization mass spectrometry (TOF-RIMS) as a detection method may allow sequencing of large numbers of DNAs faster and more comprehensively than is possible with any other technique.10-14 The major advantage of this new approach is that multiple oligonucleotides can be labeled with different enriched isotopes which can be simultaneously hybridized to

Sequencing by hybridization (SBH) promises to produce large amounts of sequence information, due to the parallel nature of the analysis, and is particularly well suited for genome diagnostics, sequencing cDNAs or partial sequencing of clones, DNA and RNA sequencing, gene polymorphism studies, and identification of expressed genes. SBH consists of hybridizing an oligonucleotide of known sequence to a DNA on a solid surface whose sequence is being sought. SBH relies on the specific base-pairing rules in duplex DNA to infer sequence information by the detection of hybridized DNA. The main variables in SBH are the length and composition of the oligonucleotide, the attachment of the DNA or the oligonucleotide to the solid surface, the method of labeling and detection of the hybridization, and the conditions for hybridization. There are two main formats for SBH: form 1 and form 2. With form 1, the DNA is attached to the solid surface and

(1) Drmanac, R.; Strezoska, Z.; Labat, I.; Drmanac S.; Crkvenjakov, R. DNA Cell Biol. 1992, 9, 527-534. (2) Drmanac, R.; Drmanac, S.; Labat, I.; Crkvenjakov, R.; Vicentic, A.; Gemmell, A. Electrophoresis 1992, 13, 566-573. (3) Drmanac, R.; Crkvenjakov, R. Int. J. Genome Res. 1992, 1, 59-79. (4) Southern, E. M; Maskos, U.; Elder, J. K. Genomics 1992, 13, 1008-1017. (5) Fodor, P. A.; Leighton Read, J.; Pirrung, M. C.; Stryer, L., Tsai Lu, A.; Solas, D. Science 1991, 251, 767-773. (6) Bains,W. Chem. Br. 1995, 31, 122-125. (7) 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. (8) Livshits, M. A.; Florentiev, V. L.; Mirzabekov, A. D. J. Biomol. Struct. Dyn. 1994, 11, 783-795. (9) Arlinghaus, H. F.; Jacobson, K. B. U.S. Patent pending application 08654181, 1996. (10) Arlinghaus, H. F.; Thonnard, N.; Spaar, M. T.; Sachleben, R. A.; Larimer, F. W.; Foote, R. S.; Woychik, R. P.; Brown, G. M. Sloop, F. V.; Jacobson, K. B. Anal. Chem. 1991 63, 402-407. (11) Arlinghaus, H. F.; Thonnard, N.; Spaar, M. T.; Sachleben, R. A.; Brown, G. M.; Foote, R. S.; Sloop, F. V.; Peterson, J. R.; Jacobson, K. B. J. Vac. Sci. Technol. 1991, A9, 1312-1319.

1510 Analytical Chemistry, Vol. 69, No. 8, April 15, 1997

S0003-2700(96)01206-1 CCC: $14.00

© 1997 American Chemical Society

immobilized DNA on a genosensor matrix. TOF-RIMS can then be used to detect them simultaneously with very high selectivity, sensitivity, and efficiency. By using isotopically enriched tin labels, up to 10 labeled oligonucleotides could be examined in a single hybridization to the DNA matrix. Furthermore, since these 10 isotopes of tin do not decay, the labeled oligonucleotides may be stored indefinitely until time for their use. Ten enriched tin isotopes are available (Isotec, Inc., Miamisburg, OH) with high enrichment (>90% for most of them).15 In addition to tin, it is possible to use rare earth and other elements for labels, thus tremendously expanding multiplexing possibilities. The new SBH method, employing stable isotopic labeling of probe oligonucleotides and detection by TOF-RIMS, will be a very extensive multiplexing method. It not only offers a significant increase in analysis speed but also will present a unique opportunity to establish rules of specificity for hybridization in complex mixtures. Doktycz et al.16 have reported that hybrid stability varies when given base pairs are located terminally and at various positions internally in short probes. Additional experiments have evaluated the position effects of mismatched pairs as well. To evaluate such complexity, utilizing multiple enriched isotope labels will be advantageous so that the competition among several oligomers for hybridization at common sites may be studied. In addition to studying hybridization processes, multiplexing SBH form 1 procedures would be effective for production, organization, and storage of genome and cDNA libraries3,17 as well as for screening genomic DNA from tens of thousands of individuals for several genetic mutations. This technique also includes, among its possibilities, error checking on DNA sequence data in combination with an alternative technique.3,18 Again, the use of multiple isotopes would greatly expedite the error checking procedure. SBH could also be applied to supplement electrophoresis data so that compressions and other areas of difficulty could be analyzed by an alternate technique. Other uses of this technique could be for development of the following: (a) RNA chips for search of ribozymes; (b) protein chips for studies of protein-protein interactions and search for specific ligands and drugs; (c) peptide chips for search for specific peptides, ligands, and proteins; and (d) antibody chips for identification of expressed proteins in different cell types. RIMS is critical for the advancement of hybridization using stable isotope-labeled probes. A unique advantage of resonance ionization (RI) is the extremely high element selectivity. In RI, tunable lasers are used to count neutral atoms (usually ground state atoms) of the gas phase element that is selected for analysis (12) 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. (13) Jacobson, K. B.; Arlinghaus, H. F. Anal. Chem. 1992, 64, 315A-327A. (14) Arlinghaus, H. F.; Whitaker, T. J.; Joyner, C. F.; Kwoka, M.; Jacobson, K. B.; Tower, J. In ProceedingssSIMS X; Benninghoven, A., Hagenoff, B., Werner, H. W., Eds.; John Wiley and Sons: New York, 1997; pp 123-130. (15) 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. (16) Doktycz, M. J.; Morris, M. D.; Dormady, S. J.; Beattie K. L.; Jacobson, K. B. J. Biol. Chem. 1995, 15, 8439-8445. (17) Drmanac, R.; Drmanac, S.; Strezoska, Z.; Paunesku, T.; Labat, I.; Zeremski, M.; Snoddy, J.; Funkhouser, W. K.; Koop, B.; Hood, L.; Crkvenjakov, R. Science 1993, 260, 1649-1652. (18) Drmanac, S.; Drmanac, R. BioTechniques 1994, 17, 328-336.

by sequentially exciting and then ionizing the atoms. Typically, the ions are then detected in a mass spectrometer. The energy spectrum of discrete excited states is unique to each element, so that selection of particular excited states for RI analysis provides great elemental selectivity. We demonstrated earlier that the ionization efficiency for the selected element can be as much as 109 times higher than for the other elements in the sample.19 Since the isotope shifts of most elements are small in comparison to the bandwidth of the RI lasers used in our experiments (7-12 GHz), all isotopes of a chosen element will be ionized with essentially equal sensitivity. If a time-of-flight mass spectrometer (TOF-MS) is used, all isotopes of an element can be detected simultaneously. If several RI laser beams are used to resonantly ionize several elements, all the isotopes of the selected elements can be detected simultaneously. The mass spectrometer requirements are therefore reduced to the resolution of neighboring isotopes of a single element; the high ionization selectivity of RI and the suppression of the secondary ions virtually eliminate interferences from molecular ions, isobars, or scattered ions from the major constituents of the sample. RI is also extremely sensitive and efficient. The intensity of modern pulsed dye lasers is sufficient to saturate both the boundbound transitions and the ionization step, thereby assuring nearunit probability of ionizing all atoms of the selected element that are in the volume intersected by the RI laser beams. RI was shown to be potentially feasible with current laser techniques for all elements of the periodic table except He and Ne.20 Simple RI schemes, such as visible (Vis) + ultraviolet (UV), UV + infrared (IR), and Vis + UV + IR, yield near-uniform sensitivity down to the few-atom level for 85% of the elements in the periodic system.19,21 For most elements, useful yields between 2 and 10% can be achieved and subzeptomole detection limits are possible.12,22-25 RI requires free atoms in the gas phase. One atomization method, sputter-initiated resonance ionization microprobe (SIRIMP),10,14,23 provides an analytical technique that avoids many of the disadvantages of nonresonant and electron impact secondary neutral mass spectrometry (lack of sensitivity and selectivity),26,27 while making significant improvements over secondary ion mass spectrometry (SIMS)28 through improved efficiency, matrix independence, and sensitivity with virtually no isobaric and molecular interferences. In the SIRIMP technique, a pulsed focused ion beam (typically ∼5 × 106 ions/pulse) is used to sputter (19) Thonnard, N.; Parks, J. E.; Willis, R. D.; Moore, L. J.; Arlinghaus, H. F. Surf. Interface Anal. 1989, 14, 751-759. (20) Hurst, G. S.; Payne, M. G.; Kramer S. D.; Young, J. P. Rev. Mod. Phys. 1979, 51, 767-819. (21) Chen, C. H.; Payne, M. G.; Hurst, S.; Kramer, S. D.; Allman, S. L.; Phillips, R. C. In Laser and Mass Spectroscopy; Lubman, D., Ed.; Oxford University Press: London, 1990; pp 3-36. (22) 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. (23) Arlinghaus, H. F.; Joyner, C. F. J. Vac. Sci. Technol. 1996, B14 (1), 294300. (24) Pappas, D. L.; Hrubowchak, D. M.; Ervin, M. H.; Winograd, N. Science 1989, 243, 64-66. (25) Pellin, M. J.; Young, C. E.; Calaway, W. F.; Whitten, J. E.; Gruen, D. M.; Blum, J. B.; Hutcheon I. D.; Wasserburg, G. J. Phil. Trans. R. Soc. London 1990, A333, 133-146. (26) Becker, C. H. J. Vac. Sci. Technol. 1987, A5, 1181-1185. (27) Oechsner, H. In Thin Film and Depth Profile Analysis; Oechsner, H., Ed.; Springer-Verlang: Berlin, 1984. (28) See articles in: Secondary Ion Mass SpectrometrysSIMS X, Benninghoven, A., et al., Eds.; John Wiley and Sons: New York, 1997, and previous proceedings.

Analytical Chemistry, Vol. 69, No. 8, April 15, 1997

1511

constituents from the surface of a solid sample. The expanding cloud of sputtered material, mostly neutral particles, is probed by the RI laser beams that ionize all the atoms of the selected element within the volume intersected by the laser beams. Suppression of secondary ions produced by the sputtering process is achieved by a combination of the relative timing between the ion sputtering pulse and the firing of the RI lasers, timed extraction voltage switching, and electrostatic energy analysis. The ions are then detected in a TOF mass spectrometer. Charge compensation is possible using pulsed low-energy electrons in combination with pulsed extraction and target voltages. The nonspecific background can always be determined by detuning the laser wavelength by a few tenths of a nanometer and measuring the signal under the same experimental conditions. Much lower detection limits in the same analysis time can be achieved by replacing the ion beam with a laser beam, i.e., laser atomization resonance ionization microprobe (LARIMP).11,12,29,30 By use of a separate laser pulse instead of an ion pulse for the atomization process, several orders of magnitude more material can be released from the sample. Thus, with LARIMP several monolayers can be removed with a single atomizing laser pulse, while with SIRIMP thousands of bombarding ion pulses are required to remove one monolayer of atoms from a sample. Therefore, LARIMP is a very powerful technique for fast trace element analysis over large areas. However, the LARIMP method has not been studied as thoroughly as SIRIMP and is often less quantitative because of shot-to-shot fluctuations in laser intensity and changes of the surface morphology with consecutive laser shots. LARIMP must continue to be refined so that the faster analysis time that it offers can be utilized. The practical lateral resolution limit of LARIMP is ∼1 µm. Imaging is achieved by changing the x and y target positions while the atomization laser beam and the RI laser beam positions remain fixed. EXPERIMENTAL SECTION Detector. Figure 1 shows a schematic diagram of the imaging instrument. It consists mainly of a microbeam ion gun, a pulsed flood electron gun for charge compensation, an excimer laser system for sample atomization, a resonance postionization laser (repetition rate 30 Hz), 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 provides 25 mm motion in x and y with a resolution step size of 0.25 µm, 200 mm motion in z with resolution of 1 µm, and 360° motion in φ. The manipulator is positioned by stepper motors driven under computer control which can change positions at a speed of 10 000 steps/s. The sample holder is a carousel arrangement which can hold eight samples up to 40 mm × 40 mm in size. For LARIMP, a microscope objective mounted in the vacuum system focuses the output of the excimer onto the sample, with spot sizes down to 3-5 µm diameter using unstable resonator optics. The focus and positioning of this lens are accomplished by external manipulations. All the important instrument functions are controlled by an IBM-compatible computer. (29) Arlinghaus, H. F.; Thonnard, N. In Laser Ablation Mechanisms and Applications; Miller, J. C., Haglund, R. F., Jr., Eds.; Springer-Verlag: Berlin, 1991; pp 165-173. (30) Arlinghaus, H. F.; Spaar, M. T.; Thonnard, N.; Holloway, P. H.; Kabalka, G. W.; Switzer, R. C. In Resonance Ionization Spectroscopy; Miller, C. M., Parks, J. E., Eds.; Inst. Phys. Conf. Series 128; Institute of Physics Publishing: Philadelphia, PA, 1992; pp 275-278.

1512 Analytical Chemistry, Vol. 69, No. 8, April 15, 1997

Figure 1. Experimental arrangement of the SIRIMP/LARIMP instrument.

Sample Preparation. M13mp18 single-stranded DNA was obtained from United States Biochemical Corp. (Cleveland, OH). Two plasmid vectors, pSP70 and pBR322, were obtained from Promega Corp. (Madison, WI). Tin-labeled 17-mer oligonucleotides were prepared at Oak Ridge National Laboratory (ORNL). Two oligonucleotide primers were prepared, M13(-20) and T7, with sequences of 5′-GTA AAA CGA CGG CCA GT-3′ and 5′-AAT ACG ACT CAC TAT AG-3′, respectively. The oligonucleotides, with hexylamine on the 5′-end, were purified and reacted with the N-hydroxysuccinimide (NHS) ester of triethylstannylpropionic acid (TESPA) that contained isotopically enriched tin (>90%).31 Separate preparations of each of the oligonucleotides were labeled with different isotopes of tin. Maximum Strength Nytran nylon membranes were obtained from Schleicher & Schuell, Inc. (Keene, NH). The membrane that was used for these experiments had a net neutral charge and a 0.2 µm pore size. Test matrices were produced at Atom Sciences. High-purity chemicals were used, along with ultrapure deionized water produced with a Barnstead E-Pure water purification system. The buffer solutions were further purified by passing them through a column containing Chelex 100 chelating ion exchange resin from Bio-Rad Laboratories (Hercules, CA) removing trace level metal contamination. The hybridization and washing solutions were applied in a class 100 clean hood. Care was taken at every step to reduce or eliminate trace level tin contamination as much as possible. These precautions may not prove necessary ultimately, but they are very useful in the initial experimentation when the effects of any contamination may be misleading in determining sensitivity and other experimental parameters. Eventually, contamination causes can be evaluated individually, and the offending reagents or procedures can be modified or eliminated. The nylon membrane was cut into pieces with ceramic scissors, ∼1 × 1.5 cm for most applications. The nylon membrane pieces were wet with a solution of 0.5 M NaOH/1.5 M NaCl in H2O and were allowed to air-dry in the clean hood. The target DNA, diluted in the NaOH/NaCl solution, was pipetted onto the nylon. Generally, 4 µL of the 4 fmol/µL target DNA solution was pipetted in 1 µL increments. The spots were ∼3 mm in diameter. The samples were air-dried, and then 30 µL aliquots of a neutralization buffer (31) Sloop, F. V.; Brown, G. M.; Foote, R. S.; Jacobson, K. B.; Sachleben, R. Bioconjugate Chem. 1993, 4, 406-409.

(0.05 M Na2HPO4 in H2O, pH 6.5) were applied. The membranes were placed under a UV lamp at 254 nm and 120 000 µJ/cm2 to bind the DNA to the membrane. Next, they were dried loosely covered for 1 h in an oven at 80 °C. The tin-labeled probe was diluted in hybridization buffer (0.5 M Na2HPO4 in H2O, pH 7.2, 1% sodium lauroyl sarcosine, 5% dextran sulfate). The resulting probe concentration was 10 or 100 nM for most of the studies. After wetting and soaking the membrane at 25 °C with the hybridization buffer, hybridization was begun by covering the 1 × 1.5 cm sample with 30 µL of the tin-labeled probe solution. The samples were placed in the incubator at 25 °C for 2 h for hybridization, except for the studies in which these parameters were varied. After hybridization, the samples were washed with 5 mL of wash buffer (0.9 M NaCl and 0.09 M sodium citrate in H2O, pH adjusted to 7.0, 1% sodium lauroyl sarcosine). Two successive washes were performed for 5 min each at 25 °C. The samples were air-dried in the clean hood. RESULTS AND DISCUSSION Determination of Detection and Hybridization Parameters. Nylon samples, spotted with Sn isotope-labeled oligonucleotide primers (17-mer), were used to measure experimental parameters for SIRIMP and LARIMP analysis. To obtain a signal using either of these techniques, the bond between Sn and the organic molecule must be broken so that free Sn atoms are available for detection. For LARIMP, which uses a laser beam to vaporize and atomize the Sn-labeled oligonucleotide, 193 nm (ArF) atomization laser light results in acceptable Sn fragmentation, but longer wavelengths do not provide atomization necessary for RI detection. This short wavelength is necessary because the Sncontaining labeling compound, TESPA, only absorbs light at