Analysis of DNA Fragments from Conventional and Microfabricated

Anal. Chem. 1998, 70, 2067-2073

Analysis of DNA Fragments from Conventional and Microfabricated PCR Devices Using Delayed Extraction MALDI-TOF Mass Spectrometry Philip L. Ross,*,† P. Ann Davis, and Phillip Belgrader‡

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

Applications of polymerase chain reaction (PCR) product analysis using rapid affinity capture followed by delayed extraction (DE) MALDI-TOFMS is presented. Such applications include multiplex short tandem repeat (STR) typing, which is demonstrated for STR systems from conventional and microchip-based thermal cycling instruments. Using the combination of the microfabricated PCR instrument and DE-MALDI-TOFMS, a complete genotyping assay can be performed in under 50 min with a resultant molecular weight accuracy approaching or exceeding 100 ppm through external calibration. The observed resolution and mass accuracy for a 69-base PCR product enables identification of single base substitutions by direct molecular weight determination. The polymerase chain reaction (PCR)1,2 has been a revolutionary tool in the ongoing effort to map the various polymorphisms within the complete human genome. As the bulk of genomic information is assembled, there will be an increasing need for suitable analytical tools that, in conjunction with PCR, permit effective utilization of such information. Such tools must be adaptable to the miniaturized, high-throughput analysis platforms demanded by complex biological problems. Mass spectrometry, aided by electrospray ionization (ESI)3 or matrix-assisted laser desorption/ionization (MALDI),4 is one such technology which holds considerable potential for high-performance nucleic acid analysis. The unparalleled accuracy and precision with which molecular weight determinations are made may fundamentally alter the means by which routine DNA-based assays are performed. The challenge of PCR product analysis by mass spectrometry is 2-fold. First, ionization processes are extremely intolerant to * Corresponding author: (e-mail) [email protected]; (phone) 508-3837679. † Current address: Perseptive Biosystems, Inc., 500 Old Connecticut Path, Framingham, MA 01701. ‡ Current address: Lawrence Livermore National Laboratory, L-452, Livermore, CA 94550. (1) Saiki, R. K.; Scharf, S. J.; Faloona, F.; Mullis, K. B.; Horn, G. T.; Erlich, H. A.; Arnheim, N. Science 1985, 230, 1350-1354. (2) Saiki, R. K.; Gelfand, D. H.; Stoffel, S.; Scharf, S. J.; Higuchi, R.; Horn, G. T.; Mullis, K. B. Science 1988, 239, 487-491. (3) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37-70. (4) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. S0003-2700(97)01256-0 CCC: $15.00 Published on Web 04/16/1998

© 1998 American Chemical Society

the presence of salts, detergents, and other PCR components. As a result, reported purification procedures have been inefficient and cumbersome and have yielded data with poor resolution and sensitivity.5-10 One promising approach makes use of DNA immobilization through the streptavidin-biotin interaction.11-16 The potential effectiveness of this approach has been established for analysis or sequencing of synthetic oligonucleotides. Initial application of this approach for amplified DNA proved successful only for synthetic or reamplified targets and required additional manipulation involving centrifugation or lyophilization.15,16 The second challenge, encountered with MALDI in particular, is the inherently poor performance owing to extensive fragmentation of oligonucleotides larger than 30 bases. Application of delayed extraction or related pulsed extraction techniques has clearly improved oligonucleotide resolution in MALDI-TOFMS analysis. However, the benefits of such technology for PCR product analysis have not been explored. Recently, we demonstrated a solid-phase approach to PCR product purification17 which exhibits a number of desirable features as a means of sample preparation prior to mass spectrometry. It can be performed as a single-step, single-tube procedure using realistic quantities of PCR product amplified from crude human DNA extracts. Since the simple operations of (5) Chang, 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. (6) Hurst, G. B.; Doktycz, M. J.; Vass, A. A.; Buchanan, M. V. Rapid Commun. Mass Spectrom. 1996, 10, 377-382. (7) Tang, K.; Taranenko, N. I.; Allman, S. L.; Chang, L. Y.; Chen, C. H. Rapid Commun. Mass Spectrom. 1994, 8, 727-730. (8) 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. (9) Liu, Y.-H.; Bai, J.; Liang, X.; Lubman, D. M.; Venta, P. J. Anal. Chem. 1995, 67, 3482-3490. (10) Liu, Y.-H.; Bai, J.; Liang, X.; Semeniak, D.; Venta, P. J.; Lubman, D. M. Rapid Commun. Mass Spectrom. 1995, 9, 735-743. (11) Uhlen, M. Nature 1989, 340, 733-744. (12) Tang, K.; Fu, D.; Ko ¨tter, S.; Cotter, R. J.; Cantor, C. R.; Ko ¨ster, H. Nucleic Acids Res. 1995, 23, 3126-3131. (13) Chou, C.-W.; Bingham, S. W.; Williams, P. Rapid Commun. Mass Spectrom. 1996, 10, 1410-1414. (14) Ko ¨ster, H.; Tang, K.; Fu, D.-J.; Braun, A.; van den Boom, D.; Smith, C. L.; Cotter, R. J.; Cantor, C. R. Nature Biotechnol. 1996, 14, 1123-1128. (15) Jurinke, C.; van den Boom, D.; Jacob, A.; Tang, K.; Wo¨rl, R.; Ko¨ster, H. Anal. Biochem. 1996, 237, 174-181. (16) Jurinke, C.; van den Boom, D.; Collazo, V.; Luchow, A.; Jacob, A.; Ko ¨ster, H. Anal. Chem. 1997, 69, 904-910. (17) Ross, P. L.; Belgrader, P. Anal. Chem. 1997, 69, 3966-3972.

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pipetting, magnetic separation, and heating are involved, the procedure is amenable to parallel processing through automation. The protocol yields a single-stranded sample, thereby alleviating interpretation ambiguity associated with unresolved mass spectra of double strands. The procedure also yields an immobilized single strand which can be utilized for other diagnostic purposes involving MALDI-MS. Such applications include enzymatic oligonucleotide extension or peptide nucleic acid probe-based assays to identify point mutations.18,19 In this report, we describe several direct PCR product sizing applications made possible by affinity capture and delayed extraction with MALDI-TOFMS. A long-term goal in this work is development of time-minimized, generally applicable modes of obtaining informative genotyping results from a crude DNA extract. As clearly established in recent ESI-FTICR work,20,21 mass spectrometry can maximize the information gathered from a single, highly accurate, molecular weight measurement of a DNA sample. MALDI-MS has significant practical advantages over ESI-FTICR, but as yet, the full capabilities of MALDI-MS for direct PCR product analysis remain unknown. The use of delayed extraction22 substantially improves performance in synthetic oligonucleotide analysis but has not been shown to facilitate characterization of significantly larger PCR products. It is shown here to dramatically improve mass resolution and mass measurement precision of PCR products with widely varying compositions. The improvement in instrument performance facilitates new applications for MALDI based on direct molecular weight determination of PCR products. These developments are consistent with the ongoing trend toward miniaturized, multiplexed, highthroughput analysis of biological samples. Analysis of a variety of representative PCR products is described here. With these, we illustrate the use of MALDI-TOFMS for analysis of single-locus and multiplex short tandem repeat (STR) fragments. In addition, encouraging results are obtained from analysis of DNA fragments produced from a rapid, portable, microfabricated PCR amplification device. Finally, the ability to identify the presence of a single base substitution by direct molecular weight measurement of a PCR product is demonstrated, indicating the possibility of avoiding postPCR assays in diagnostic applications. These experiments definitively illustrate advanced capabilities of MALDI-TOFMS as a high-performance genetic analysis tool in the context of the ongoing progression toward miniaturized, automated, high throughput genotyping. EXPERIMENTAL SECTION Polymerase Chain Reaction. A number of PCR products were generated for analysis by mass spectrometry. Primer pairs (Life Technologies, Gaithersburg, MD) and product molecular weight ranges for the STR loci, THO1,23 COL1A,24 and TPOX25 loci have been described previously. A 69-base PCR fragment (18) Braun, A.; Little, D. P.; Ko¨ster, H. Clin. Chem. 1997, 43, 1151-1158. (19) Ross, P. L.; Lee, K.; Belgrader, P. Anal. Chem. 1997, 69, 4197-4202. (20) Muddiman, D. C.; Wunschel, D. S.; Liu, C.; Pasa-Tolic, L.; Fox, K. F.; Fox, A.; Anderson, G. A., Smith, R. D. Anal. Chem. 1996, 68, 3705-3712. (21) Muddiman, D. C.; Anderson, G. A.; Hofstadler, S. A.; Smith, R. D. Anal. Chem. 1997, 69, 1543-1549. (22) Juhasz, P. Roskey, M. T.; Smirnov, I. P.; Haff, L. A.; Vestal, M. L.; Martin, S. A. Anal. Chem. 1996, 68, 941-946. (23) Polymeropoulos, M. H.; Xiao, H.; Rath, D. S.; Merril, C. R. Nucleic Acids Res. 1991, 19, 3753. (24) Pepe, G. Hum. Mutat. 1993, 2, 300-305.

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(mtDNA) containing a single base substitution (T f C) was also prepared for a number of individuals. Primers for the expanded TPOX system (for biplex PCR) and the 69-base PCR amplifications were synthesized using an Expedite 8909 (PerSeptive Biosystems, Framingham, MA) synthesizer and were used without additional purification. All amplifications used DNA polymerase from cloned Pyrococcus furiosus (Pfu, Stratagene, LaJolla, CA), using accompanying 10× buffer according to the manufacturer’s instructions. Typical PCR reagent conditions were (50-µL volumes) as follows: template, 10-50 ng; primers, 0.5-1.0 µM, Mg, 2.0 mM; dNTPs, 160 µM; Pfu, 1 unit. PCR reactions were performed on a Perkin-Elmer 9600 thermal cycler with the following program: 96 °C for 1 min; 10 cycles of 96 °C, 30 s, 62 °C, 30 s, 72 °C, 30 s; 22 cycles of 90 °C, 30 s, 62 °C, 30 s, 72 °C, 30 s; and a final 5-min extension at 72 °C. THO1-TPOX biplex amplifications were carried out under similar conditions, with magnesium ion concentrations increased to 3.0 mM. Human DNA template was extracted from whole blood using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). PCR Amplification and MALDI-MS Using a Microfabricated Thermal Cycler. The miniature analytical thermal cycling instrument (MATCI) used in some of the present experiments has been described in detail elsewhere.26-28 Briefly, thermal cycling is performed in passively cooled etched silicon chambers heated electronically using a pulsed width modulator. Thin-walled, 1-mm-i.d. polypropylene liners inserted into the chambers of the PCR device served as reaction chambers. Reaction mixtures were prepared and added to the tube inserts, which were then placed into the device. Mineral oil was placed on top of the reaction mixtures to prevent evaporation. Typical reaction volumes used were 18 µL. An aliquot (3-5 µL) was then removed to check PCR yields on agarose gels, leaving approximately 12-13 µL of usable PCR mixture for purification and mass spectrometry. PCR reagent concentrations were used as described above with the appropriate volume scaledown. The thermal cycling programs used temperatures similar to that for conventional PCR amplifications, but the times of individual steps were reduced to take advantage of the improved thermal coupling provided by the narrow dimension of the silicon chambers. Investigation of time parameters in the PCR protocol is described in the Results and Discussion section. Affinity Purification of PCR Products. A detailed description of the procedure used for PCR product purification for MALDI-TOFMS analysis can be found elsewhere.17 Crude PCR mixtures were treated with washed streptavidin-coated magnetic beads (BioMag Streptavidin, PerSeptive Biosystems) suspended in 0.5 M NaCl, 0.005 M EDTA. After incubation (5-10 min) and several washes of the magnetic beads with water and 0.1 M ammonium acetate, nonbiotinylated DNA strands were heatdenatured into 2-6-µL volumes of water. Aliquots of the ssDNA were then combined with matrix solution and taken for MALDIMS analysis. In the case of PCR samples prepared using the (25) Bikker, H.; Baas, F.; de Vijlder, J. J. M. Hum. Mol. Genet. 1992, 1, 137. (26) Woolley, A. T.; Hadley, D.; Landre, P.; deMello, A. J.; Mathies, R. A.; Northrup, M. A. Anal. Chem. 1996, 68, 4081-4086. (27) Northrup, M. A.; Bennet, B.; Hadley, D.; Landre, P.; Lehew, S.; Richards, S.; Stratton, P. A. Anal. Chem., in press. (28) Belgrader, P.; Smith, J. K.; Weedn, V. W.; Northrup, M. A. J. Forensic Sci., in press.

Figure 1. DE-MALDI-TOFMS analysis of PCR products containing STR polymorphisms at the (A) THO1, (B) COL1A, and (C) TPOX loci. Numbers in parentheses indicate percentage deviation from calculated values. THO1 and TPOX genotypes were verified using Geneprint (Promega, Madison, WI) STR typing kits. Spectra in (A) were calibrated using programmed instrument calibation parameters and spectra in (B) and (C) used external calibration files generated in separate experiments. Peaks labeled with an asterisk in (A) indicate minor quantities of released biotinylated strands.

microfabricated thermal cycler, an acetone wash was included after product capture onto the magnetic beads. Mass Spectrometry. All analyses described here used a Voyager DE-RP workstation with a flight path length of 1.35 m operating in linear, pulsed extraction mode. Negative and positive ion modes gave comparable results, with somewhat more intense signal observed for negative ions. Matrix solutions were typically a 4:1 molar ratio of 3-hydroxypicolinic acid/picolinic acid in 50:50 (v/v) acetonitrile/water buffered with 0.05 M ammonium acetate or ammonium citrate. Spectra shown are typically averages of 20-80 individual spectra. Spectra were externally calibrated

(Figure 1A uses a calibration equation based on instrument dimensions and voltages) using PCR products from DNA extracts of known sequence, as described in Results and Discussion. RESULTS AND DISCUSSION Analysis of STR Loci. To explore the use of DE-MALDI for direct analysis of PCR fragments, model trinucleotide and tetranucleotide loci were prepared. Figure 1 shows analysis of affinitypurified STR fragments at three loci using DE-MALDI. In all cases, a peak molecular weight value is observed, making possible a precise molecular weight determination. The observed peak Analytical Chemistry, Vol. 70, No. 10, May 15, 1998

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molecular weight values and deviation from calculated values is indicated for each spectrum. As contrasted with rapid electrophoretic approaches such as capillary electrophoresis (CE)29 and microchip CE,30 a very important advantage of DE-MALDI is the minimal dependence on size standards to yield reliable data. This latter advantage is illustrated in Figure 1, where high resolution yields accurate numerical results from fragments close to 100 bases (Figure 1C) with minimal attention paid to calibration. The spectra shown were obtained on different days with different sample plates and slightly varying source region voltages while using either programmed instrument calibration parameters (Figure 1A) or external calibration files generated for separate experiments. Some variability in mass accuracy was observed, which was a reflection of the different analysis conditions used between various samples. Nevertheless, these findings, particularly Figure 1A, illustrate that reasonable molecular weight accuracy can be obtained with virtually no effort spent on calibration, a feature that eliminates the dependence on DNA size standards for each analysis. The accuracy demonstrated in Figure 1 greatly exceeds that necessary for analysis of length polymorphisms. This unique capability of DE-MALDI can have a profound impact in sensitive applications such as identity testing or diagnostics by eliminating possible uncertainties in allele designation The ability to obtain molecular weight values from PCR products was previously demonstrated using static MALDI-TOF at an accuracy of approximately 0.2% or better. The results in Figure 1 show an order of magnitude improvement in this accuracy. This improvement is a consequence of the ability to resolve molecular ion peaks from largely unresolved background. Earlier DE-MALDI-TOFMS reports documented substantial improvements in resolution for analysis of mixed base oligodeoxynucleotides up to a 50-mer. The present results show that resolution enhancement occurs for significantly larger ssDNA fragments of varying composition up to 92 bases. Multiplex STR Analysis. Conventional analysis of short tandem repeat loci is made significantly more efficient by multiplexing, where primer sets are used to simultaneously amplify several polymorphic loci in a single PCR.31,32 Multiplexing minimizes the amount of original DNA template required while increasing the information content derivable from a single PCR. Representative data from a THO1-TPOX biplex experiment using DE-MALDI-MS are shown in Figure 2. The same samples produced poorly resolved, low-intensity spectra under static acceleration conditions. The most significant drawbacks to multiplex analysis are the significantly lower product yield and considerable disparity in amplification efficiency between individual loci. For MALDI-MS, difficulty can be encountered with analyte mixtures due to suppression of individual components in the sample. Such suppression is not observed for the multiplex analysis presented here. Using an external calibration file from (29) Wang, Y.; Ju, J.; Carpenter, B. A.; Atherton, J. M.; Sensabaugh, G. F.; Mathies, R. A. Anal. Chem. 1995, 67, 1197-1203. (30) Schmalzing, D.; Koutney, L.; Adourian, A.; Belgrader, P.; Matsudaira, P.; Ehrlich, D. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10279-10284. (31) Lins, A. M.; Sprecher, C. J.; Puers, C.; Schumm, J. W. Biotechniques 1996, 20, 882-889. (32) Sparkes, R.; Kimton, C.; Watson, S.; Oldroyd, N.; Clayton, T.; Barnett, L.; Arnold, J.; Thompson, C.; Hale, R.; Chapman, J.; Urquhart, A.; Gill, P Int. J. Legal Med. 1996, 109, 186-194.

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Figure 2. Analysis of THO1-TPOX biplex products using DEMALDI-TOFMS. Products were amplified using conventional thermal cycling, and mass spectra were calibrated using separately generated external calibration parameters.

a separate experiment, molecular weight values taken from peak maximums remain reasonably accurate. The demonstrated feasibility to analyze PCR products from multiple loci gives promise for expansion to triplex or quadruplex systems. Ideally, product sizes should be minimized to enhance MALDI sensitivity while still maintaining nonoverlapping allelic ladders. For the THO1-TPOX system here, increasing the magnesium ion concentration (1.7×) and modifying the forward TPOX primer composition improved the yield and gave spectra with more equivalent intensity of THO1 and TPOX products. At this point, successful development of higher order multiplexing would require effort mostly directed toward PCR optimization rather than optimization of the mass spectrometry. Analysis of STRs and Multiplex STRs from a Rapid Microfabricated Thermal Cycling Device. The application of mass spectrometry for PCR product analysis has been widely documented for situations where large quantities of product can be collected and pooled together. The present results represent an advance toward minimizing the amount of PCR product required (10-20% of a single PCR reaction used) while still capitalizing on the desirable features of MALDI. With the technological drive toward detection and analysis of biological systems on increasingly miniaturized platforms, the use of mass spectrometry as a suitable tool for direct DNA sizing requires continued refinement. Here, we have explored the feasibility of utilizing affinity capture purification with DE-MALDI-TOFMS in a miniaturized context through a series of experiments performed using a rapid, miniaturized PCR device based on a micromachined thermal cycling chamber. The MATCI is equipped with in situ optical detection, such that fluorescent probe-based assays can be used to discriminate single-base polymorphisms.28 However, characterization of length polymorphisms such as tandem repeats requires an analysis platform with reasonably accurate PCR

Figure 4. Analysis of MATCI-amplified THO1-TPOX biplex PCR products (affinity purified) using DE-MALDI-TOFMS. A 35 min cycling program, as shown in Figure 3 and described in the text, was used, immediately followed by a 20 min affinity capture purification step.

Figure 3. Amplification of THO1 6,9 sample using a microfabricated PCR thermal cycling instrument, with analysis using affinity capture and DE-MALDI-TOFMS. A 15-min affinity capture/denature protocol was used directly from the final crude PCR reaction mixture. For the 32-min sample, 10 individual spectra were collected and externally calibrated using a THO1 6,9 sample. From the 10 spectra, average Mr values for six and nine alleles were 20 214.0 (SD ) 2.9; Mr (calc) ) 20 215.4) and 23 847.8 (SD ) 4.2; Mr (calc) ) 23 848.5).

product sizing, as can be accomplished using MALDI-TOFMS. Figure 3 shows a series of experiments performed to optimize amplification at the THO1 locus. The program used temperatures of 94 °C denaturing, 64 °C annealing, and 72 °C extension. For all experiments, denaturing was held for 4 s, while the annealing and extension temperatures were lowered from 25 s each (Figure 3A) to 15 s each (Figure 3C). Further lowering of annealing and extension times resulted in a precipitous drop in amplification yield, as observed by agarose gel electrophoresis and mass spectrometry The benefit of using such a device can be seen from the total amplification times indicated in Figure 3. These spectra were obtained from crude PCR products which were then purified by affinity capture in the normal manner. Reasonable amplification yield is acheived with a conventional thermal cycler in 2.2 h. The same can be performed with the MATCI in 32 min. A total analysis time, from a crude DNA extract, of under 50 min is achieved with molecular weight accuracy far superior to other

separation and sizing platforms (see below). This experiment also represents a substantial decrease in the amount of original PCR sample processed, since typically only 12 µL yielded sufficient sample for four 1.5-µL spots. This signifies a 4-fold improvement from our previously reported findings and over a 20-fold improvement over recently described ESI-FTICR work.20 A typical spectrum obtained from a THO1-TPOX biplex amplification is shown in Figure 4. Template and primer concentrations and thermal cycling conditions were nominally the same as used in Figure 3A. As was observed with biplex amplifications using normal thermal cycling, the mass resolution begins to decrease, although not to an extent that compromises genotyping accuracy. For the PCR samples prepared here using the MATCI, DEMALDI results indicate superior amplification yields to conventional PCR under similar template and primer concentrations. The better thermal coupling provided by the device improves product yield by reducing temperature differentials across the reaction volume as the reaction is cooled or heated. There is a synergistic relationship between adoption of mass spectrometry for DNA sizing and use of a miniaturized device as shown here. The sensitivity and accuracy of MALDI-MS increases with progressively smaller analyte molecules. Similarly, minimization of both the total amplification time and required DNA template quantity using the MATCI is most pronounced with shorter amplicons. Together, the two technologies represent a dramatic performance improvement as a complete analytical system. Turnaround time and sample consumption are reduced while the final analytical accuracy and reproducibility are substantially increased. Molecular Weight Accuracy of DE-MALDI Using MATCIPCR. Amplifications performed using the microchip PCR device gave noticeably better yields than with conventional PCR. In MALDI analysis of PCR samples from the MATCI, spectra were observed to be more intense and were produced more uniformly across single sample spots. This reproducibility afforded the opportunity to explore in more detail the molecular weight accuracy obtainable for PCR product analysis by DE-MALDI. Detailed characterization of delayed extraction and related technologies has been performed for synthetic oligonucleotide analysis.22,33 The complexities of PCR product analysis have prevented a similar evaluation for genomic DNA extracts. It was therefore (33) Dai, Y.; Whittal, R. M.; Weinberger, S. R.; Li, L. Rapid Commun. Mass Spectrom. 1996, 10, 1792-1796.

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Figure 5. Stability analysis of external calibration demonstrated for a THO 6,9 sample prepared using MATCI-PCR and affinity capture. Displayed spectrum was calibrated with a file generated six days prior to analysis. Calculated Mr values; 6 allele, 20 215.4; 9 allele, 23 848.5.

a matter of considerable interest to evaluate the molecular weight accuracy achievable with the samples prepared here. It should be acknowledged that similar investigations have been performed using representative bacterial PCR products and ESI-FTICR.20,21 The resolution and accuracy obtained therein cannot be matched by using MALDI-TOF, but such results do represent an upper limit to aim toward in the present experiments. A single sample was prepared using the microchip PCR device and processed in the normal manner. Aliquots of the resultant ssDNA were then deposited onto separate spots on the MALDI probe. A spectrum was collected from one spot, and then a series of 10 spectra were collected from a neighboring spot. In this fashion, the series of spectra were collected in approximately 10 min. The spectra obtained were similar to those shown in Figure 3, in that mass assignments could be made from a well-resolved (>700 fwhm) peak maximum. The sample homogeneity was such that many spectra could be obtained from a single spot, but only the first 10 spectra collected were saved. The spectrum from the first spot was used to generate a calibration file for external calibration of the set of 10 spectra. The molecular weight accuracies from an average of the 10 spectra were 70 and 30 ppm for the six (67 bases) and nine (79 bases) alleles, respectively. A spectrum from a THO1 6, 9 sample, acquired on a day different from the experiments above, is shown in Figure 5. The molecular weight assignments of this spectrum were made using the calibration file generated previously. The results shown in Figure 5 illustrate remarkable stability of both the calibration data and the molecular weight assignments. This intrumental stability highlights in a very definitive manner the ability of DE-MALDI to obtain precise and accurate PCR product molecular weight information routinely with relative procedural simplicity. Determining Single-Base Substitutions by Exact Mass Measurement. The results described above represent a level of precision and accuracy considerably beyond previous reports of MALDI analysis of PCR products and exceed that required for characterization of short tandem repeats. The accuracy of less than 100 ppm suggests that the occurrence of a single base substitution may be addressed by direct DNA sizing within a given size range. A number of reports illustrating the use of MALDI to 2072 Analytical Chemistry, Vol. 70, No. 10, May 15, 1998

Figure 6. Typical spectra obtained from DE-MALDI-TOFMS analysis of a 69-base PCR (human mtDNA) fragment containing a known T-C polymorphism. DNA extracts harboring T (panel A, normal, calculated Mr ) 20 909.8) or C substitution (panels B and C, mutant, calculated Mr ) 20 894.8) at position 16 362 were selected from standard cycle sequencing data. Unsmoothed, externally calibrated spectra are shown.

characterize single nucleotide polymorphisms have appeared.18,19,34,35 These assays require from 20 min to several hours of post-PCR effort to distinguish base substitutions in target DNA. Obtaining the same information by direct Mr determination represents a valuable simplification, thereby broadening the scope of MALDI as a versatile genotyping tool. As a working demonstration of the capability to discriminate base substitutions, a 69-base fragment spanning nucleotides 16 342-16 410 of human mitochondrial DNA was amplified using a biotinylated reverse primer. Individuals with a “T” (normal or “Anderson” sequence) or a “C” (mutant) at position 16 362 were selected. Following sample purification, a series of experiments was performed in a manner similar to the previous section. An external calibration spectrum was generated on a sample spot using a reference PCR sample (16362T). Series of spectra were then acquired from PCR products from the set of three individuals (34) Haff, L. A.; Smirnov, I. P. Genome Res. 1997, 7, 377-378. (35) Haff, L. A.; Smirnov, I. P. Nucleic Acids Res. 1997, 25, 3749-3750.

Table 1. Molecular Weight Data for Polymorphic 69-Base PCR Fragment n

individual 1 16362T

individual 2 16362C

∆M1

individual 3 16362C

∆M2

1 3 5 8

20 905.5 20 909.5 20 910.0 20 910.6

20 894.1 20 893.6 20 893.9 20 895.1

11.4 16.1 16.1 15.5

20 893.1 20 892.0 20 894.1 20 894.9

12.4 17.5 15.9 15.7

a

n, no. of spectra in average.

and were averaged in the order of acquisition. Representative data are shown in Figure 6 and Table 1. In general, the spectral quality observed in these experiments was superior to the analysis of short tandem repeats. Resolving power values (fwhm) for these single-component spectra ranged from 1000 to 1700, exceeding previous reports of MALDI-MS analysis of PCR products and shorter mixed-base oligonucleotides. The data in Table 1 show that with the first spectrum obtained for each sample, discrimination between the T and C forms can be made. With three measurements, each averaged result differs by less than 3 Da from the expected mass. Mass deviations of under 50 ppm are achieved for all three samples after accumulation of five spectra, and additional data have minimal effect on the average values. The best tradeoff between accuracy and experimental simplicity appears to occur after averaging three individual spectra. For products in this molecular weight range, it was not difficult to obtain multiple high-quality spectra across a single sample spot in a matter of minutes. Acquiring multiple spectra increases analysis times; however, time-consuming post PCR assays for polymorphism identification may be avoided. With the level of accuracy and precision shown here, this mode of

analysis can also distinguish T-C polymorphisms (∆m ) -15 Da) from T-A (∆m ) +9 Da) and T-G (∆m ) +25 Da) by making use of a normal “reference” sample for external instrument calibration. The performance of such an assay may be improved by calibration with an internal oligonucleotide standard. An additional area of interest would be single-base discrimination by direct MALDI analysis following multiplex PCR. CONCLUSIONS The use of DE-MALDI-TOFMS is demonstrated here to be a precise, accurate, and versatile PCR product analysis tool that is compatible with the next generation of high-performance genetic analysis technology. Analysis of STRs, multiplex STRs, and discrimination of base substitutions are demonstrated using a simple, single-tube purification step with no instrument optimization. Similarly, analysis of STRs and biplex STRs produced by a rapid, microfabricated PCR device is shown to be well suited to MALDI-TOF analysis. These findings show MALDI-TOFMS to have an expanding role as a robust, high-throughput tool capable of characterizing a wide range of polymorphisms in a direct manner. ACKNOWLEDGMENT The authors gratefully acknowledge assistance and technical support from M. Allen Northrup and Dean Hadley of Lawrence Livermore National Labs Microtechnology Center, Livermore, CA. Disclaimer: The views presented here are the opinions and observations of the authors and in no way reflect the position of the U.S. Air Force or the U.S. Department of Defense. Received for review November 17, 1997. February 26, 1998.

Accepted

AC971256Z

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