would take a long time. In addition, there was a desire to sequence the genomes of mouse, Drosophila, C. eleguns, yeast, and E. coli-organisms whose extensive genetics could be profitably correlated to detailed DNA sequence information (4,5). Resonance ionization spectroscopy (RIS) has been used to analyze a wide variety of natural and manufactured materials for trace element content a n d concentrations (6-8). This analytical procedure can, i n principle, quantitatively measure any known element (except He and Ne), and several elements can be attached to DNA. Thus RIS offers the possibility of using new labels for DNA sequencing. When RIS is used with a mass s p d r o m e t e r , each stable 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 research is to develop procedures for analyzing DNA sequences by RIS, thereby providing Human Genome Project researchers with a faster and less expensive method to accomplish the ordering of the -3 billion nucleotides in human chromosomes.
AA
Developmc t of Resonance
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 procedures have used primarily radioisotopes or fluorescent organic compounds as labels for detection of DNA 0003-270019~0364-315Ai$02.50/0 @ 1992 American Chemical Society
fragments following gel electrophoresis (1-3). Such procedures are well established: Radioisotopes a r e detected on silver emulsion films and fluorescent labels with sensitive phototubes. Hundreds of thousands of DNA base sequences have been processed by several laboratories using these two kinds of labels. However, when t h e goal of sequencing t h e -3 x lo9 bases of the human genome was under serious consideration three years ago, it was clear that sequencing with existing methods at the rate of 1million bases per year
The sequencing method I n t h e S a n g e r DNA sequencing method (1)the DNA fragment whose sequence is to be determined is used a s a template in a reaction mixture to which DNA polymerase is added. When a 17-mer (an oligonucleotide of 17 nucleotides) containing a s e quence that binds to a complement a r y sequence on t h e template is used as a primer, the polymerase will use t h e deoxynucleoside triphosphates of adenine (A), guanine (GI, cytosine (C),and thymine (T)to extend the primer and replicate the sequence 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 fragments contain the original primer, the replicated sequence of the DNA of interest, and the dideoxy terminator; when these DNA fragments are separated by polyacrylamide gel electrophoresis, a difference in length of one nucleotide can be detected. When 32Por 35Sis used as the label, the A-, G-, c-,and T-terminated fragments must be run in four adjacent 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 T lanes. Because these four different bases define t h e sequence of t h e
ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1,1992
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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 a s 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 a n instrument can detect many isotopes simultaneously rather than just the
I
four labels that are resolvable in the optical system. The use of RIS to a n alyze metal-labeled DNA fragments separated by polyacrylamide gel electrophoresis offers several advantages, 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 natural 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-thionncleotides, as is already done with 35S (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
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stable isotope approach. The percent natural abundances of 3ZS , 3SS , 34s , and 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 i t from sulfur from other sources, the isotope must be enriched. Because 32Sconstitutes 95% of the natural S abundance, the companion isotopes must be purified to > 9 9 % . At the other extreme, '"S has only a 0.11% n a t u r a l abundance so t h a t , even when purified to 30%, the commercially available isotope contains more 34S than ""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 relative 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 substrates for DNA polymerase, because enriched isotopes a r e usually supplied a s the oxides; manipulation of gel electrophoresis techniques so that the separated DNA bands can he 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 a t Oak Ridge National Laboratory. They used the method to detect a single Cs atom in a background of lo" Ar atoms and 10" CH, molecules ( 1 4 ) . Subsequently 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. Technique. Tunable lasers a r e 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 particular excited s t a t e s for RIS analysis permits great element selectivity. It has been demonstrated that the ionization efficiency for the selected element can be as much a s 10' times higher than that for the other elements i n t h e sample ( 1 5 ) . The high selectivity of the RIS process also helps to maintain linearity in the mass spectrometer ion extraction region by reducing space charge effects that would otherwise be present because of ionization of the major sample constituents. Because isotopic shifts of most elements are small in comparison to the bandwidth of the RIS lasers (712 GHz) used in our experiments, all isotopes of a chosen element will be ionized with essentially equal sensitivity. The intensity of modern pulsed dye lasers is sufficient to saturate both the bound-bound transitions a n d the ionization step, thereby assuring unit probability of ionizing all atoms of the selected element that are in the volume intersected by the RIS laser beams. Isotopic selectivity is achieved with t h e mass spectrometer. If a TOF mass spectrometer is used, all isotopes of an element 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 and the suppression of the secondary ions virtually eliminate interferences from molecular ions, isobars, or scattered ions from major sample constituents. The sensitivity and selectivity of the RIS process are especially valuable for trace element analysis in materials in which the matrix complexity is frequently a serious source of interference. The efficiencv of ionizing and then counting the atoms of the selected element in the sputtered cloud depends on the ionization effi. ciency (-loo%), the temporal and spatial overlap of the laser beam with the atomized cloud (2050%), the total transmission Of the mass spectrometer (60-SO%), a n d
'
the detector efficiency (60-80%). Recently pptr detection limits (several orders of maenitude below attomoles) were shown ?or In in Si (16,171 with a useful yield (atoms detected per atoms sputtered) of -26% (16). Another salient feature of the RIS process is its generality. Using available lasers, it is possible to ionize more than SO% of the elements, including all transition elements and lanthanides, with very high element selectivity a n d almost 100% efficiency. 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 photoionization 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 he used for the IR photon because of its low energy. This results in saturated ionization of only the selected element with essentially no ionization of other elements (as little as 1 part in 109). States in the ionization continuum (autoionizing 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
the photoionization step, further increase selectivity while reducing the uossibilitv of interference from nonresonant ionization processes. Except for He and Ne, which are not practical RIS candidates, and Ar, F, and Kr, which require very complex laser a r r a n g e m e n t s t o g e n e r a t e vacuum-UV radiation, the elements in the upper right corner of the periodic system can be ionized by the absorption 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, i t offers a significant advantage over conventional ionization and nonresonant photoionization methods. Instrumentation. RIS analysis requires free atoms in the gas phase. In the sputter-initiated resonance ionization spectroscopy (SIRIS) technique (Figure 31, a sample is placed on a planchet and inserted into a vacuum chamber, where it is hombarded with a high-energy pulsed Ar ion beam (10' 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 pm. The expanding cloud of sputtered material consists of neutral atoms, molecular fragments, and ions; the ions are removed by timed
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(a) Resonance ionization by the absorption of two resonant photons and a low-energy iR photon. (b) Photoionization by resonant excitation 01 an autoionizing state. This ionization scheme further reduces interfering ionization processes by adding Selectivity in the last Step of the RIS process. (c) ~lementswith a large energy uap to the first excited state can be ionized via atwo-photon ailowed state
ANALYTICAL CHEMISTRY, VOL. 64,NO. 5, MARCH 1,1992 * 317 A
INSTRUMENTATION extraction voltage switching a n d electrostatic energy analysis (positive ions are suppressed; negative ions are accelerated). The remaining neutral particles are then probed hy the RIS laser beams that ionize all atoms of the selected element within the volume intersected by the laser beams. Efficient overlap of t h e l a s e r beams with the cloud of desorbed material is achieved by choosing the appropriate delay t i m e between 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 beam diameter i s typically 2-3 mm. The ionized atoms are extracted into a detector system consisting of a n electrostatic energy a n alyzer and a magnetic sector mass spectrometer or a TOF mass spectrometer, a n d measurements a r e made in charge digitization (signal expressed as voltage) or single ioncounting (signal expressed as counts) mode. The mass resolution (mlAm) of 600 of the current system is sufficient to completely resolve neighboring isotopes. Much lower detection limits can be achieved in the same length of time by replacing the ion beam with a laser beam-a technique called laser atomization resonance ionization spectroscopy (LARIS),also shown in Figure 3 ( 1 1 , 16,211.By using a separate 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 x IO7 particles per 500-ns ion pulse). Thus with LARIS tens of monolayers can be removed with a single atomizing laser pulse, whereas with SIRIS many bombarding ion pulses are required 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 i t offers the potential to a t omize a much larger fraction of the Sn-labeled DNA band in the polyacrylamide gel in a given time period, especially when a small focused beam is required the ion density will decrease significantly with decreasing beam diameter, whereas the laser power density increases with decreasing beam diameter ( 1 1 ) .Most of the data we will discuss below were taken by SIRIS, b u t some of t h e LARIS results are included to demo 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 he delivered to the sample in a highly reproducible a n d controlled manner and the amount of debris from such large atomization events that can be controlled or tolerated by the subsequent events in the RIS and extraction processes. For the LARIS experiments three different wavelengths involving two different kinds of las e r s have been used to irradiate 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 NdYAG laser, and the ArF (193 nm) line of an excimer laser. The atomi-
Atomization laser beam (LARIS)
manipulator
c
zation laser output is adjustable with a variable attenuator with typical e n e r g i e s i n t h e r a n g e of 0.011000 pJ/pulse, and it can be focused with a fused-silica lens through a fused-silica window onto the sample to a spot 75-150 pm in diameter. C h a r g e compensation. The ion beam causes a charge to accumulate on the sample. If the charge is not dissipated, serious quantitation difficulties in SIRIS measurements can occur because of alteration of electrostatic potentials in the ion extraction region and thereby in the ion detection 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 eliminating t h i s charge problem. Neither the dried polyacrylamide nor the nylon membrane, usually employed for DNA sequence analysis, is a n electrical conductor. We therefore deposited a layer of Au on the surface 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 sputtered off completely; instead, cavities formed in the nylon underneath the Au film and none of the Fe label on the DNA could be detected unambiguously. 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 combination with pulsed extraction and t a r g e t voltages t o d i s s i p a t e t h e charge on nylon and gel samples (IO). Because of the long time period hetween ion pulses in SIRIS (tens of milliseconds), low-energy electrons can reach the target without being deflected or accelerated, thereby permitting self-adjustment of the surface potential without damaging the sample. Carefully timed sequential events are critical to the success of this process. This charge compensation 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 t h e process can be repeated 6000-20,000 times per second. Imaging. Imaging is achieved either by scanning the ion beam over
the sample or, as in the present case, by changing the x and y target positions (x-scan, yscan) while the atomization laser beam or the bombarding ion beam position remains fixed. The target position can be changed with a computer-controlled manipulator. Recently we installed a manipulator that provides k 25 mm motion in the x and y axes with a resolution of 0.25 pm and a repeatability of 1-pm, 200-mm motion i n the z direction with a resolution of 0.5 pm and a repeatability of 1p n , 360" rotation, and a linear drive to actuate a fifth axis. The manipulator provides stepper motors driven under computer control that can change positions a t a speed of 10,000 steps per second. A sample carousel specifically designed for DNA sequencing can hold 170mm-long planchets instead of t h e 25-mm ones used heretofore. Depth profiles. I n addition to measuring spatial concentration, SIRIS also can be used to measure element concentration as a function of depth. SIRIS depth profile measurements involve two steps: scanning the sample with a continuous ion beam to etch a series of rastered 1x 2 mm or smaller craters to a specific depth and taking data with a pulsed ion beam in the center of the crater after a specific number of raster frames (11,20).To prevent signal contribution from edges of the probing area, i t is necessary to raster away material from a n area larger than t h e probing beam diameter. Charge accumulation is avoided during depth profiling of insulating samples 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 a t levels that are significantly above the background t h a t arises from contamination from environmental 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 a t the mass number of a n isotope. The mass numbers for the S n isotopes are 112, 114, 115, 116, 117, 118, 119,120,122, and 124; the most naturally abundant isotope is '"Sn a t 32.5996, and the least abundant is '"Sn a t 0.36%. We calculated the labeling efficiency of eight S n isotopes using the commercially available enrichments of 60-95% and concluded that a t 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 multitude of paths that exist from one level to another. In practice, however, it is convenient to continually tune the resonance laser to find a n optimal resonance ionization laser scheme. Schemes for several elements have been determined (22). The scheme chosen for Sn ionization was to use a 286.332nm W photon, a 614.956-nm visible photon, and a 1064-nm IR photon ( 1 1 ) . The scheme for Fe was to use one 296.690-nm UV photon and then a second 323.8-nm W photon to excite a n 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 suitable 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 procedures to convert Fe,O, to ferrocene carboxylic acid and then to the N-hydroxysuccimide ester, because this ester has been used successfully to attach other organic ligands to oligonucleotides (9). At t h e same time Foote, a colleague in the Biology Division, used a DNA synthesizer to produce primers containing the 17 deoxynucleotides t h a t a r e appropriate for t h e M13 DNA sequence and that had a hexylamine attached to the 5' end of the primer. When the ferrocene carboxyl ester a n d t h e hexylamine moiety were mixed together in dimethylformamide at a slightly alkaline pH, a spontaneous reaction occurred that produced t h e f e r r o c e n e - l a b e l e d primer. This product was identified by HPLC, W spectra, and electrochemical 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 sequencing method. Larimer, another colleague in the Biology Division, used three forms of the primer-one containing ferrocene attached to hexylamine; one hexylamine alone; and a third, the underivatized 17-mer-to d e m o n s t r a t e t h a t t h e ferrocenelabeled 17-mer did function normally. Figure 4 shows that 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 distinguish DNA fragments that differ by one nucleotide, 300 mass units. The existence of 10 stable isotopes for S n was the catalyst for developing a synthetic route for a Sn label (23). Sachleben, Brown, and Sloop established a scheme to convert SnO, to the N- hydroxysuccinimide ester of triethylstannyl propanoic acid (TESPA), which was then reacted with the 5'-hexylamine derivative of oligonucleotides (Figure 1).As shown by Foote, this labeling reaction requires more alkaline conditions and a different solvent than does the corresponding ferrocene reaction. Products are more hydrophobic than the underivatized oligonucleotide and are readily purified by chromatography on C,, reversed-phase columns using a methanol gradient. When tested as DNA polymerase primers, the Sn-labeled oligonucleotides functioned normally, as demonstrated by the ghost-free autoradiograms ob-
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Fig , Con ?el 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 S-hexylamine(I-&),and S-ferrocenecarboxaminohexylamine(I-C,-Fc). The label was ["Slthymidine S-(a-thia)triphosphate.Alter eleclrapharesis on an 8% pdyacryiamide gal, an autoradiogram was obtained tram the dded gel. (Adapted with permission tmm Reference 9.)
ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1.1992 * 319 A
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INSIRLJMENTATION tained in the Sauger sequencing procedure (23). Sensitivity and selectivity Sensitivity will be addressed i n many ways as this study proceeds. Initially a series of 5-pL drops of “Fe-labeled DNA (20-mer) were deposited on a clean Au foil (9). Each drop represented a step in a series of 1 / 2 0 dilutions. A SIRIS beam of 100-pm diameter detected the “6Fe throughout the dilution series until the signal could not be distinguished from the Fe signal coming from the “clean” Au foil surface. The lowest M. concentration used was 2 x Because only a small fraction of the dried 5-pL drop was intersected by the ion beam, and because only a fraction of a monolayer is sputtered into the gas phase, the amount actually measured was 100 ppm S and 8-10 ppm Fe but less than the detection limit of 15 ppb S n (10).This high Fe content, which could be a result of t h e manufacturing made - Drocess. . this element unsuitable for use as a DNA label. Because SIRIS can he used to obtain depth profiles and thus localize layers of an element a t any position in the sample, we used it to determine that the Fe on the membrane was primarily on the surface. With this additional information, our enthusiasm for Fe-labeled DNA waned considerably and will remain low until materials with greatly reduced amounts of Fe become available, However, because Sn concentrations were below the detection limit, it appeared 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 bands after electrophoresis was evaluated by comparing DNA that had been transferred 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 colleague i n t h e Biology Division, helped to devise and execute such experiments i n ways t h a t would be analogous to standard blotting practices for DNA sequencing. The nylon surface is not nearly a s smooth and featureless as one might think (10). A scanning electron microscope image showed a fibrous network with irregularly shaped filaments of nylon and relatively large a r e a s between the flattened filaments. This produces a very irregular sample surface for SIRIS analysis. Even if the DNA were to hind exclusively to the nylon surface there would he many opportunities for i t to reside in the shadow of other fibers, on the backside of the fibers, or in
. 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 pm, wide enough to even out some of the irregularities of the nylon. However, if the beam diameter were reduced to 30-50 pm for resolving narrowly separated DNA fragments after electrophoresis, the irregularities would likely be more of a problem. Given that polyacrylamide could have a different type of irregular surface feature and should not he contaminated on its surface, its applicability to this method was evaluated. One concern with the analysis of Fe- or Sn-labeled DNA was t h e amount of DNA that would reside on the surface of the dried gel. ““Sn-labeled 17-mer DNA was allowed to migrate electrophoretically for several centimeters into a standard polyacrylamide gel (400 pm thick). Sufficient DNA was used (0.5 nmol) so that it could he located by placing a fluorescent plate behind the gel and illuminating it through the gel with short-wavelength LN light. After the DNA band was located, the section of gel containing the band was cut from the gel using a glass coverslip (to minimize metal contamination) and the gel was placed on a 1 x 2 cm piece of acid-washed paper, covered with plastic wrap, and dried a t SO “C under vacuum, the standard procedure in handling DNA sequencing gels (IO,11).When the dried gel
Fi ure 5. SIRIS determination of in a “6Sn-labeled 17-mer DNA band on polyacrylamide gel.
”83
Data were obtained from the (a) first and (b) second scan of a single track on the gel. (Adapted with permission from Reference 10.)
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was scanned with SIRIS, a “%n signal a t the site of the UV band was found (Figure 5). The116Sn-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 similar 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 hands were still readily detectable. Because polymerase chain reaction (PCR) products may be advantageous for genome mapping and characterization, a set of PCR products of 100, 200, and 800 base pairs was prepared using Snlabeled primers. After gel electrophoresis the DNA bands were detected by SIRIS and LARIS on the dried polyacrylamide gel.
tively); only the latter wavelength was effective in detecting the ”‘Snlabeled DNA, but the signal was still no larger than that obtained from SIRIS. DNA is transparent at 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 (Figure 6e and 60, was used to detect ‘%h-labeled DNA t h a t had been hybridized to complementary DNA that had been chemically attached, by Foote, to a 25-pm-diameter fiber. When the laser beam traversed the DNA-coated fiber, a strong signal was obtained repeatedly with very
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 diameter was approximately 130 pn. For this short atomization laser wavelength (193 nm) a much higher signal was obtained with M I S than had been obtained with SIRIS. Not only the DNA but the Sn reagent, TESPA, absorbs strongly a t 193 nm, leading to greater atomization of the Sn. The comparison between SIRIS and M I S for different atomization laser wavelengths demonstrates the importance of finding the right laser wavelengths and fluence for optimization of the LARIS yield. Further analysis using other wavelengths
Depth analysis The DNA is probably distributed nniformly throughout the 400-pm thickness of the dried gel. Further drying reduces the gel thickness to -40 pm. Distributions of the DNA as a function of depth were measured with SIRIS by using the ion beam first in a rastering mode to etch out a n area of the dried gel and then in a n analytical mode to analyze the center of the rastered area (11).The ‘“h-labeled 17-mer concentrations were significantly higher (approximately 20-100-fold) at the surface. The integrated 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 the 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 concentration 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 l16Snlabeled 11-mers were electrophoretically migrated into two polyacrylamide gels a n d located by SIRIS (Figure 6a and 6c). The samples were then analyzed by LARIS a t 532 and 266 n m atomization l a s e r wavelengths (Figure 6b and 6d, respec-
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polyacrylamide gel (total amount is -500 pmol). Scans Obtained by SiRlS (a, c) and LARIS (b, d) methads. SiRiS (e) and LARiS (1) scans over a single fiber that has been incubated in 1-wM “%.labeled DNA soiution. Solid and dashed lines are y-scans through two dinerent x-positions showing the reproducibility 01 the signal. (Adapted with permission from Reference 21.1
322 A * ANALYTICAL C H E M I S T R Y , V O L . 64, NO. 5, MARCH 1,1992
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INSTRUMENTATION and matrices will be carried out in the near future. Multlple DNA labels An experiment was designed to demonstrate the advantage of using multiple DNA labels (10).Three oligonucleotides of different lengths were labeled with "'Sn and one independently with "'Sn. Figure 7 shows that after electrophoresis the "'Sn occurred in only one of the three peaks of ""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 observed in the gel. Much narrower bands will be analyzed in DNA sequencing experiments. We h a v e demonstrated that a 0.2-mm band of a 28-mer labeled with lZ4Sncan be defined by SIRIS (11).This last experiment was performed by Koons on the "open-faced'' gels of Allen et al. (24) as part of a n 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 ""Sn-labeled-24-mer were placed into 20-mm wells of t h e electrophoresis gel (400 pm 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
Fi ure 7 SIRIS determination of "&n in "'Sn-labeled 24-, 25-, and 26-mer (solid line) and "'Sn in "'Sn-labeled 26-mer (dotted line) DNA bands on polyacrylamide gel, -50 prnoWoligomer in a 20-mm band. For the analysis, 100 shots per data point were taken using an ion current of 2 fl.The data were binomially smoothed.
in each case. The log-log plot of the signal versus amount of DNA at the peak position was linear down to 0.4 pmol(l1). The lowest amount has 400 fmoll 20-mm width band or 20 fmollmm. This result confirmed earlier experiments 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 extending t h i s 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 SIRISlLARIS are far in the subattomole region. To approach this level of DNA detection the amount of background signal attributable to Sn contamination must be lowered. Accomplishing 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 procedure the amount of each DNA frag-
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ment produced by the DNA polymerase is a direct function of the amount of DNA template present. In our experiment the amount of template (10 nmol) was about 30 times greater than that used normally. The primer contained [1Z0Sn15'-triethylstannyl propionamide, with u-thi~[~'Sl-dTTPand low levels of dATP, dCTP, and dGTP as substrates. After a short incubation to allow primer extension and incorporation of all the "S, more dATP, dCTP, dGTF', and dTTP were added to each of the four tubes. In addition, each tube received one of the dideoxy analogues of each nucleotide. These dideoxy analogues cause termination of synthesis of the DNA that is complementary to the DNA template (1). The sites of such termination occur randomly, and the resulting DNA fragments occur i n progressively longer forms and represent all possible locations of the A, G, C, and T bases on the growing DNA molecule. The positions of these DNA fragments after electrophoresis on polyacrylamide gel can be interpreted directly to define the sequence of these four bases.
After electrophoresis this gel was placed on acid-washed paper so that it would not shrink and become distorted during the drying process. The gel was then placed on a n X-ray fdm to locate all the "S-containing DNA bands. Because our SIRIS apparatus at that time required the sample to be no larger than 1x 2 em, a piece of t h e gel containing several DNA bands, as shown by 36S, was mounted on a planchet and analyzed by SIRIS to locate the "'Sn. The correlation between the positions of 55S and '"Sn was excellent (Figure 8). This result provided the first real s e quencing data obtained in this study.
Potentialfor rapid DNA sequence analysis The width of the DNA bands is a function of electrophoresis parameters such as gel concentration and thickness, m a t r i x modifiers, a n d DNA fragment sizes. The lateral resolution of adjacent DNA bands by RIS depends on the adjustable diameter of the ion or laser beam used to sputter or atomize the sample. For routine measurement the beam diameter has been 150-300 pm, but it could be reduced to 3-5 p.
Given that several hundred DNA bands become separated on a n electrophoresis gel, a n a r r o w beam would be advantageous. The standard electrophoresis gel separates 300-400 DNA bands on a gel 40 cm long. However, if 500 bands were each 0.1 mm wide and were separated by 0.1 mm, the entire pattern could be contained in 10 cm. An ion beam with a spot size diameter of 50 pm can resolve bands with 100 p between them on much smaller gels than are currently required for standard DNA sequencing procedures. A recent report by Heller et al. (25)described the separation of restriction fragments ranging from 72 to 1353 base pairs in a distance of less than 5 mm on a 1-cm polyacrylamide gel. In the past the gel electrophoresis step was the rate-limiting factor in DNA, whereas now some of the earlier steps and the subsequent data processing steps may be the bottleneck. Innovations in robotics are being applied to the processing steps, and new d a t a management techniques are emerging. With such modifications it is expected that the gel electrophoresis step will again be limiting.
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INSTRUMENTATION The SIRISlLARIS procedures will make a contribution a t this point. The rate a t which RIS can analyze Sn-labeled DNA fragments on polyacrylamide gel can he estimated (91. If the following assumptions were true, the rate of analysis would be 2.2 x lo7 hands per day: The SIRISl LARIS techniques can employ a Cu vapor l a s e r t h a t c a n o p e r a t e at 10,000 Hz; the atomizing beam can he 50 pm diameter; the DNA bands contain four isotopes of Sn that represent the A, G, C, and T DNA fragments in a single gel lane; the electrophoretic DNA hands are 0.1 mm wide and separated by 0.1 mm so that 500 hands 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 a t 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 a n other eight, and so forth. This rapid analytical rate would require 10-20 electrophoresis i n struments performing DNA separations and feeding the dried gels into n single SIRIS/LARISinstrument. If
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ANALYTICAL CHEMISTRY. VC
Figure 8. G e l E sis pa (a) and SIRIS analysis ( 0 ) of 'ZoSn-labeledDNA fragments formed during Sanger DNA sequencing. ''Sn-labeled 17-mer was used aslhe M13 pr mer 10 p w oe a SIR S s gnal. ano u-Ino [ 3 ] . a T T P*as .sea lor a-iorao ograpny Aoaplea WII perm %!on tiom Relerence 23
NO. 5, MARCH 1,1992
we could transfer the separated DNA hands onto a metal, Si, glass, or plastic surface, we could increase sensitivity by orders of magnitude because the polyacrylamide gel dilutes the labeled DNA, thereby decreasing the signal obtained per atomization pulse. The nylon membrane is too irregular to offer any improvement over the gel; therefore, a desirable surface should he much smoother than nylon and must hind the DNA efficiently. Potential for genome mapping Scattered among the chromosomes of mammals a r e various kinds of repetitive sequences such a s (dC-dA1, (dG-dTl,, which have no codon function for protein synthesis hut act more or less a s spacers. Among different species or inbred strains, the value of n may be different (261. Portions of the chromosomes can he ohtained as DNA molecules that contain the repetitive sequence along with 200-400 other base pairs. The sequence of these DNAs i s determined, a n d t h e appropriate PCR primers are synthesized and used to make probes t h a t a r e distributed throughout the genome-several to a chromosome. By locating the sites on the chromosome from which each DNA arose, a map of the entire genome is ohtained. Such a map simply designates known landmarks a t several positions on each chromosome to which genes, such a s those for hypertension (271, can he shown to he linked. This information helps to determine 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 recognizable trait. The offspring with the trait are then compared with those that do not possess i t by isolating DNA from each individual and testing each with a set of labeled DNAs that recognize the regions that contain the (dC-dA1. repeats. Each such site h a s a DNA sequence t h a t i s unique as compared with the other sites in the genome. This comparison is performed by gel electrophoresis of fragments of genomic DNA and hybridization of t h e resulting DNA hands with each of the -100-200 labeled DNA probes. The multiplexing possibilities that arise from the use of stable isotopes a s the DNA label reduce the number of such tests by allowing many to he r u n simultaneously. We a r e currently exploring these possibilities.
Current and future directions Other experiments have yet to be done before this procedure can be used to perform reliable DNA sequencing. For example, when t h e electrophoresis gel is 400 pm thick most of the DNA is below the surface and unavailable to the current standard SIRIS procedure, whereas more DNA is available for analysis by M I S . Thinner gels and more powerful atomization beams are desirable. 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 resolution between DNA bands; this might allow the linear dimensions of the gel to be reduced a n d still retain the ability t o resolve 300-500 DNA bands. We are also trying to develop ways to use those rare earth elements that have multiple isotopes so t h a t the multiplex nature of the analysis can be expanded. Also, it would be advantageous to have the stable isotope labels on the dideoxy terminator or on interior nucleotides. In yet a n other area RIS has several features t h a t could be modified to provide higher detection limits and more precise localization of the labeled DNA. A sample planchet that can acwmmodate 17-cm-long gel strips has been acquired recently; heretofore, only 2-cm samples of gel could be examined. As other aspects of RIS become established it will be necessary to install 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 modeled after those t h a t process data from the automated fluorescence a n alyzers (28).Several of these studies are under way.
We wish to emphasize the mntrihutions of m r colleagues in the Biology and Chemistry Divisions of O W L and the work of N. Thonnard of Atom Sciences, h e . This research is supported by the O W L Exploratory Studies Pmgram and the Office of Health and Environmental Research, U S Department of Energy, under contract DE-AC05-840R21400 with the Martin Marietta Energy Systems, h e . , and contract DE-AC-05-89ER80735 to Atom Sciences. h e .
References (1) Sanger, F.; Nickelen, S.; Coulson, A. R. h o c . NaN Acad. Sci. USA 1977, 94, 5463.
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INSTRUMENTA~ION 12) Prober, J. M.; Trainor, G. L.; Darn, R. J.: Hohbs. F. W.; Robertson, C. W.; Zogursky, R. J.: Cocuzza, A. J.; J e n s e n , M. A,; Haumeister, K. Science 1987,238,
:mi
(31 S m i t h , L.; Saunders, J. 2.; Kaiser,
R. J . ; H u g h e s , P.; Dodd, C.; Connel, C. R.; Heiner. C.; Kent, S.R.H.: Hood. L. E. Nati,rp 1986. 321. 674. 14) Cantor, C. R. Science 1990, 248. 49. ( 5 ) Watson. J. I). Science 1990. 248. 44. ifi) H ~ s o n o n cIonization ~ Sppctroscopy; Parks. J. E.; Omenetto, N., Eds.; Inst. Phys. Coni. S e r i e s 114; Institute of Physics: Rristol. AI,. 1991 (71 Hurst. G. S.; Payne, M. G.; Kramer. S. D.; Young, J. P. HPU.Mod. Phys 1979, 51. 7fi7. it31Letokhov, V. S. l a s ~ rPhotninnization Sppctroscnpy; Academic Press: New Ynrk, ,on7 ."11..
( 9 ) Jacobson, K. R.; Arlinghaus, H. F.;
Polymer Characterization Physical Property, Spectroscopic, and ChromatographicMethods
T
he development of new polymeric products is integrally related to the ability to characterize polymeric systems. This new vciume explores the ways in which characteri. zation is part of the production process. Presenting the significant advances in polymer characterization methodology. Poyner Characterization offers reports from recognized experts in the field. Its 26 chapters are divided into four sections covering wlymei fisaionalion and particle size
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S c h m i t t , H. W.; S a e h l e h e n , R . A,; 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.;. Rurehett, K. L.; Jacobson. I).A. Gmnmrcs 1991, 9. 51. (101 A r l i n g h a u s , H. F.; T h o n n a r d . N.; Spaar, M. T.; Saehleben, R. A,; Larimer, F. W.; Foote, R. S.; Woyehik, R. P . ; Brown, G. M.; Sloop, F. V.; Jacobson, K. R.Anal. ClrPm. 1991. 63. 402. ( 1 1 ) Arlinghaus. H. F.; T h o n n a r d , N.; Spaar, M. T.; Saehleben, R. A,; Drown, n. s.;SI^^^. F. v.; peterG. M.; son. J. R.: Jaeohson. K. R. I. Vac. Sci. Techno/. 1991. A9. 1312. (12, Shaw, K., New England Nuclear, per sonal communication. (131Rrennan, T.; Chakel, J.; Rente, P.; Field. M. In Hinlogical Mass Spectroscopy; Hurlingame. A. L.; McCloskey, d . A., Eds.; Elsevier Science Publishing: Ams t e r d a m . T h e N e t h e r l a n d s . 1990:
ics: Bristol. AL. 1989: D. 163. 116) A r l i n g h a u s . H. F'.; Spaar, M. T . ;
Thonnard. N.; MeMahan, A. W.; Jaenhson. K. B. In Optical M e t h o e f i r Ultrasenr i f i w 1)pfectinn and Analvsis: Teclrnmtps &d Applications; Fearey,'B. L., Ed.; T h e International Society for Optical Engineering: Washington, DC, 1991: Vol. 1435, pp. 26-35. (171 Pappas. D. L.: Hruhowehak. D. M.; E r v i n . M . H.; W i n o g r a d . N . Scipnre 1989. 243, 64. l 1 R 1 Chen, C . H . : Payne, P . G . ; Hurst, S.; Kramer. S. I).; Allman, S. L.; Phillips, H.C. In l a s w and Mass Spprtroscnpy; Lobman. D., Ed.; Oxford University Press: London, 1990; pp. :3-36. 1191 Thonnard. N.: Parks. d . E.: Willis. R. D.; Moore,' L . J . ; Arlinghnus, H. F. Surf Interfacp A n d 1989, 14. I S I . (201 A r l i n g h a u s . H. F.; S p a a r , M. T.; Thonnard. N. J. Vac. Sri. Tkltnnl. 1990, AX, 231R. I211 Arlinghaus. H. F.; Thonnard. N. In Iaspr Ablation Mechanisms and Applications; Miller. J. C.; Hngland, R. F.,Jr.. Eds.; Springer Verlag: Ilerlin. 1991; p. ~~
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( 2 2 1 Saloman, E . R. Sppctrochim. Acta 1990. 45H. 37. (231 S a c h l e h e n , R . A , ; B r o w n . G . M . ; Sloop, F. V.; Arlinghaus, H., F.; England, W. M . ; Foote, R. S.; Lnrimer, F. W.; Woychik. R: P.; Thonnard, N.; Jacohsnn. K. 13, Genptic Analysis Tklmiqim and Application; Elsevier: New Yurk. 1991; Vol. H. p. 167. ( 2 4 ) Allen, R. C.; Graves. G.; Rudoule, R. HioTdmiqws 19R9. 7, 736. ( 2 5 ) Heller. M. J.; Tullis, R. H.; Wick. R. A. Presented at t h e Genome Mapping and Sequencing Meeting. Cold Spring Harbor Lahoratory. San Iliegn. CA, May 1991. 1261 Weber, J. L.; May, P. E . Am. J. Hum. Gen~r.1989, 44, 3RR. I271 Jacoh. H. %I.;Lindpainter. .I.; Lin-
coln, S.E.; Kusumi. K.; Runkcr, R. K.; Man, Y-P.; Gnntem. D.; Drau, V , d . ; Lander, E . S. Cell 1991. 67. 213.. (2x1 Tibbitts C.. Vanderhilt University. personal communication.
bisiribulion dynamic mechanical analysis and rhwlcq
.,
IDecllOYOD"
iorpholqy
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 lnll be a useful reference for research. ers in polymer chemistly 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 DIMsion of Polymeric Materiais: kience and Engineering of the American Chemicai Smew Advances In Chemistry Series No 227 544 pages 1990 Cothbo-nn ISBN 0 8412 1651 7 .C 99 47157 elno 09 Arneriian Chemical Soiirri
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ti i f r ~ /w~ ~ ~ o / i s(I'iKlitJ r~ii wwiwd his I'ii./). Iii,ni 711,'/ih,>s Hopkiirs U i ~ i ~ ' r I s /,ii t ~ I!l:jS. fidlox'ing a postdnrtorr,l appni,itmoit rit ('01 7 i d r idiere he workrd with /.itins Painling, IIP brcamp a staff mrmbrr oftkr /Moa1)ivisioii of Oak Ridge Natioiial Irlhorotor)', Hr holds an additional appointment as profmsor in flip Graduate Sckool of Hiomedical Sciences of tlir University of Tennessee-Oak Ridge. His research interests inclttde the mechanism ofenzyme action, the structure-fnnction relationshipsfor transfer RNA, pteridine m~tabolism,and d~velopmentof new technologies for DNA sequencing. H. I.: Arlinghaus received his Ph.D. in 1986 under theguidance o f A . Henninghovpn from the Westfdlische Wilhelms-Uniuersitut of Munster, Germany. Following a postdnctoral appointmpnt at Argonne National laboratory. he became a staffmember ofAtom Sciences, Inc. His research interests include trace element analysis and dptection tpchniques 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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