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Anal. Chem. 1991, 63, 402-407
Potential Application of Sputter- Initiated Resonance Ionization Spectroscopy for DNA Sequencing Heinrich F. Arlinghaus,*J Norbert Thonnard,’ Michael T. Spaar,’ Richard A. Sachleben,2 Frank W. Larimer? ,~ M. Brown,2Fred V. and K. Bruce Jacobson3 Robert S. Foote,*Richard P. W ~ y c h i kGilbert A t o m Sciences, Inc., 1I4 Ridgeway Center, Oak Ridge, Tennessee 37830, Chemistry and Biology Divisions, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, a n d Graduate School of Biomedical Sciences, L’niuersity of Tennessee, Oak Ridge, Tennessee 37831
To improve the technique for DNA sequencing, large numbers of stable isotopes could be used to label DNA, thereby multiplexing the separation process and providing a new and much faster procedure for localizing DNA after electrophoresis. Sputter-lnitlated resonance ionization spectroscopy ( S I R I S ) was used to demonstrate detection of subattomoie quantities of isotopically enriched iron- and tin-labeled DNA with excellent lateral and m a s resolution. The organometallic compounds, ferrocenecarboxyllc acid and (triethylstanny1)alkanecarboxylic acid, were synthesized and attached through the amine group of a 5’-hexylamine on the terminal position of an oligonucleotide. The adaptation of S I R I S for detecting samples containing 50 pmols of either iron or tin attached to DNA is described. Uslng high repetition rate lasers in SIRIS, It should eventually be posslble to detect more than lo7 bases per day if other preparation steps in the procedure do not become rate limlng. The results of analysis of iron- and tin-labeled DNA on polyacrylamide gels and Nylon membrane indicate that S I R I S has the potential to make a strong contribution to DNA sequencing and also for other methods that requires detection of the location and amount of DNA or an oligonucleotide that hybridizes to DNA.
INTRODUCTION Since the genetic makeup of an organism can be defined by the sequence of its DNA and as current methods are relatively slow and labor intensive, there is a critical need to develop improvements over current sequence determination methods. The sequence of the human genome consists of approximately 3 x IO9 nucleotide pairs, that of the mouse, 2 x IO9,and those of other organisms with well-defined genetic characteristics, to */loath as many. The human genome alone will require an enormous effort; therefore, together with the other genomes, a considerable challenge is present. Current methods of DNA sequencing rely on gel electrophoresis to resolve, according to their size, DNA fragments that were produced from a larger DNA segment. The fragments are generated chemically or enzymatically to terminate in all possible positions of each of the four nucleotides (A, G, C, and T) in the original DNA segment. In the MaxamGilbert ( I ) chemical procedure, the DNA segment is labeled with a radioisotope (‘*P or 35S)on the 5’-end; in the Sanger ( 2 ) procedure the label may occupy the 5’-end position, an internal position, or be incorporated in the dideoxynucleotide (on the 3’-terminus of the DNA fragments). Localization of these labels after gel electrophoresis, usually by autoradiographic exposures of many hours or even days, allows the Atom Sciences, Inc. *ChemistryDivision, Oak Rid e National Laboratory. Biology Division, Oak Ridge kational Laboratory. University of Tennessee.
sequence to be read directly from the gel by noting the relative electrophoretic migration distances for the fragments. Commonly, four lanes of the gel are required to separate the fragments that terminate in A, G, C, or T that come from a given DNA segment. Multiplexing has been introduced to reduce the steps in electrophoresis and in analysis. Church and Kieffer-Higgins (3)have demonstrated a method of combining the A-, G-, C-, and T-terminated fragments of 40 or more DNAs on the same four adjacent gel lanes for electrophoresis. Since the fragments were prepared so that each DNA was associated with a unique sequence of 20 nucleotides at the 5‘-end, identification of each DNA after electrophoresis was accomplished by hybridization to a 20-nucleotide complementary DNA that was labeled with 32P.No loss of resolution in the electrophoretic pattern occurred with the use of 40 DNAs as compared to one DNA. Another multiplex method is employed in DNA sequencing in which the labels are fluorescent molecules on the 5’-end or the dideoxynucleotideof the DNA fragments that terminate in A, G, C, and T ( 4 , 5 ) . Four fluorescent compounds were chosen that could be spectroscopically resolved and that had no differential effect on the electrophoretic behavior of the DNAs to which they were attached. By this strategy all four types of DNA fragments were combined together in one gel lane to reduce the number of gels and to improve the precision of locating the electrophoretic bands and determining their order. This paper describes a method that combines aspects of both of these multiplexing methods by utilizing stable isotopes instead of radioisotopes or fluorescent labels. Four isotopes from one element, used on the A-, G-, C-, and T-terminated fragments of the DNA, allow these fragments to be combined in one gel lane since the isotopes could have no differential effect during electrophoresis. Using sets of four isotopes from several elements allows several DNAs to be combined together in that same gel lane. Since the isotopes could be analyzed in the electrophoresis gel directly there would be no need for subsequent hybridization, as in the Church and KiefferHiggins method. For more details about this approach and the strategy of isotope selection, see ref 6. A multispectral multiplexing method using a large number of fluorescent labels was proposed (7),but stable isotopes would be advantageous since spectral overlap is a serious problem in fluorescence analysis. Detection of isotopes of iron and tin on DNA was achieved by sputter-initiated resonance ionization spectroscopy (SIRIS). We describe the SIRIS technique and initial tests of the sensitivity, selectivity, reproducibility, and background in these complex biochemical matrices. We will demonstrate that SIRIS can localize and quantify isotope-labeled DNA bands both in the electrophoresis gels and on material transferred onto Nylon membranes.
EXPERIMENTAL SECTION SIRIS Instrument. A schematic of the SIRIS instrument used to analyze a variety of sample types is shown in Figure 1.
0003-2700/91/0363-0402$02,50/00 199 1 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 5, MARCH 1, 1991
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The present system consists of a Perkin-Elmer duoplasmatron microbeam ion gun (incident angle = 60° from normal, smallest beam diameter = 5 pm, maximum current = 10 PA), a pulsed-flood electron gun from Kimball Physics, an RIS laser system, consisting of a Quanta Ray DCR-SA pulsed Nd:YAG laser (repetition rate 30 Hz) pumping two Quanta Ray PDL-1 pulsed dye lasers having a typical bandwidth of 0.3 cm-', a computer-controlled ( x , y , z , $1 sample holder, which can hold up to ten 3/4 in. x 3/4 in. targets, two HeNe lasers for exact target positioning, a video imaging system, a sample interlock system, and a detection system. For the SIRIS experiments a pulsed Ar' ion beam with an energy of 9 keV, an ion current of 2-6 pA, and a spot size of 50-200 pm diameter has been used to bombard various isotope-labeled DNA samples. The primary ion beam pulse was in the 0.3-1-ps range, allowing efficient sample utilization. The majority of sputtered particles (neutral and ionized atoms and molecules as well as molecular fragments) removed from the surface are largely ground-state neutral atoms; any free sputtered ions and other charged particles are suppressed by a combination of the relative timing between the ion-sputtering pulse and the firing of the RIS lasen, timed extraction voltage switching, and electrostatic energy analysis. After 1-2 ps the expanding cloud of neutral particles is probed by the RIS laser beams that ionize selectively all the atoms of a chosen single element within the volume intersected by the laser beams. All isotopes of that selected free element will respond equally, in most cases; the same element bound in a molecular complex should not ionize at all unless the complex is dissociated by the laser beam in which case the element would be ionized immediately. Interferences by isobaric molecular fragments are virtually eliminated by the selective ionization process. The selectively ionized atoms are extracted and directed through an electrostatic analyzer and a magnetic-sector mass spectrometer onto an ion detector system, and measurements are made in charge digitization (signal is expressed as voltage) or single-ion-counting (signal is expressed as counts) mode. The efficiency of ionizing the selected element from the sputtered cloud and then counting it depends on the ionization efficiency ( w loo%), the temporal and spatial overlap of the laser beam with the atomized cloud (20-50%), the total transmission of the double-focusing mass spectrometer (Z30%), and the detector efficiency (6040%). Efficient overlap of the laser beams with the sputtered material is achieved by choosing the appropriate delay time between firing the ion gun and the RIS laser and carefully positioning the RIS laser beams with the ion beam/ sample surface intersection. Recently, sub-ppb detection limits for indium in silicon have been shown (8)along with a selectivity of more than lo9 (9). SIRIS depth profiles are obtained by scanning the sample with a continuous ion beam to etch a series of 1 mm X 2 mm craters to a specific depth (a single ion beam scan of the 1 mm X 2 mm area is expressed as one raster frame), after which the data are taken with a pulsed ion beam having a beam diameter of
