Development of Resonance Ionization Spectroscopy for DNA

INSTRUMENTATION

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K. Bruce Jacobson Biology Division Oak Ridge National Laboratory Oak Ridge, TN 37831-8077

H. F. Arlinghaus Atom Sciences, Inc. 114 Ridgeway Center Oak Ridge, TN 37830

Until recently, DNA sequencing pro­ cedures have used primarily radio­ isotopes or fluorescent organic com­ pounds as labels for detection of DNA 0003 - 2700/92/0364 -315A/$02.50/0 © 1992 American Chemical Society

would take a long time. In addition, there was a desire to sequence the genomes of mouse, Drosophila, C. elegans, yeast, and E. coli—organisms whose extensive genetics could be profitably correlated to detailed DNA sequence information (4, 5). Resonance ionization spectroscopy (RIS) h a s been used to analyze a wide variety of natural and manufac­ tured materials for trace element content a n d concentrations (6-8). This analytical procedure can, i n principle, q u a n t i t a t i v e l y m e a s u r e any known element (except He and Ne), and several elements can be at­ tached to DNA. Thus RIS offers the possibility of using new labels for DNA sequencing. When RIS is used with a mass spectrometer, each sta­ ble isotope can be a DNA label. Also, because of recent improvements in the rate of RIS analysis, the method can be very fast. The goal of our re­ search is to develop procedures for analyzing DNA sequences by RIS, thereby providing H u m a n Genome Project researchers with a faster and less expensive method to accomplish the ordering of the ~ 3 billion nucle­ otides in human chromosomes.

fragments following gel electrophore­ sis (1-3). Such procedures are well established: Radioisotopes a r e de­ tected on silver emulsion films and fluorescent labels with sensitive pho­ totubes. Hundreds of thousands of DNA base sequences have been pro­ cessed by several laboratories using these two kinds of labels. However, when t h e goal of sequencing t h e ~ 3 χ 10 9 bases of the human genome was u n d e r s e r i o u s c o n s i d e r a t i o n three years ago, it was clear that se­ quencing with existing methods a t the rate of 1 million bases per year

In t h e S a n g e r DNA s e q u e n c i n g method (i) the DNA fragment whose sequence is to be determined is used as a template in a reaction mixture to which DNA polymerase is added. When a 17-mer (an oligonucleotide of 17 nucleotides) c o n t a i n i n g a s e ­ quence that binds to a complemen­ t a r y sequence on t h e template is used as a primer, the polymerase will use t h e deoxynucleoside t r i p h o s ­ phates of adenine (A), guanine (G), cytosine (C), and thymine (T) to ex­ tend the primer and replicate the se­ quence in the template DNA. Furthermore, the enzyme will use the dideoxynucleoside triphosphates of the same four bases to terminate the newly formed DNA. The new enzymatically synthesized DNA frag­ ments contain t h e original primer, the replicated sequence of the DNA of interest, and the dideoxy termina­ tor; when these DNA fragments are separated by poly aery lamide gel elec­ trophoresis, a difference in length of one nucleotide can be detected. When 3 2 P or 3 5 S is used as the la­ bel, the A-, G-, C-, and T-terminated fragments must be run in four adja­ cent lanes of the electrophoresis gel, and the sequence is read from the autoradiogram by noting the position of the bands in the A, G, C, and Τ lanes. Because these four different bases define t h e sequence of t h e

ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992 · 315 A

INSTRUMENTATION DNA, the use of isotopes in groups of four is particularly advantageous. Multiplexing results from the use of four different fluorescent labels that can be combined in a single gel electrophoresis lane. The wavelength discrimination of the fluorescent detector is used to identify the individual bases (2, 3). We proposed the possibility that four stable isotopes of a single element could be used as a label that would allow the combination of all four types of DNA fragments into one gel channel, because a mass spectrometer would distinguish each isotope (9). Figure 1 illustrates the use of stable isotopes of Sn for DNA sequencing with RIS. Use of stable isotopes offers the advantage that the four labels will not have any differential mobility effect in gel electrophoresis; fluorescent labels may be sufficiently different in mass to cause migration differences. Furthermore, the resolution of a time-of-flight (TOF) mass spectrometer is greater than that of a spectrofluorometer, and such an instrument can detect many isotopes simultaneously rather than just the

four labels that are resolvable in the optical system. The use of RIS to analyze metal-labeled DNA fragments separated by polyacrylamide gel electrophoresis offers several a d v a n tages, including extreme sensitivity, very high selectivity, high spatial resolution, and a very rapid rate of analysis (10, 11). To be a useful DNA label, a stable isotope must have these properties: sufficient stability in an organometallic compound to survive all steps of the sequencing procedure, no adverse effect on the sequencing reactions or on electrophoretic mobility, a multiplicity of four or more, low n a t u r a l abundance to avoid background problems, and enough natural abundance that separation does not become too expensive. Sulfur isotopes were attractive label possibilities because they could be incorporated into a - t h i o n u c l e otides, as is already done with 3 5 S (12). A mass spectrometer-based procedure for DNA sequencing specifically uses the sulfur isotopes (13). A discussion of their use will illustrate the strengths and weaknesses of the

Convert isotopically enriched Sn0 2 to TESPA

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Polyacrylamide gel electrophoresis to separate DNA fragments according to size in one gel lane

Employ RIS and TOFMS for very sensitive location and differentiation of the Sn isotopes

Feed RIS output into computer program to determine nucleotide sequence; store in database

Figure 1. Flow chart for use of stable Sn isotopes for DNA sequencing. 316 A · ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992

stable isotope approach. The percent natural abundances of 32 S, 3 3 S , 3 4 S , and 3 6 S are 95.0, 0.75, 4.22, and 0.11, respectively. These isotopes can be readily separated in standard mass spectrometers. To discern the sulfur isotope attached to the DNA fragment and discriminate it from sulfur from other sources, the isotope must be enriched. Because i2 S constitutes 95% of the natural S abundance, the companion isotopes must be purified to > 99%. At the other extreme, 3(i S has only a 0.11% n a t u r a l a b u n d a n c e so t h a t , even when purified to 30%, the commercially available isotope contains more 34 S than 3fi S. Further purification is very expensive. The most desirable elements for DNA sequencing by RIS should have isotopes that are neither very abundant nor very rare in the natural distribution. Sulfur is less attractive than some elements for use in DNA sequencing, for three reasons: First, there is the distribution argument just presented; second, for RIS, many metallic elements are much easier to ionize than sulfur; and third, the rel ative abundance in nature is higher than that of many alternative elements. Advancement of this RIS application will require four steps: development of methods to convert metal oxides to organometallic compounds that can be attached to the primers or nucleotide s u b s t r a t e s for DNA polymerase, because enriched isotopes are usually supplied as the oxides; manipulation of gel electrophoresis techniques so that the separated DNA bands can be displayed for analysis; adaptation of RIS methods to detect metal atoms attached to DNA fragments; and enlargement of data management systems to handle the enormous rate of data acquisition that will be necessary. Our major research efforts have been on the first and third components of the project using standard gel electrophoresis procedures. RIS The concept of RIS was introduced by Hurst and co-workers at Oak Ridge National Laboratory. They used the method to detect a single Cs atom in a background of 10 19 Ar atoms and 10 1 8 C H 4 molecules (14). S u b s e quently the RIS concept was generalized, and Hurst et al. showed that it could be incorporated with current laser techniques for all elements except He and Ne (7). RIS has been successfully applied in a variety of fields, including semiconductors,

geo- and cosmochemistry, hydrology, and environmental and biomedical sciences. T e c h n i q u e . Tunable l a s e r s are used to count ground-state neutral atoms of the element selected for analysis 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 t h a t selection of p a r t i c u l a r excited s t a t e s for RIS analysis permits great element selec­ tivity. It has been demonstrated that the ionization efficiency for the se­ lected element can be as much as 10 9 times higher than that for the other elements in the sample (15). The high selectivity of the RIS process ' also helps to maintain linearity in the mass spectrometer ion extraction region by reducing space charge ef­ fects that would otherwise be present because of ionization of the major sample constituents. Because isotopic shifts of most ele­ ments are small in comparison to the bandwidth of the RIS lasers (7— 12 GHz) used in our experiments, all isotopes of a chosen element will be ionized with essentially equal sensi­ tivity. The intensity of modern pulsed dye lasers is sufficient to saturate both the b o u n d - b o u n d t r a n s i t i o n s and the ionization step, thereby assuring unit probability of ionizing all atoms of the selected element that are in the volume intersected by the RIS la­ ser beams. Isotopic selectivity is achieved with the m a s s spectrometer. If a TOF mass spectrometer is used, all iso­ topes of an element can be detected simultaneously. The mass spectrom­ eter requirements are therefore re­ duced to the resolution of neighbor­ ing isotopes of a single element; the high ionization selectivity and the suppression of the secondary ions virtually eliminate interferences from molecular ions, isobars, or scat­ tered ions from major sample con­ stituents. The sensitivity and selectivity of the RIS process are especially valu­ able for trace element analysis in materials in which the matrix com­ plexity is frequently a serious source of interference. The efficiency of ion­ izing and then counting the atoms of the selected element in the sputtered cloud depends on the ionization effi­ ciency ( — 100%), the temporal and spatial overlap of the resonance laser beam with the atomized cloud ( 2 0 50%), the total transmission of the mass spectrometer (60-80%), and

the detector efficiency (60-80%). Re­ cently pptr detection limits (several orders of magnitude below attomoles) were shown for In in Si (16, 17) with a useful yield (atoms detected per atoms sputtered) of —26% (16). Another salient feature of the RIS process is its generality. Using avail­ able lasers, it is possible to ionize more than 80% of the elements, in­ cluding all transition elements and lanthanides, with very high element selectivity a n d almost 100% effi­ ciency. Simple RIS schemes, such as UV + vis + IR, UV + UV + IR, vis + vis + IR, and UV + IR, yield nearuniform sensitivity down to the level of few atoms (18, 19). Figure 2 presents simple energylevel diagrams illustrating the pho­ toionization of neutral ground-state atoms. Figure 2a shows resonance ionization by the absorption of two resonant photons and a low-energy IR photon. The resonant transitions are saturated with low laser power, whereas high power can be used for the IR photon because of its low en­ ergy. This results in saturated ion­ ization of only the selected element with e s s e n t i a l l y no ionization of other elements (as little as 1 part in 10 9 ). States in the ionization continuum ( a u t o i o n i z i n g s t a t e s , F i g u r e 2b), which can be excited resonantly and thereby allow lower laser intensity in

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the photoionization step, further in­ crease selectivity while reducing the possibility of interference from nonresonant ionization processes. Except for He and Ne, which are not practi­ cal RIS candidates, and Ar, F, and Kr, which require very complex laser a r r a n g e m e n t s to g e n e r a t e v a c u ­ um-UV radiation, the elements in the upper right corner of the periodic system can be ionized by the absorp­ tion of two photons to reach a twophoton resonance, with consequent ionization from the absorption of a third photon (Figure 2c). Although this third process is less selective and less sensitive than the first two, it offers a significant advantage over conventional ionization and nonresonant photoionization methods. I n s t r u m e n t a t i o n . RIS analysis requires free atoms in the gas phase. In the sputter-initiated resonance ionization spectroscopy (SIRIS) tech­ nique (Figure 3), a sample is placed on a planchet and inserted into a vacuum chamber, where it is bom­ barded with a high-energy pulsed Ar ion beam (10 7 ions per pulse) (20). The diameter of the bombarding ion beam can be focused between a few and several hundreds of micrometers and typically measures between 50 and 200 μπι. The expanding cloud of sputtered material consists of neu­ tral atoms, molecular fragments, and ions; the ions are removed by timed

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Hi Figure 2. Simplified energy-level diagrams illustrating the photoionization of ground-state atoms. (a) Resonance ionization by the absorption of two resonant photons and a low-energy IR photon. (b) Photoionization by resonant excitation of an autoionizing state. This ionization scheme further reduces interfering ionization processes by adding selectivity in the last step of the RIS process. (c) Elements with a large energy gap to the first excited state can be ionized via a two-photon allowed state.

ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992 · 317 A

INSTRUMENTATION extraction voltage switching a n d electrostatic energy analysis (posi­ tive ions are suppressed; negative ions are accelerated). The remaining neutral particles are then probed by the RIS laser beams that ionize all atoms of the selected element within the volume intersected by the laser beams. Efficient o v e r l a p of t h e l a s e r beams with the cloud of desorbed material is achieved by choosing the a p p r o p r i a t e delay t i m e b e t w e e n firing the ion gun and firing the RIS laser, and carefully positioning the RIS laser beams with the ion beam/ sample surface intersection. The RIS laser b e a m d i a m e t e r is typically 2 - 3 mm. The ionized atoms are ex­ tracted into a detector system con­ sisting of an electrostatic energy an­ alyzer and a magnetic sector mass spectrometer or a TOF mass spec­ t r o m e t e r , and m e a s u r e m e n t s are made in charge digitization (signal expressed as voltage) or single ioncounting (signal expressed as counts) mode. The mass resolution (m/Am) of 600 of the current system is suffi­ cient to completely resolve neighbor­ ing isotopes. Much lower detection limits can be achieved in the same length of time by replacing the ion beam with a la­ ser beam—a technique called laser atomization resonance ionization spectroscopy (LARIS), also shown in Figure 3 (11, 16, 21). By using a sep­ arate laser pulse instead of an ion pulse for the atomization process, 3 or more orders of magnitude more material can be released from the sample (the limit with the ion source

on the present SIRIS system is 5 χ 10 7 particles per 500-ns ion pulse). Thus with LARIS tens of monolayers can be removed with a single atomiz­ ing laser pulse, whereas with SIRIS many bombarding ion pulses are re­ quired to remove one monolayer of atoms from a sample. Because the SIRIS mode has been studied much more extensively, most of our initial work employed the ion beam. LARIS is attractive, however, because it offers the potential to at­ omize a much larger fraction of the Sn-labeled DNA band in the polyacrylamide gel in a given time pe­ riod, especially when a small focused beam is required; the ion density will decrease significantly with decreas­ ing beam diameter, whereas the la­ ser power density increases with de­ creasing beam diameter (11). Most of the data we will discuss below were t a k e n by SIRIS, but some of the LARIS results are included to dem­ o n s t r a t e t h e f e a s i b i l i t y of t h i s method. The main unknowns for LARIS are the amount of power that can be de­ livered to the sample in a highly re­ producible and controlled m a n n e r and the amount of debris from such large atomization events t h a t can be controlled or tolerated by the subse­ quent events in the RIS and extrac­ tion processes. For the LARIS exper­ iments three different wavelengths involving two different kinds of la­ sers have been used to i r r a d i a t e samples: the second (532 nm) and f o u r t h (266 n m ) h a r m o n i c of a Nd:YAG laser, and the ArF (193 nm) line of an excimer laser. The atomi­

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Figure 3. Schematic of the SIRIS/LARIS technique used for DNA sequencing. 318 A · ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992

zation laser output is adjustable with a variable attenuator with typical e n e r g i e s in t h e r a n g e of 0 . 0 1 1000 μ^Ι/ρ^βε, and it can be focused with a fused-silica lens through a fused-silica window onto the sample to a spot 75-150 μπι in diameter. Charge c o m p e n s a t i o n . The ion beam causes a charge to accumulate on the sample. If the charge is not dissipated, serious quantitation diffi­ culties in SIRIS measurements can occur because of alteration of electro­ static potentials in the ion extraction region and thereby in the ion detec­ tion efficiency. One advantage of the LARIS mode is that there is usually no charge accumulation. Because much more developmental study has been devoted to the SIRIS mode, we tried to find a means of e l i m i n a t i n g t h i s charge problem. Neither the dried polyacrylamide nor the nylon membrane, usually em­ ployed for DNA sequence analysis, is an electrical conductor. We therefore deposited a layer of Au on the sur­ face of a nylon membrane on which Fe-labeled DNA had already been deposited. We expected t h a t t h e analysis would cause a small area of the Au film to be sputtered off by the ion beam and that the adjacent Au film would carry away the charge that accumulated from the ion beam. However, the Au film was not sput­ tered off completely; instead, cavities formed in the nylon underneath the Au film and none of the Fe label on the DNA could be detected unambig­ uously. Substitution of the Au with C, Al, and a variety of grids still did not produce satisfactory results. We finally abandoned the idea of metal films and decided to use low-energy pulsed electrons (20-70 eV) in com­ bination with pulsed extraction and t a r g e t v o l t a g e s to d i s s i p a t e t h e charge on nylon and gel samples (10). Because of the long time period be­ tween ion pulses in SIRIS (tens of milliseconds), low-energy electrons can reach the target without being deflected or accelerated, thereby per­ mitting self-adjustment of the sur­ face potential without damaging the sample. Carefully timed sequential events are critical to the success of this process. This charge compensa­ tion method is extremely successful. In the current instrument, charge compensation by low-energy, pulsed electrons occurs 30 times per second; for devices that use a Cu vapor laser to pump dye lasers, we anticipate t h a t the process can be repeated 6000-20,000 times per second. Imaging. Imaging is achieved ei­ ther by scanning the ion beam over

the sample or, as in the present case, by changing the χ and y target posi­ tions (A;-scan, y-scan) while the atomization laser beam or the bombarding ion beam position remains fixed. The target position can be changed with a computer-controlled m a n i p u l a t o r . Recently we installed a manipulator that provides ± 25 mm motion in the χ and y axes with a resolution of 0.25 μπι and a repeatability of l-pm, 200-mm motion in the ζ direction with a resolution of 0.5 μπι and a re­ peatability of 1 pm, 360° rotation, and a linear drive to actuate a fifth axis. The manipulator provides step­ per motors driven under computer control that can change positions at a speed of 10,000 steps per second. A sample carousel specifically designed for DNA sequencing can hold 170mm-long planchets instead of the 25-mm ones used heretofore. D e p t h p r o f i l e s . In addition to measuring spatial concentration, SIRIS also can be used to measure element concentration as a function of depth. SIRIS depth profile mea­ surements involve two steps: scan­ ning the sample with a continuous ion beam to etch a series of rastered 1 x 2 mm or smaller craters to a spe­ cific depth and taking data with a pulsed ion beam in the center of the crater after a specific number of ras­ ter frames (11, 20). To prevent signal contribution from edges of the prob­ ing area, it is necessary to raster away material from an area larger t h a n t h e probing beam diameter. Charge accumulation is avoided dur­ ing depth profiling of insulating sam­ ples if the sample is continuously flooded with low-energy electrons. Choosing the label Both Fe and Sn have been employed as DNA labels (9-11). When SIRIS or LARIS is used, these elements are detected at levels t h a t are signifi­ cantly above the background t h a t arises from contamination from envi­ ronmental sources. In the sputtering process only the free neutral atom of the chosen element will be detected, and ions of that or other elements or organic complexes should not give a signal at the mass number of an iso­ tope. The mass numbers for the Sn isotopes are 112, 114, 115, 116, 117, 118, 119, 120, 122, and 124; the most naturally abundant isotope is 1 2 0 Sn at 32.59%, and the least abundant is 115 Sn at 0.36%. We calculated the la­ beling efficiency of eight Sn isotopes using the commercially available en­ richments of 6 0 - 9 5 % and concluded that at 90 atom percent excess these isotopes could perform well as DNA

sequencing labels (9). Selection of the laser energies to accomplish resonance ionization can begin with Grotrian or other energylevel diagrams that define the energy levels and the m u l t i t u d e of p a t h s that exist from one level to another. In practice, however, it is convenient to continually tune the resonance la­ ser to find an optimal resonance ion­ ization laser scheme. Schemes for several elements have been deter­ mined (22). The scheme chosen for Sn ionization was to use a 286.332nm UV photon, a 614.956-nm visible photon, and a 1064-nm IR photon (11). The scheme for Fe was to use one 296.690-nm UV photon and then a second 323.8-nm UV photon to ex­ cite an autoionizing state. Organometallic chemistry Each time a new element is selected for use as a label, a chemical route for transforming its oxide into a suit­ able organometallic compound must be found. Ferrocene was chosen as the first DNA label because synthetic methods were already available. Our colleagues Sachleben and Brown in the Chemistry Division of Oak Ridge National Laboratory developed pro­ cedures to convert F e 2 0 3 to ferrocene carboxylic acid and then to the 7V-hydroxysuccimide ester, because this ester has been used successfully to attach other organic ligands to oligo­ nucleotides (9). At the same time Foote, a col­ league in the Biology Division, used a DNA synthesizer to produce primers containing the 17 deoxynucleotides t h a t are a p p r o p r i a t e for the M13 DNA sequence and that had a hexylamine attached to the 5' end of the primer. When the ferrocene carboxyl ester and the hexylamine moiety were mixed together in dimethylformamide at a slightly alkaline pH, a spontaneous reaction occurred that produced the ferrocene-labeled primer. This product was identified by HPLC, UV spectra, and electro­ chemical properties. The ferrocene-labeled 17-mer was then compared with the underivatized 17-mer for its ability to serve as primer in the Sanger DNA sequenc­ ing method. Larimer, another col­ league in the Biology Division, used three forms of the primer—one con­ taining ferrocene attached to hexyl­ amine; one hexylamine alone; and a third, the underivatized 17-mer—to demonstrate t h a t the ferrocenelabeled 17-mer did function nor­ mally. Figure 4 shows t h a t the standard DNA sequencing ladders were ob­

tained in all three cases. The primers with substituents gave rise to DNA bands that migrated somewhat more slowly, as would be expected from the ability of gel electrophoresis to dis­ tinguish DNA fragments that differ by one nucleotide, 300 mass units. The existence of 10 stable isotopes for Sn was the catalyst for developing a synthetic route for a Sn label (23). Sachleben, Brown, and Sloop estab­ lished a scheme to convert S n 0 2 to the TV-hydroxysuccinimide ester of t r i e t h y l s t a n n y l propanoic acid (TESPA), which was then reacted with the 5'-hexylamine derivative of oligonucleotides (Figure 1). As shown by Foote, this labeling reaction re­ quires more alkaline conditions and a different solvent than does the cor­ responding ferrocene reaction. Prod­ ucts are more hydrophobic than the u n d e r i v a t i z e d oligonucleotide and are readily purified by chromatogra­ phy on C | 8 reversed-phase columns using a methanol gradient. When tested as DNA polymerase primers, the Sn-labeled oligonucleotides func­ tioned normally, as demonstrated by the ghost-free autoradiograms ob-

Figure 4. Comparison of gel electrophoresis patterns of differently substituted primers obtained from the Sanger sequencing procedure. The template was M13mp18 DNA, and the primers were normal (I), derivatized with 5'-hexylamine (l-C 6 ), and 5'ferrocene carboxaminohexylamine (l-C6-Fc). The label was [35S]thymidine 5'-(a-thio)triphosphate. After electrophoresis on an 8% polyacrylamide gel, an autoradiogram was obtained from the dried gel. (Adapted with permission from Reference 9.)

ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992 · 319 A

INSTRUMENTATION tained in the Sanger sequencing pro­ cedure (23). Sensitivity and selectivity S e n s i t i v i t y will be a d d r e s s e d in many ways as this study proceeds. Initially a series of 5-μίι drops of 56 Fe-labeled DNA (20-mer) were de­ posited on a clean Au foil (9). Each drop represented a step in a series of 1/20 dilutions. A SIRIS beam of 100 -μηι diameter detected the 5 e Fe throughout the dilution series until the signal could not be distinguished from the Fe signal coming from the "clean" Au foil surface. The lowest concentration used was 2 χ 10~ 17 Μ. Because only a small fraction of the dried 5-μίι drop was intersected by the ion beam, and because only a fraction of a monolayer is sputtered into the gas phase, the amount actu­ ally measured was « 1 amol. This measurement demonstrated that the SIRIS ion beam caused a significant number of neutral Fe atoms to be re­ leased from the DNA, a necessary event for RIS a n a l y s e s . Keep in mind, however, that the analysis will be done on organic matrices, such as polyacrylamide gel or nylon filter membranes, rather than on Au foil. DNA fragments undergo electrophoretic separation in polyacryl­ amide gel and, in some cases, are subsequently "blotted" onto the nylon membrane. To evaluate detection on a gel, 57 Fe-labeled and 1 1 6 Sn-labeled 17-mers were dissolved in gelatin or polyacrylamide and the conditions for SIRIS adjusted to maximize sen­ sitivity and selectivity and minimize the secondary ion mass spectrometry (SIMS) background. SIRIS is nor­ mally operated in a SIMS suppres­ sion mode. When the SIMS suppres­ s i o n w a s t u r n e d off a n d t h e resonance ionization l a s e r s were blocked (SIMS mode of analysis), we observed relatively high SIMS sig­ nals at most mass positions for the Fe or Sn isotopes—in some cases much higher t h a n t h e observed SIRIS signal. Such SIMS signals were observed from gelatin, nylon membrane, and polyacrylamide. If SIMS had been used, these isobaric contributions from various organic complexes that happen to have these mass values would have constituted a debilitating background problem. The resonance laser must be tuned precisely to the characteristics of the element under analysis, and when it is detuned even by 1A the signal may be lost. This requirement illustrates the highly selective analytical capa­ bilities of RIS to detect, in this case, Sn or Fe in the presence of isobaric

fragments. In addition, these results demonstrate that the RIS technique is necessary to detect isotope-labeled DNA without interferences. Another important consideration is endogenous contamination, which can vary with different elements. Fe, for example, is a common element in the Earth's crust and is often used in the manufacture of industrial equip­ ment. When we subjected a nylon membrane—onto which we planned to transfer DNA from polyacrylamide gels — to e l e m e n t a l a n a l y s i s , we found it contained > 100 ppm S and 8-10 ppm Fe but less than the detec­ tion limit of 15 ppb Sn (10). This high Fe content, which could be a result of the m a n u f a c t u r i n g process, made this element unsuitable for use as a DNA label. Because SIRIS can be used to ob­ tain depth profiles and thus localize layers of an element at any position in the sample, we used it to deter­ mine that the Fe on the membrane was primarily on the surface. With this additional information, our en­ thusiasm for Fe-labeled DNA waned considerably and will remain low un­ til materials with greatly reduced a m o u n t s of Fe become available. However, because Sn concentrations were below the detection limit, it ap­ peared to be quite attractive for use as a label. Detecting metal-labeled DNA on polyacrylamide and nylon with RIS The utility of RIS for detecting Feand Sn-labeled DNA b a n d s after electrophoresis was e v a l u a t e d by comparing DNA that had been trans­ ferred to a nylon membrane with DNA that had been dried in place in the gel. We assumed that the former procedure would be better because the DNA should bind primarily on the nylon surface. Woychik, a col­ l e a g u e in t h e Biology D i v i s i o n , helped to devise and execute such ex­ p e r i m e n t s in ways t h a t would be analogous to standard blotting prac­ tices for DNA sequencing. The nylon surface is not nearly as smooth and featureless as one might think (10). A scanning electron mi­ croscope image showed a fibrous net­ work with irregularly shaped fila­ ments of nylon and relatively large a r e a s between the flattened fila­ ments. This produces a very irregu­ lar sample surface for SIRIS analy­ sis. Even if the DNA were to bind exclusively to the nylon surface there would be many opportunities for it to reside in the shadow of other fibers, on the backside of the fibers, or in

320 A · ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992

"deep pits" among fibers, where it would be partly undetectable by the ion beam. The ion beam diameter is normally 150-300 μπι, wide enough to even out some of the irregularities of the nylon. However, if the beam diame­ ter were reduced to 3 0 - 5 0 μπι for re­ solving n a r r o w l y s e p a r a t e d DNA fragments after electrophoresis, the irregularities would likely be more of a problem. Given that polyacryl­ amide could have a different type of irregular surface feature and should not be contaminated on its surface, its applicability to this method was evaluated. One concern with the analysis of F e - or S n - l a b e l e d DNA was t h e amount of DNA that would reside on the surface of the dried gel. ' ^ S n - l a ­ beled 17-mer DNA was allowed to migrate electrophoretically for sev­ eral c e n t i m e t e r s into a s t a n d a r d polyacrylamide gel (400 μπι thick). Sufficient DNA was used (0.5 nmol) so that it could be located by placing a fluorescent plate behind the gel and illuminating it through the gel with short-wavelength UV light. Af­ ter the DNA band was located, the section of gel containing the band was cut from the gel using a glass coverslip (to minimize metal contam­ ination) and the gel was placed on a 1 χ 2 cm piece of acid-washed paper, covered with plastic wrap, and dried at 80 °C under vacuum, the standard procedure in handling DNA sequenc­ ing gels (10, 11). When the dried gel

2

4 6 8 y-Axis (mm)

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Figure 5. SIRIS determination of 116 Sn in a 1,6Sn-labeled 17-mer DNA band on polyacrylamide gel. Data were obtained from the (a) first and (b) second scan of a single track on the gel. (Adapted with permission from Reference 10.)

INSTRUMENTATION was scanned with SIRIS, a l l e S n sig­ nal at the site of the UV band was found (Figure 5). The 1 1 6 Sn-labeled DNA concentration on the gel surface was very low; analysis of the same track a second time gave a detectably lower signal, whereas investigation of a new track produced a signal sim­ ilar to the first. Direct analysis on the gel provided data demonstrating the feasibility of the SIRIS method. When samples were stored for 12 months the Sn-labeled DNA bands were still readily detectable. Because polymerase chain reaction (PCR) products may be advantageous for genome map­ ping and characterization, a set of PCR products of 100, 200, and 800 base pairs was prepared using Snlabeled primers. After gel electro­ phoresis t h e DNA bands were de­ tected by SIRIS and LARIS on the dried polyacrylamide gel.

501

Depth analysis The DNA is probably distributed uni­ formly throughout the 400-μπι thick­ ness of the dried gel. Further drying reduces the gel thickness to —40 μπι. Distributions of the DNA as a func­ tion of depth were measured with SIRIS by using the ion beam first in a rastering mode to etch out an area of the dried gel and then in a n ana­ lytical mode to analyze the center of the rastered area (11). The 1 1 6 Sn-labeled 17-mer concentrations were significantly higher (approximately 20-100-fold) at the surface. The in­ tegrated DNA signal over the first few nanometers corresponds to only a fraction of the total amount of DNA distributed throughout the bulk. The concentration of Sn a t t h e surface may be caused by the drying process of the gel, in which water is removed from t h e gel t h r o u g h t h e a c i d washed paper by a vacuum pump. It is fortunate that the DNA concentra­ tion is elevated at the surface; with the standard SIRIS scan the signal is obtained only from t h e surface, whereas with LARIS the main bulk of the material can contribute to the signal. SIRIS LARIS comparison The wavelength of the atomization laser in the LARIS procedure was shown to be a critical factor in the analysis (21). I n Figure 6 " u n ­ labeled 17-mers were electrophoretically migrated into two polyacryl­ amide gels a n d located by SIRIS (Figure 6a and 6c). The samples were then analyzed by LARIS at 532 and 266 nm a t o m i z a t i o n l a s e r w a v e ­ lengths (Figure 6b and 6d, respec­

large signal-to-noise ratios. Because the diameter of the fiber is obviously much smaller than that of the atomizing laser beam, the peak width implies that the beam diame­ ter was approximately 130 μπι. For this short atomization laser wave­ length (193 nm) a much higher sig­ nal was obtained with LARIS than had been obtained with SIRIS. Not only t h e DNA but the Sn reagent, TESPA, absorbs strongly a t 193 nm, leading to greater atomization of the Sn. The comparison between SIRIS and LARIS for different atomization laser wavelengths demonstrates the importance of finding the right laser wavelengths and fluence for optimi­ zation of the LARIS yield. Further analysis u s i n g other wavelengths

tively); only t h e latter wavelength was effective in detecting the " u n ­ labeled DNA, but the signal was still no larger t h a n t h a t obtained from SIRIS. DNA is transparent a t 532 nm, and no prominent bands could be found. The signal from the 532-nm scan by LARIS represents random detection of Sn background, giving values that are very low compared with the SIRIS data. A third wavelength, 193 nm (Fig­ ure 6e and 6f), was used to detect 116 Sn-labeled DNA t h a t had been hybridized to complementary DNA that had been chemically attached, by Foote, to a 25-μπι-diameter fiber. When the laser beam traversed the DNA-coated fiber, a strong signal was obtained repeatedly with very

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322 A · ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992

INSTRUMENTATION and matrices will be carried out in the near future. Multiple DNA labels An experiment was designed to dem­ onstrate the advantage of using mul­ tiple DNA labels (10). Three oligonu­ cleotides of different lengths were labeled with l i e S n and one indepen­ dently with 1 1 8 Sn. Figure 7 shows that after electrophoresis the 1 1 8 Sn occurred in only one of t h e three peaks of 116 Sn, which demonstrates the potential for multiplexing as well as the mass spectrometric resolution of the two isotopes. The position of the Sn signal was correlated exactly with the position of the UV bands ob­ served in the gel. Much narrower bands will be analyzed in DNA se­ q u e n c i n g e x p e r i m e n t s . We h a v e demonstrated that a 0.2-mm band of a 28-mer labeled with I 2 4 Sn can be defined by SIRIS (11). This last ex­ periment was performed by Koons on the "open-faced" gels of Allen et al. (24) as part of an exploration of new electrophoretic procedures. Signal linearity The linearity of the RIS signal with the amounts of Sn-labeled DNA was

assessed with a dilution experiment in which different amounts (50, 10, and 2 pmol and 400 fmol) of solutions of 1 1 6 Sn-labeled-24-mer were placed into 2 0 - m m wells of t h e electro­ phoresis gel (400 μιη thick) and electrophoresed about 15 cm into the gel. The gel was then dried on paper and analyzed by SIRIS. A signal of more than 20 times background was found

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in each case. The log-log plot of the signal ver­ sus amount of DNA at the peak posi­ tion was linear down to 0.4 pmol (11). The lowest amount h a s 400 fmol/ 20-mm width band or 20 fmol/mm. This result confirmed earlier experi­ ments with SIRIS that had shown linear detection of other elements in gelatin and semiconductor samples over a concentration range of several orders of magnitude (16). In extend­ ing this study more recently, we demonstrated t h a t a SIRIS signal approximately three times that of the background can be detected after electrophoresis u s i n g 1 fmol S n DNA/mm. The detection limits of SIRIS/LARIS are far in the subattomole region. To approach this level of DNA detection the amount of back­ ground signal attributable to Sn con­ tamination must be lowered. Accom­ plishing this goal will require cleaner reagents and a cleaner environment, problems we are trying to solve. Sanger sequencing experiment We decided to determine what could be seen on an actual sequencing gel (23). In the Sanger sequencing proce­ dure the amount of each DNA frag-

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324 A · ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992

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INSTRUMENTATION

What Every Chemist Should Know about the U.S. Patent System!

Understanding Chemical Patents A Guide for the Inventor, Second Edition

L

earn how to read and understand pat­ ents, how to use patents as a source of information, how to recognize that an in­ vention has been made, and how to work with attorneys or agents in seeking patent protec­ tion for inventions. Gain enough familiarity with the special terminology of patents to be able to deal comfortably with patent attor­ neys, agents, and technical liaison personnel. Understanding Chemical Patents answers the questions of not only practicing chemists and chemical engineers, but also people in other fields who need to understand the patent system.

The SIRIS/LARIS procedures will make a contribution at this point. The rate at which RIS can analyze Sn-labeled DNA fragments on polyacrylamide gel can be estimated (9). If the following assumptions were true, the rate of analysis would be 2.2 χ 10 7 bands per day: The SIRIS/ LARIS techniques can employ a Cu vapor l a s e r t h a t can o p e r a t e a t 10,000 Hz; the atomizing beam can be 50 μπι diameter; the DNA bands contain four isotopes of Sn that rep­ resent the A, G, C, and Τ DNA frag­ ments in a single gel lane; the electrophoretic DNA bands are 0.1 mm wide and separated by 0.1 mm so that 500 bands are contained in a 10cm-long pattern; four positions are probed to locate the Sn on the DNA and four are used to locate the space between bands; and the resonance laser fires five times at each locus. By adding other sets of four isotopes to multiplex the analysis, this rate would double by adding four more isotopes, double again by adding an­ other eight, and so forth. This rapid analytical rate would require 1 0 - 2 0 electrophoresis in­ struments performing DNA separa­ tions and feeding the dried gels into a single SIRIS/LARIS instrument. If

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Contents • • • • • • • • • • • • • • • •

Introduction: The Purpose of Patents How to Read a Patent Patents as an Information Source Deciding Whether to File a Patent Application The Independent Inventor Preparation of the Patent Application Prosecuting the Patent Application Interferences and the Importance of Records Patent Infringement and Patent Claims Making Use of Patents The Employed Inventor Copyrights, Trademarks, and Trade Secrets Recent Biotechnology-Related Patent Law Changes in U.S. Patent Laws: 1980-1990 Trends in U.S. and World Patent Law Representative U.S. Patent Fees and Payment of Money

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Figure 8. Gel electrophoresis pattern (a) and SIRIS analysis (b) of 120 Sn-labeled DNA fragments formed during Sanger DNA sequencing. 120 Sn-labeled 1 7 - m e r w a s used as the M13 primer to provide a SIRIS signal, and α-thiol 35 [ S]-dTTP was used for autoradiography. (Adapted with permission from Reference 23.)

326 A · ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992

we could transfer the separated DNA bands onto a metal, Si, glass, or plas­ tic surface, we could increase sensi­ tivity by orders of magnitude be­ cause the polyacrylamide gel dilutes the labeled DNA, thereby decreasing the signal obtained per atomization pulse. The nylon membrane is too ir­ regular to offer any improvement over the gel; therefore, a desirable surface should be much smoother than nylon and must bind the DNA efficiently. Potential for g e n o m e mapping

Scattered among the chromosomes of mammals are various kinds of repet­ itive sequences such as (dC-dA)„ (dG-dT)„, which have no codon func­ tion for protein synthesis b u t act more or less as spacers. Among dif­ ferent species or inbred strains, the value of η may be different (26). Por­ tions of the chromosomes can be ob­ tained as DNA molecules that con­ tain the repetitive sequence along with 200-400 other base pairs. The sequence of these DNAs is deter­ mined, a n d t h e a p p r o p r i a t e PCR primers are synthesized and used to make probes t h a t are distributed throughout the genome—several to a chromosome. By locating the sites on the chro­ mosome from which each DNA arose, a map of the entire genome is ob­ tained. Such a map simply desig­ nates known landmarks at several positions on each chromosome to which genes, such as those for hyper­ tension (27), can be shown to be linked. This information helps to de­ termine the relative location of any given gene in the overall genome. Experimentally, two strains of the animal that have different lengths of the repetitive sequence are bred and the offspring sorted for some recog­ nizable trait. The offspring with the trait are then compared with those that do not possess it by isolating DNA from each individual and test­ ing each with a set of labeled DNAs that recognize the regions that con­ tain the (dC-dA)M repeats. Each such site h a s a DNA sequence t h a t is unique as compared with the other sites in the genome. This comparison is performed by gel electrophoresis of fragments of genomic DNA and hy­ bridization of t h e r e s u l t i n g DNA bands with each of the - 1 0 0 - 2 0 0 la­ beled DNA probes. The multiplexing possibilities that arise from the use of stable isotopes as the DNA label reduce the number of such tests by allowing many to be r u n simultaneously. We are cur­ rently exploring these possibilities.

Current and future directions Other experiments have yet to be done before this procedure can be used to perform reliable DNA se­ quencing. For example, when the electrophoresis gel is 400 μπι thick most of the DNA is below the surface and unavailable to the current stan­ dard SIRIS procedure, whereas more DNA is available for analysis by LARIS. Thinner gels and more pow­ erful atomization beams are desir­ able. Perhaps a method can be found to cause more of the DNA to migrate to the surface layer of the gel and thus be available for analysis. Also, thinner gels may give better resolu­ tion between DNA bands; this might allow the linear dimensions of the gel to be reduced and still retain the ability to resolve 3 0 0 - 5 0 0 DNA bands. We are also trying to develop ways to use those rare earth elements that have multiple isotopes so that the multiplex nature of the analysis can be expanded. Also, it would be ad­ vantageous to have the stable isotope labels on the dideoxy terminator or on interior nucleotides. In yet an­ other area RIS has several features t h a t could be modified to provide higher detection limits and more pre­ cise localization of the labeled DNA. A sample planchet that can accom­ modate 17-cm-long gel strips has been acquired recently; heretofore, only 2-cm samples of gel could be ex­ amined. As other aspects of RIS become es­ tablished it will be necessary to in­ stall a TOF mass spectrometer so that all isotopes of an element can be analyzed simultaneously. Finally, processing the data obtained from rapid analyses will require specially designed computer programs mod­ eled after those that process data from the automated fluorescence an­ alyzers (28). Several of these studies are under way.

We wish to emphasize the contributions of our colleagues in the Biology and Chemistry Divi­ sions of ORNL and the work of N. Thonnard of Atom Sciences, Inc. This research is supported by the ORNL Exploratory Studies Program and the Office of Health and Environmental Re­ search, U.S. Department of Energy, under con­ tract DE-AC05-84OR21400 with the Martin Marietta Energy Systems, Inc., and contract DE-AC-05-89ER80735 to Atom Sciences, Inc.

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References (1) S a n g e r , F . ; N i c k e l e n , S.; C o u l s o n , A. R. Proc. Natl. Acad. Set. USA 1977, 94, 5463.

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ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992 · 327 A

INSTRUMENTATION

Physical

Polymer Characterization Physical Property, Spectroscopic, and Chromatographic Methods

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he development of new polymeric prod­ ucts is integrally related to the ability to characterize polymeric systems. This new volume explores the ways in which characteri­ zation is part of the production process. Pre­ senting the significant advances in polymer characterization methodology. Polymer Charac­ terization offers reports from recognized ex­ perts in the field. Its 26 chapters are divided into four sections covering • polymer fractionation and particle size distribution • dynamic mechanical analysis and rheology • spectroscopy • morphology

(2) Prober, J. M.; Trainor, G. L.; Darn, R. J.; Hobbs, F. W.; Robertson, C. W.; Zogursky, R. J.; Cocuzza, A. J.; Jensen, Μ. Α.; Baumeister, K. Science 1987, 238, 336. (3) Smith, L.; S a u n d e r s , J. Z.; Kaiser, R. J.; Hughes, P.; Dodd, C ; Connel, C. R.; Heiner, C ; Kent, S.B.H.; Hood, L. E. Nature 1986, 321, 674. (4) Cantor, C. R. Science 1990, 248, 49. (5) Watson, J. D. Science 1990, 248, 44. (6) Resonance Ionization Spectroscopy; Parks, J. E.; Omenetto, N., Eds.; Inst. Phys. Conf. Series 114; I n s t i t u t e of Physics: Bristol. AL. 1991. (7) Hurst, G. S.; Payne, M. G.; Kramer, S. D.; Young, J. P. Rev. Mod. Phys. 1979, 51, 767. (8) Letokhov, V. S. leaser Photoionization Spectroscopy; Academic Press: New York, 1987. (9) Jacobson, K. B.; Arlinghaus, H. F.; S c h m i t t , H. W.; S a c h l e b e n , R. Α.; Brown, G. M.; T h o n n a r d , N.; Sloop, F. V.; Foote, R. S.; Larimer, F. W.; Woychik, R. P.; England, W. M.; Burchett, K. L.; Jacobson, D. A. Genomics 1991, 9, 51. (10) Arlinghaus, H. F.; T h o n n a r d , N.; Spaar, M. T.; Sachleben, R. Α.; Larimer, F. W.; Foote, R. S.; Woychik, R. P.; Brown, G. M.; Sloop, F. V.; Jacobson, Κ. Β. Anal. Chem. 1991, 63, 402. ( 1 1 ) Arlinghaus, H. F.; T h o n n a r d , N.; Spaar, M. T.; Sachleben, R. Α.; Brown, G. M.; Foote, R. S.; Sloop, F. V.; Peter­ son, J. R.; Jacobson, Κ. Β. /. Vac. Set. Technol. 1991, A9, 1312. (12) Shaw, R., New England Nuclear, per­ sonal communication. (13) Brennan, T.; Chakel, J.; Bente, P.; Field, M. In Biological Mass Spectroscopy; Burlingame, A. L.; McCloskey, J . Α., Eds.; Elsevier Science Publishing: Am­ s t e r d a m , The N e t h e r l a n d s , 1990; pp. 219-27. (14) Hurst G. S.; Nayfeh, M. H.; Young, J. P. Appl. Phys. /^«.'1977, 30, 229. (15) Beekman, D. W.; Thonnard, N. In Resonance Ionization Spectroscopy; Lucatorto, T. B.; P a r k s , J . E., Eds.; Inst. Phys. Conf. Series 94; Institute of Phys­

ics: Bristol, AL, 1989; p. 163. (16) A r l i n g h a u s , H. F.; S p a a r , M. T.; Thonnard, N.; McMahan, A. W.; Jacobson, Κ. Β. In Optical Methods for Ultrasen­ sitive Detection and Analysis: Techniques and Applications; Fearey, B. L., Ed.; The International Society for Optical Engi­ neering: Washington, DC, 1991; Vol. 1435, pp. 2 6 - 3 5 . (17) Pappas, D. L.; Hrubowchak, D. M.; E r v i n , M. H.; Winograd, N. Science 1989 243 64. (18) Chen, C. H.; Payne, P. G.; Hurst, S.; Kramer, S. D.; Allman, S. L.; Phillips, R. C. In iMser and Mass Spectroscopy; Lubman, D., Ed.; Oxford University Press: London, 1990; pp. 3 - 3 6 . (19) Thonnard, N.; Parks, J. E.; Willis, R. D.; Moore, L. J.; Arlinghaus, H. F. Surf. Interface Anal. 1989, 14, 751. (20) A r l i n g h a u s , H. F.; S p a a r , M. T.; Thonnard, N. / Vac. Sci. Technol. 1990, A8, 2318. (21) Arlinghaus, H. F.; Thonnard, N. In leaser Ablation Mechanisms and Applica­ tions; Miller, J. C ; Hagland, R. F., Jr., Eds.; Springer Verlag: Berlin, 1991; p. 165. (22) Saloman, Ε. Β. Spectrochim. Acta 1990, 45B, 37. (23) Sachleben, R. Α.; Brown, G. M. Sloop, F. V.; Arlinghaus, H. F.; England W. M.; Foote, R. S.; Larimer, F. W. Woychik, R. P.; Thonnard, N.; Jacobson Κ. Β. Genetic Analysis Techniques and Ap plication; Elsevier: New York, 1991; Vol 8, p. 167. (24) Allen, R. C ; Graves, G.; Budoule, B. HioTechniques 1989, 7, 736. (25) Heller, M. J.; Tullis, R. H.; Wick, R. A. Presented at the Genome Mapping and Sequencing Meeting. Cold Spring Harbor Laboratory, San Diego, CA, May 1991. (26) Weber, J. L.; May, P. E. Am. J. Hum. Genet. 1989, 44, 388. (27) Jacob, H. J.; Lindpainter, J.; Lin­ coln, S. E.; Kusumi, K ; Bunker, R. K.; Mao, Y-P.; Gantem, D.; Dzau, V. J.; Lander, E. S. Cell 1991, 67, 213. (28) Tibbitts C , Vanderbilt University, personal communication.

Many of the chapters report on the combined use of several characterization methods in order to elucidate the relationship between polymer structure-morphology and polymer performance. This will be a useful reference for research­ ers in polymer chemistry and physics, mate­ rials science, and analytical chemistry. Clara D. Craver. Editor. Chemir Laboratories Theodore Provder. Editor. The Glidden Company Developed from a symposium sponsored by the Divi­ sion of Polymeric Materials: Science and Engineering of the American Chemical Society Advances in Chemistry Series No. 227 544 pages (1990) Clothbound ISBN 0-8412-1651-7 LC 90-47157 $109.95 Ο

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K. Bruce Jacobson (right) received his Ph.D. from The Johns Hopkins University. In 1958, following a postdoctoral appointment at Cal Tech where he worked with Linus Pauling, he became a staff member of the Biology Division of Oak Ridge National IMbo­ ratory. He holds an additional appointment as professor in the Graduate School of Bio­ medical Sciences of the University of Tennessee-Oak Ridge. His research interests in­ clude the mechanism of enzyme action, the structure-function relationships for transfer RNA, pteridine metabolism, and development of new technologies for DNA sequencing. H. F. Arlinghaus received his Ph.D. in 1986 under the guidance of A. Benninghoven from the Westfdlische Wilhelms-Universitat of Munster, Germany. Following a postdoc­ toral appointment at Argonne National IMboratory, he became a staff member of Atom Sciences, Inc. His research interests include trace element analysis and detection tech­ niques for sputtered particles from ion-bombarded and laser-irradiated solid surfaces, depth profile analysis, and DNA sequencing with multiple stable isotopes.

328 A · ANALYTICAL CHEMISTRY. VOL. 64. NO. 5. MARCH 1. 1992

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