CA2153387A1 - Dna sequencing by mass spectrometry - Google Patents

Abstract

The invention describes a new method to sequence DNA. The improvements over the existing DNA sequencing technologies are high speed, high throughout, no electrophoresis and gel reading artifacts due to the complete absence of an electrophoretic step, and no costly reagents involving various substitutions with stable isotopes. The invention utilizes the Sanger sequencing strategy and assembles the sequence information by analysis of the nested fragments obtained by base-specific chain termination via their different molecular masses using mass spectrometry, as for example, MALDI or ES mass spectrometry. A further increase in throughpout, can be obtained by introducing mass-modifications in the oligonucleotide primer, chain-terminating nucleoside triphosphates and/or in the chain-elongating nucleoside triphosphates, as well as using integrated tag sequences which allow multiplexing by hybridization of tag specific probes with mass-differentiated molecular weights.

Description

_ 0 94/16101 215 3 3 8 7 PCT/US94100193 DNA SEQUENCING BY MASS SPECTROMETRY

Back~round of the Invention Since the genetic information is represented by the sequence of the four s DNA building blocks deoxyadenosine- (dpA), deoxyguanosine- (dpG), deoxycytidine-(dpC) and deoxythymidine-5'-phosphate (dpT), DNA sequencing is one of the most fundamental technologies in molecular biology and the life sciences in general. The ease and the rate by which DNA sequences can be obtained greatly affects related technologies such as development and production of new therapeutic agents and new and useful 0 varieties of plants and microorg~ni~ms via recombinant DNA technology. In particular, unraveling the DNA sequence helps in underst~ntling human pathological conditions including genetic disorders, cancer and AIDS. In some cases, very subtle differences such as a one nucleotide deletion, addition or substitution can create serious, in some cases even fatal, consequences. Recently, DNA sequencing has become the core technology of the Human Genome Sequencing Project (e.g., J.E. Bishop and M. Waldholz, 1991, Genome:
The Story of the Most Astorli.shing Scientific Adventure of Our Time - The Attempt to Map All the Genes in the Human Body~ Simon & Schuster, New York). Knowledge of the complete human genome DNA sequence will certainly help to understand, to diagnose, to prevent and to treat human ~lise~ies- To be able to tackle successfully the dçtçrrnin~tion of the approximately 3 billion base pairs of the human genome in a reasonable time frame and in an economical way, rapid, reliable, sensitive and inexpensive methods need to be developed, which also offer the possibility of automation. The present invention provides such a technology.
Recent reviews of today's methods together with future directions and trends 2s are given by Barrell (The FASFR Journal 5, 40-45 (1991)), and Trainor (Anal. Chem. 62, 41 g-26 (1990)).
Currently, DNA sequencing is performed by either the chemical degradation method of Maxam and Gilbert (Methods in Enzymology 65, 499-560 (1980)) or the enzymatic dideoxynucleotide termination method of Sanger et al. (Proc. Natl. Acad. Sci.
USA 74, 5463-67 (1977)). In the chemical method, base specific modifications result in a base specific cleavage of the radioactive or fluorescently labeled DNA fragment. With the four separate base specific cleavage reactions, four sets of nested fragments are produced which are separated according to length by polyacrylamide gel electrophoresis (PAGE).
After autoradiography, the sequence can be read directly since each band (fragment) in the gel originates from a base specific cleavage event. Thus, the fragment lengths in the four "ladders" directly translate into a specific position in the DNA sequence.
In the enzymatic chain termination method, the four base specific sets of DNA fragments are formed by starting with a primer/template system elongating the primer into the unknown DNA sequence area and thereby copying the template and wo 94/16101 -2- PCT/US94/00193 synthesizing a complementary strand by DNA polymerases, such as Klenow fragment of E. coli DNA polymerase I, a DNA polymerase from Thermus aquaticus, Taq DNA
polymerase, or a modified T7 DNA polymerase, Sequenase (Tabor et al., Proc. Natl.
Acad. Sci. USA 84, 4767-4771 (1987)), in the presence of chain-termin~ting reagents.
s Here, the chain-termin~tinp~ event is achieved by incorporating into the four separate reaction mixtures in addition to the four normal deoxynucleoside triphosphates, dATP, dGTP, dTTP and dCTP, only one of the chain-termin~ting dideoxynucleoside triphosphates, ddATP, ddGTP, ddTTP or ddCTP, Ie~e~ ,rely, in a limiting small concentration. The four sets of resulting fr~gments produce, after electrophoresis, four 0 base specific ladders from which the DNA sequence can be determinPcl A recent modification of the Sanger sequencing strategy involves the degradation of phosphorothioate-cont~ining DNA fragments obtained by using alpha-thio dNTP instead of the norrnally used ddNTPs during the primer extension reaction mediated by DNA polymerase (Labeit et al., DNA 5, 173-177 (1986); Amersham, PCT-Application GB86/00349; Eckstein et al., Nucleic Acids Res. 16, 9947 (1988)). Here, the four sets of base-specific sequencing ladders are obtained by limited digestion with exonuclease III or snake venom phosphodiesterase, subsequent separation on PAGE and vi~ li7~tion byradioisotopic labeling of either the primer or one of the dNTPs. In a further modification, the base-specific cleavage is achieved by alkylating the sulphur atom in the modified phosphodiester bond followed by a heat tre~trnent (Max-Planck-Gesell~ch~ft, DE 3930312 Al). Both methods can be combined with the amplification of the DNA via the Polymerase Chain Reaction (PCR).
On the upfront end, the DNA to be sequenced has to be fragmented into sequencable pieces of currently not more than 500 to 1000 nucleotides. Starting from a 2s genome, this is a multi-step process involving cloning and subcloning steps using different and appropliate cloning vectors such as YAC, cosmids, plasmids and M13 vectors (Sambrook et al., Molecular Clonin.~: A T ~horatory Manual, Cold Spring Harbor Laboratory Press, 1989). Finally, for Sanger sequencing, the fragments of about 500 to 1000 base pairs are integrated into a specific restriction site of the replicative form I (RF I) of a derivative of the M13 bacteriophage (Vieria and Messing, Gene 19, 259 (1982)) and then the double-stranded form is transforrned to the single-stranded circular form to serve as a template for the Sanger sequencing process having a binding site for a universal primer obtained by chemical DNA synthesis (Sinha, Biernat, McManus and Koster, Nucleic Acids Res. 12, 4539-57 (1984); U.S. Patent No. 4725677 U~ lealll of the 3s restriction site into which the unknown DNA fragment has been inserted. Under specific conditions, unknown DNA sequences integrated into supercoiled double-stranded plasmid DNA can be sequenced directly by the Sanger method (Chen and Seeburg, DNA 4, 165-170 (1985)) and Lim et al., Gene Anal. Techn. 5, 32-39 (1988), and, with the Polymerase Chain Reaction (PCR) (PCR Protocols: A Guide to Methods and Applications. Innis et al., ~O 94/16101 215 3 3 ~ 7 PCT/US94/00193 editors, Academic Press, San Diego (1990)) cloning or subcloning steps could be omitted by directly sequencing off chromosomal DNA by first amplifying the DNA segment by PCR and then applying the Sanger sequencing method (Innis et a/., Proc. Natl. Acad. Sci.
USA 85, 9436-9440 (1988)). In this case, however, the DNA sequence in the interested s region most be known at least to the extent to bind a sequencing primer.
In order to be able to read the sequence from PAGE, detectable labels have to be used in either the primer (very often at the 5'-end) or in one of the deoxynucleoside triphosphates, dNTP. Using radioisotopes such as 32p, 33p, or 35S is still the most frequently used technique. After PAGE, the gels are exposed to X-ray films and silver grain exposure is analyzed. The use of radioisotopic labeling creates several problems.
Most labels useful for autoradiographic detection of sequencing fragements have relatively short half-lives which can limit the useful time of the labels. The emission high energy beta radiation, particularly from 32p, can lead to breakdown of the products via radiolysis so that the sample should be used very quickly after labeling. In addition, high energy radiation can also cause a deterioration of band sharpness by scattering. Some of these problems can be reduced by using the less energetic isotopes such as 33P or 35S (see, e.g., Ornstein et a/., Biotechniques 3, 476 (1985)). Here, however, longer exposure times have to be tolerated. Above all, the use of radioisotopes poses significant health risks to the experimentalist and, in heavy sequencing projects, decont~rnin~tion and h~n~lling the radioactive waste are other severe problems and burdens.
In response to the above mentioned problems related to the use of radioactive labels, non-radioactive labeling techniques have been explored and, in recent years, integrated into partly automated DNA sequencing procedures. All these improvements utilize the Sanger sequencing strategy. The fluorescent label can be tagged to the primer (Smith et al., Nature ~., 674-679 (1986) and EPO Patent No. 87300998.9; Du Pont De Nemours EPO Application No. 0359225; Ansorge et al. J. Biochem. Biophys. Methods13, 325-32 (1986)) or to the chain-tennin~ting dideoxynucloside triphosphates (Prober et a/. Science 238, 336-41 (1987); Applied Biosystems, PCT Application WO 91/05060).
Based on either labeling the primer or the ddNTP, systems have been developed byApplied Biosystems (Smith et a/., Science ~, G89 (1987); U.S. Patent Nos. 570973 and 689013), Du Pont De Nemours (Prober et al. Science ~, 336-341 (1987); U.S. Patents Nos. 881372 and 57566), Pharmacia-LKB (Ansorge et a/. Nucleic Acids Res. 15, 4593-4602 (1987) and EMBL Patent Application DE P3724442 and P3805808.1) and Hitachi (JP 1-90844 and DE 4011991 A1). A somewhat similar approach was developed by 3s Brumbaugh et al. (Proc. Natl. Sci. USA 85, 5610-14 (1988) and U.S. Patent No.
4,729,947). An improved method for the Du Pont system using two electrophoretic lanes with two dirrelelll specific labels per lane is described (PCT Application W092/02635).
A different approach uses fluorescently labeled avidin and biotin labeled primers. Here, the sequencing ladders ending with biotin are reacted during electrophoresis with the wo 94/16101 2~ ~33a 4 PCT/US94/00193 labeled avidin which results in the detection of the individual sequencing bands(Brumbaugh et al, U.s. Patent No. 594676).
More recently even more sensitive non-radioactive labeling techniques for DNA using chemiluminescence triggerable a~d amplifyable by enzymes have been s developed (Beck, O'Keefe, Coull and Koster, Nucleic Acids Res. 17, 5115-5123 (1989) and Beck and Koster, Anal. Chem. 62, 2258-2270 (1990)). These labeling methods were combined with multiplex DNA sequencing (Church et a/. Science 240, 185-188 (1988) to provide for a strategy aimed at high throughput DNA seqllçnçing (Koster et al., Nucleic Acids Res. Symposium Ser. No. 24, 318-321 (1991), University of Utah, PCT
Application No. WO 90/15883); this strategy still suffers from the disadvantage of being very laborious and difficult to automate.
In an attempt to simplify DNA sequencing, solid supports have been introduced. In most cases published so far, the template strand for sequencing (with or without PCR amplification) is immobilized on a solid support most frequently lltili7ing the strong biotin-avidin/streptavidin interaction (Orion-Yhtyma Oy, U.S. Patent No. 277643;
M. Uhlen et a/. Nucleic Acids Res. 16, 3025-38 (1988); Cemu Bioteknik, PCT
Application No. WO 89/09282 and Medical Research Council, GB, PCT Application No.
WO 92/03575). The primer extension products synthesi7ed on the immobilized template strand are purified of en_ymes, other sequencing reagents and by-products by a washing step and then released under de~ ,g conditions by loosing the hydrogen bonds between the Watson-Crick base pairs and subjected to PAGE separation. In a different approach, the primer extension products (not the template) from a DNA sequencingreaction are bound to a solid support via biotin/avidin (Du Pont De Nemours, PCTApplication WO 91/11533). In contrast to the above mentioned methods, here, the 2s interaction between biotin and avidin is overcome by employing denaturing conditions (formamide/EDTA) to release the primer extension products of the sequencing reaction from the solid support for PAGE separation. As solid supports, beads, (e.g., magnetic beads (Dynabeads) and Sepharose beads), filters, capillaries, plastic dipsticks (e.g., polystyrene strips) and microtiter wells are being proposed.
All methods discussed so far have one central step in common:
polyacrylamide gel electrophoresis (PAGE). In many instances, this represents a major drawback and limitation for each of these methods. Preparing a homogeneous gel by polymerization, loading of the samples, the electrophoresis itself, detection of the sequence pattem (e.g., by autoradiography), removing the gel and cleaning the glass plates 3s to prepare another gel are very laborious and time-consuming procedures. Moreover, the whole process is error-prone, difficult to automate, and, in order to improve reproducibility and reliability, highly trained and skilled personnel are required. In the case of radioactive labeling, autoradiography itself can consume from hours to days. In the case of fluorescent labeling, at least the detection of the sequencing bands is being -21~3387 io 94/16101 -5- PCT/US94/00193 performed automatically when using the laser-sc~nning devices integrated into commercial available DNA sequencers. One problem related to the fluorescent labeling is the influence of the four different base-specific fluorescent tags on the mobility of the fragment~ durin g electrophoresis and a possible overlap in the spectral bandwidth of the s four specific dyes reducing the discrimin~ting power between neighboring bands, hence, increasing the probability of sequence ambiguities. Artifacts are also produced by base-specific interactions with the polyacrylamide gel mah:ix (Frank and Koster, Nucleic Acids Res. 6, 2069 (1979)) and by the formation of secondary struch~res which result in "band conll)le3sions" and hence do not allow one to read the sequence. This problem has, 0 in part, been overcome by using 7-de~7~deoxyguanosine triphosphates (Barr et al., Biotechniques 4, 428 (1986)). However, the reasons for some artifacts and conspicuous bands are still under investigation and need further improvement of the gel electrophoretic procedure.
A recent innovation in elechrophoresis is capillary zone elechrophoresis (CZE) (Jorgenson et al., J. Chromatography ~, 337 (1986); Gesteland et al., Nucleic Acids Res. 18, 1415-1419 (1990)) which, compared to slab gel elechrophoresis (PAGE), significantly increases the resolution of the separation, reduces the time for an electrophoretic run and allows the analysis of very small samples. Here, however, other problems arise due to the mini~hlri7~tion of the whole system such as wall effects and the necessity of highly sensitive on-line detection methods. Compared to PAGE, another drawback is created by the fact that CZE is only a "one-lane" process, whereas in PAGE
samples in multiple lanes can be electrophoresed simultaneously.
Due to the severe limitations and problems related to having PAGE as an integral and central part in the standard DNA sequencing protocol, several methods have 2s been proposed to do DNA sequencing without an electrophoretic step. One approach calls forhybridizationorfr~gment~tionsequencing(Bains,Biote~hnologyl0,757-58(1992) and Mirzabekov et a/., FEBS T etters ~, 118-122 (1989)) lltili7ing the specific hybridization of known short oligonucleotides (e.g., octadeoxynucleotides which gives 65,536 dirr~lell~ sequences) to a complementary DNA sequence. Positive hybridization reveals a short stretch of the unknown sequence. Repeating this process by performing hybridizations with all possible octadeoxynucleotides should theoretically determine the sequence. In a completely different approach, rapid sequencing of DNA is done byunilaterally degrading one single, immobilized DNA fragment by an exonuclease in a moving flow stream and detecting the cleaved nucleotides by their specific fluorescent tag 3s via laser excitation (Jett et al., J. Biolnolecular Structure & Dynamics 7, 301-309, (1989);
United States Department of Energy, PCT Application No. WO 89/03432). In anothersystem proposed by Hyman (Anal.Biochem. 174, 423-436 (1988)), the pyrophosphate generated when the correct nucleotide is attached to the growing chain on a primer-template system is used to determine the DNA sequence. The enzymes used and the DNA

2 ~S 3 3 ~ -6- PCT/US94/00193 are held in place by solid phases (DEAE-Sepharose and Sepharose) either by ionicinteractions or by covalent attachment. In a continuous flow-through system, the amount of pyrophosphate is determined via bioluminescence (luciferase). A synthesis approach to DNA sequencing is also used by Tsien et a/. (PCT Application No. WO 91/06~78). Here, the incoming dNTP's are protected at the 3'-end by various blocking groups such as acetyl or phosphate groups and are removed before the next elongation step, which makes this process very slow compared to standard sequencing methods. The template DNA is immobilized on a polymer support. To detect incorporation, a fluorescent or radioactive label is additionally incorporated into the modified dNTP's. The same patent application 0 also describes an app~lus de~ign~ d to automate the process.
Mass spectrometry, in general, provides a means of "weighing" individual molecules by ionizing the molecules in vacuo and making them "fly" by vol~fili7~tion.
Under the influence of combinations of electric and magnetic fields, the ions follow trajectories depending on their individual mass (m) and charge (z). In the range of molecules with low molecular weight, mass spectrometry has long been part of the routine physical-organic repertoire for analysis and characterization of organic molecules by the determination of the mass of the parent molecular ion. In addition, by arranging collisions of this parent molecular ion with other particles (e.g., argon atoms), the molecular ion is fragmented forming secondary ions by the so-called collision in(lllced dissociation (CID).
The fr~gment~tion pattern/l ~lhw~y very often allows the derivation of detailed structural information. Many applications of mass spectrometric methods in the known in the art, particularly in biosciences, and can be found summarized in Methods in Fn7ymolo~y~
Vol. 193: "Mass Spectrometry" (J.A. McCloskey, editor), 1990, Academic Press, New York.
Due to the a~clll analytical advantages of mass spectrometry in providing high detection sensitivity, accuracy of mass measurements, detailed structural information by CID in conjunction with an MS/MS configuration and speed, as well as on-line data transfer to a computer, there has been considerable interest in the use of mass spectrometry for the structural analysis of nucleic acids. Recent reviews summarizing this field include K. H. Schram, "Mass Spectrometry of Nucleic Acid Components, Biomedical Applications of Mass Spectrometry" 34, 203-287 (1990); and P.F. Crain, "Mass Spectrometric Techniques in Nucleic Acid Research," Mass Sylectrometry Reviews 2, 505-554 (1990). The biggest hurdle to applying mass spectrometry to nucleic acids is the difficulty of vol~tili7ing these very polar biopolymers. Therefore, "sequencing" has been limited to low molecular weight synthetic oligonucleotides by det~ g the mass of the parent molecular ion and through this, confinning the already known sequence, oralternatively, confinning the known sequence through the generation of secondary ions (fragment ions) via CID in an MS/MS configuration l1tili7ing, in particular, for the ionization and vol~tili7~tion, the method of fast atomic bombardment (FAB mass 21~3387 ~,~ro 94/16101 -7- PCT/US94/00193 spectrometry) or plasma desorption (PD mass spectrometry). As an example, the application of FAB to the analysis of protected dimeric blocks for chemical synthesis of oligodeoxynucleotides has been described (Koster et al. Biomedical Environmental Mass Spectrometry 14, 111-116 (1987)).
s Two more recent ionization/desorption techniques are electrospray/ionspray (ES) and matrix-assisted laser desorption/ionization (MALDI). ES mass spectrometry has been introduced by Fenn et al. (J. Phys. Chem. 88, 4451-59 (1984); PCT Application No.
WO 90/14148) and current applications are summarized in recent review articles (R.D.
Smith et al., Anal. Chem. 62, 882-89 (1990) and B. Ardrey, Electrospray Mass 0 Spectrometry, Spectroscopy Furope, _, 10-18 (1992)). The molecular weights ofthe tetradecanucleotide d(CATGCCATGGCATG) (SEQ ID NO:1) (Covey et al. "The Det~.,.lill;1lion of Protein, Oligonucleotide and Peptide Molecular Weights by Ionspray Mass Spectrometry," Rapid Communications in Mass Spectrometry, 2, 249-256 (1988)), of the 21-mer d(AAATTGTGCACATCCTGCAGC) (SEQ ID NO:2) and without giving details of that of a tRNA with 76 nucleotides (Methods in Fn7,ymolo~y, 193, "Mass Spectrometry" (McCloskey, editor), p. 425, 1990, Academic Press, New York) have been published. As a mass analyzer, a quadrupole is most frequently used. The detçnnin~tion of molecular weights in femtomole amounts of sample is very accurate due to the presence of multiple ion peaks which all could be used for the mass calculation.
MALDI mass spectrometry, in contrast, can be particularly attractive when a time-of-flight (TOF) configuration is used as a mass analyzer. The MALDI-TOF mass spectrometry has been introduced by Hillenkamp et al. ("Matrix Assisted W-Laser Desorption/Ionization: A New Approach to Mass Spectrometry of Large Biomolecules,"
Biolo~ical Mass Spe~ ullletry (Burling~ne and McCloskey, editors), Elsevier Science 2s Publishers, Amsterdam, pp. 49-60, 1990.) Since, in most cases, no mul~iple molecular ion peaks are produced with this technique, the mass spectra, in principle, look simpler compared to ES mass spectrometry. Although DNA molecules up to a molecular weight of 410,000 daltons could be desorbed and vol~tili7Pcl (Williams et al., "Vol~tili7~tion of High Molecular Weight DNA by Pulsed Laser Ablation of Frozen Aqueous Solutions,"Science, ~, 1585-87 (1989)), this technique has so far only been used to determine the molecular weights of relatively small oligonucleotides of known sequence, e.g., oligothymidylic acids up to 18 nucleotides (Huth-Fehre et al., "Matrix-Assisted Laser Desorption Mass Spectrometry of Oligodeoxythymidylic Acids,"
Rapid Communications in Mass Spectrometry, 6, 209-13 (1992)) and a double-stranded 3s DNA of 28 base pairs (Williams et al., "Time-of-Flight Mass Spectrometry of Nucleic Acids by Laser Ablation and Ionization from a Frozen Aqueous Matrix," ~
Communications in Mass Spectrometry~ 4, 348-351 (1990)). In one publication (Huth-Fehre et al., 1992, supra), it was shown that a mixture of all the oligothymidylic acids from n=12 to n=18 nucleotides could be resolved.

wo 94/16101 2~3~ 8- PCT/US94/00193 In U.S. Patent No. 5,064,754, RNA transcripts extended by DNA both of which are complementary to the DNA to be sequenced are prepa~ed by incorporatingNTP's, dNTP's and, as tçr~nin~ting nucleotides, ddNTP's which are substituted at the 5'-position of the su~ar moiety with one or a combination of the isotopes 12C, 13C, 14C, lH, 2H, 3H, 160, 17O and 180. The polynucleotides obtained are degraded to 3'-nucleotides, cleaved at the N-glycosidic linkage and the isotopically labeled 5'-functionality removed by periodate oxidation and the resulting formaldehyde species determined by massspectrometry. A specific combination of isotopes serves to discriminate base-specifically between internal nucleotides origin~tin~ from the incorporation of NTP's and dNTP's and o terminal nucleotides caused by linking ddNTP's to the end of the polynucleotide chain. A
series of RNA/DNA fragments is produced, and in one embodiment, separated by electrophoresis, and, with the aid of the so-called matrix method of analysis, the sequence is deduced.
In Japanese Patent No. 59-131909, an instrument is described which detects nucleic acid fragments separated either by electrophoresis, liquid chromatography or high speed gel filtration. Mass spectrometric detection is achieved by incoll,old~ g into the nucleic acids atoms which normally do not occur in DNA such as S, Br, I or Ag, Au, Pt, Os, Hg. The method, however, is not applied to sequencing of DNA using the Sanger method. In particular, it does not propose a base-specific correlation of such elements to an individual ddNTP.
PCT Application No. WO 89/12694 (Brennan et al., Proc. SPIE-Int. Soc.
Opt. Fn~. 1206, (New Technol. Cytom. Mol. Biol.), pp. 60-77 (1990); and Brennan, U.S.
Patent No. 5,003,059) employs the Sanger methodology for DNA sequencing by using a combination of either the four stable isotopes 32S, 33S, 34S 36S or 35C1 37C1 79Br 2s 81Br to specifically label the chain-terrnin~ting ddNTP's. The sulfur isotopes can be located either in the base or at the alpha-position of the triphosphate moiety whereas the halogen isotopes are located either at the base or at the 3'-position of the sugar ring. The seqllenring reaction mixtures are ~epa~dled by an electrophoretic technique such as CZE, transferred to a combustion unit in which the sulfur isotopes of the incorporated ddNTP's are transformed at about 900C in an oxygen atmosphere. The SO2 generated with masses of 64, 65, 66 or 68 is ~let~rmined on-line by mass spectrometry using, e.g., as mass analyzer, a quadrupole with a single ion-multiplier to detect the ion current.
A similar approach is proposed in U.S. Patent No. 5,002,868 (Jacobson et al., Proc. SPE-Int. Soc. Opt. Fn~ 1435, (Opt. Methods Ultrasel ~itive Detect. Anal. Tech.
~L), 26-35 (1991)) using Sanger sequencing with four ddNTP's specifically substituted at the alpha-position of the triphosphate moiety with one of the four stable sulfur isotopes as described above and subsequent separation of the four sets of nested sequences by tube gel electrophoresis. The only difference is the use of resonance ionization spectroscopy (RIS) in conjunction with a magnetic sector mass analyzer as disclosed in U.S. Patent No.

_ ~O 94/16101 9 ~ 3 ~ 7 PCT/US94/00193 4,442,354 to detect the sulfur isotopes corresponding to the specific nucleotideterminators, and by this, allowing the ~ignment of the DNA sequence.
EPO Patent Applications No. 0360676 Al and 0360677 A1 also describe Sanger sequencing using stable isotope substitutions in the ddNTP's such as D, 13c, 15N, 170 180 32S 33S 34S 36s, I9F, 35Cl, 37Cl, 79Br, 81Br and 1271 or functional groups such as CF3 or Si(CH3)3 at the base, the sugar or the alpha position of the triphosphate moiety according to chemical functionality. The Sanger sequencing reaction mixtures are separated by tube gel electrophoresis. The effluent is converted into an aerosol by the electrospray/thermospray nebulizer method and then atomized and ionized by a hot plasma (7000 to 8000K) and analyzed by a simple mass analyzer. An instrument is proposed which enables one to automate the analysis of the Sanger sequencing reaction mixture consisting of tube electrophoresis, a nebulizer and a mass analyzer.
The application of mass spectrometry to perform DNA sequencing by the hybridization/fragment method (see above) has been recently suggested (Bains, "DNA
Sequencing by Mass Spectrometry: Outline of a Potential Future Application,"
Chimicaoggi 9, 13-16 (1991)).

Summary of the Invention The invention describes a new method to sequence DNA. The improvements over the existing DNA sequencing technologies include high speed, high throughput, no required electrophoresis (and, thus, no gel reading artifacts due to the complete absence of an electrophoretic step), and no costly reagents involving various substitutions with stable isotopes. The invention utilizes the Sanger sequencing strategy and assembles the sequence information by analysis of the nested fragments obtained by base-specific chain termin~tion via their different molecular masses using mass spectrometry, for example, MALDI or ES mass spectrometry. A further increase in throughput can be obtained by introducing mass modifications in the oligonucleotide primer, the chain-termin~ting nucleoside triphosphates and/or the chain-elongating nucleoside triphosphates, as well as using integrated tag sequences which allow multiplexing by hybridization of tag specific probes with mass differenti~te(l molecular weights.

Brief Description of the FIGURES
FIGURE 1 is a representation of a process to generate the samples to be analyzed by mass spectrometry. This process entails insertion of a DNA fragment of unknown sequence into a cloning vector such as derivatives of M13, pUC or phagemids;
transforming the double-stranded form into the single-stranded form; performing the four Sanger sequencing reactions; linking the base-specifically termin~te~l nested fragment family temporarily to a solid support; removing by a washing step all by-products;

WO 94/16101 X~S'3~ 10 - PCT/US94/00193 conditioning the nested DNA or R~A fragments by, for example, cation-ion exchange or modification reagent and presenting the immobilized nested fragments either directly to mass spectrometric analysis or cleaving the purified fragment family off the support and evaporating the cleavage reagent.
FIGURE 2A shows the Sanger sequencing products using ddTTP as termin~ting deoxynucleoside triphosphate of a hypothetical DNA fragment of 50 nucleotides (SEQ ID NO:3) in length with approximately equally balanced base composition. The molecular masses of the various chain tçrrnin~te-l fragments are given.
FIGURE 2B shows an ide~li7~cl mass spectrum of such a DNA fragment 1 0 mixture.
FIGURES 3A and 3B show? in analogy to FIGURES 2A and 2B, data for the same model sequence (SEQ ID NO:3) with ddATP as chain terrnin~tor.
FIGURES 4A and 4B show data, analogous to FIGURES 2A and 2B when ddGTP is used as a chain terminator for the same model sequence (SEQ ID NO:3).
1 j FIGURES SA and 5B illustrate the results obtained where chain termination is performed with ddCTP as a chain terminator, in a similar way as shown in FIGURES 2A and 2B for the same model sequence (SEQ ID NO:3).
FIGURE 6 summarizes the results of FIGURES 2A to 5B, showing the correlation of molecular weights of the nested four fragment families to the DNAsequence (SEQ ID NO:3).
FIGURES 7A and 7B illustrate the general structure of mass-modified sequencing nucleic acid primers or tag sequencing probes for either Sanger DNA or Sanger RNA sequencing.
FIGURES 8A and 8B show the general structure for the mass-modified triphosphates for either Sanger DNA or Sanger RNA sequencing. General formulas of the chain-elongating and the chain-terrnin~ting nucleoside triphosphates are demonstrated.
FIGURE 9 outlines various linking chemistries (X) with either polyethylene glycol or terminally monoalkylated polyethylene glycol (R) as an example.
FIGURE 10 illustrates similar linking chemistries as shown in FIGURES
8A and 8B and depicts various mass modifying moieties (R).
FIGURE 11 outlines how multiplex mass spectrometric sequencing can work using the mass-modified nucleic acid primer (UP).
FIGURE 12 shows the process of multiplex mass spectrometric sequencing employing mass-modified chain-elongating and/or termin~ting nucleoside triphosphates.
FIGURE 13 shows multiplex mass spectrometric sequencing by involving the hybridization of mass-modified tag sequence specific probes.
FIGURE 14 shows a MALDI-TOF spectrum of a mixture of oligothymidylic acids. d(pT) 12-18 FIGURE 15 shows a superposition of MALDI-TOF spectra of the 50-mer RECTIFIED SHI~ET (RULE 91) ISA/EP

21~33~7 _ ~0 94/16101 PCTtUS94/00193 d(TAACGGTCATTACGGCCATTGACTGTAGGACCTGCATTACATGACTAGCT) (SEQ
ID NO:3) (500 fmol) and dT(pdT)gg (500 fmol).
FIGURES 16A- 16M show the MALDI-TOF spectra of all 13 DNA sequences representing the nested dT-terminated fragments of the Sanger DNA sequencing simulation 5 of Figure 2, 500 fmol each as follows: 16A is a 7-mer; 16B is a 10-mer; 16C is a 11-mer;
16D is a 19-mer; 16E is a 20-mer; 16F is a 24-mer; 16G is a 26-mer; 16H is a 33-mer; 16I is a 37-mer; 16J is a 38-mer; 16K is a 42-mer; 16L is a 46-mer and 16M is a 50-mer.FIGURES 17A and 17B show the superposition of the spectra of FIGURE 16.
The two panels show two different scales and the spectra analyzed at that scale. Figure 17A
10 shows the superposition of the spectra of 16A- 16F. The letter above each peak corresponds to the original spectra of the fragment in FIGURE 16. For example, peak B corresponds to FIGURE 16B; peak C corresponds to FIGURE 16C, etc.
FIGURE 18 shows the superimposed MALDI-TOF spectra from MALDI-MS
analysis of mass-modified oligonucleotides as described in Example 21.
FIGURE 19 illustrates various linking chemistries between the solid support (P) and the nucleic acid primer (NA) through a strong electrostatic interaction.FIGURES 20A and 20B illustrate various linking chemistries between the solid support (P) and the nucleic acid primer (NA) through a charge transfer complex of a charge transfer acceptor (A) and a charge transfer donor (D).
FIGURE 21 illustrates various linking chemistries between the solid support (P) and the nucleic acid primer (NA) through a stable organic radical.
FIGURE 22 illustrates a possible linking chemistry between the solid support (P) and the nucleic acid primer (NA) through Watson-Crick base pairing.
FIGURE 23 illustrates linking the solid support (P) and the nucleic acid 25 primer (NA) through a photolytically cleavable bond.

Detailed Description of the Invention This invention describes an improved method of sequencing DNA. In particular~ this invention employs mass spectrometry, such as matrix-assisted laser 30 desorption/ionization (MALDI) or electrospray (ES) mass spectrometry (MS), to analyze the Sanger sequencing reaction mixtures.
In Sanger sequencing, four families of chain-terrnin~te~l fragments are obtained. The mass difference per nucleotide addition is 289.19 for dpC, 313.21 for dpA, 329.21 for dpG and 304.2 for dpT, respectively.
In one embodiment, through the separate determination of the molecular weights of the four base-specifically terminated fragment families, the DNA sequence can be assigned via superposition (e.g.. interpolation) of the molecular weight peaks of the four individual experiments. In another embodiment, the molecular weights of the four specificallv terminated fragment families can be determined simultaneously by MS, either RECTIFIED SHEET (RULE 91 ) ISA/EP

-~3~1 - 11.1 -by mixing the products of all four reactions run in at least two separate reaction vessels (i.e., all run separately, or two together, or three together) or by running one reaction having all four chain-terrnin~ting nucleotides (e.g., a reaction mixture comprising dTTP, ddTTP, dATP, ddATP. dCTP. ddCTP, dGTP, ddGTP) in one reaction vessel. By 5 simultaneously analyzing all four base-specifically terrnin~tecl reaction products, the RECT~FIED SHEET (RULE 91 ) ISA/EP

~o 94/16101 -12- 215 3 3 8 7 PCT/US94100193 molecular weight values have been, in effect, interpolated. Comparison of the mass difference measured between fragments with the known masses of each chain-te~"~ ting nucleotide allows the assignment of sequence to be carried out. In some in~t~nres, it may be desirable to mass modify, as ~ cl-csed below, the ~hain-te.",il-~ting nucleotides so as to s expand the dirr~lence in molecular weight between each nucleotide. It will be apparent to those skilled in the art when mass-modification of the chain-te"llill~ti,-g nucleotides is desirable and can depend, for inct~nre, on the resolving ability of the particular spectlolll~Lel employed. By way of example, it may be desirable to produce four chain-tel",in~li,-g nucleotides, ddTTP, ddCTP1, ddATP2 and ddGTP3 where ddCTP1, ddATP2o and ddGTP3 have each been mass-modified so as to have molecular weights resolvable from one another by the particular spectrometer being used.
The terms chain-elongating nucleotides and chain-l~.",i,-~ting nucleotides are well known in the art. For DNA, chain-elongating nucleotides include 2'-deoxyribonucleotides and chain-t~.,llil-~li,-g nucleotides include 2', 3'-dideoxyribonucleotides. For RNA, chain-elongating nucleotides include ribonucelotides and chain-terrnin~ting nucleotides include 3'-deoxyribonucleotides. The term nucleotide is also well known in the art. For the purposes of this invention, nucleotides include nucleoside mono-, di-, and triphosphates. Nucleotides also include modified nucleotides such as phosphorothioate nucleotides.
Since mass spectrometry is a serial method, in contrast to cull~;lllly used slab gel electrophoresis which allows several samples to be processed in parallel, in another embodiment of this invention, a further improvement can be achieved by multiplex mass spectrometric DNA sequencing to allow simultaneous sequencing of more than one DNA or RNA fragment. As described in more detail below, the range of about 300 mass units between one nucleotide addition can be utilized by employing either mass-modified nucleic acid sequencing primers or chain-elongating and/or tçrrnin~tingnucleoside triphosphates so as to shift the molecular weight of the base-specifically termin~tecl fr~gment~ of a particular DNA or RNA species being sequenced in a predetermined manner. For the first time, several sequencing reactions can be mass spectrometrically analyzed in parallel. In yet another embodiment of this invention, multiplex mass spectrometric DNA sequencing can be performed by mass modifying the fragment families through specific oligonucleotides (tag probes) which hybridize to specific tag sequences within each of the fragment families. In another embodiment, the tag probe can be covalently attached to the individual and specific tag sequence prior to mass spectrometry.
In one embodiment of the invention, the molecular weight values of at least two base-specifically tel.l~ t~d fragments are delelmilled con.;ull~,.lLly using mass spectrometry. The molecular weight values of preferably at least five and more preferably at least ten base-specifically t~ cl fragments are delelll~illed by mass spectrometry.

Wo 94/16101 ?~j3f3a -13- PCT/US94/00193 Also included in the invention are detelll~inations of the molecular weight values of at !east 20 base-specifically te~ t~d fr~gmPnt~ and at least 30 base-specifically termin~tecl fragments. Further, the nested base-specifically termin~ted fragments in a specific set can be purified of all reactarts and by-products but are not separated from one another. The entire set of nested base-specifically termin~ted fragments is analyzed con~;ullellLly and the molecular weight values are delelll~ ed. At least two base-specifically te.~ tedfragments are analyzed coll~;ul,~l,lly by mass specllo"~ when the fr~met~t~ are contained in the same sample.
In general, the overall mass spectrometric DNA sequencing process will start lo with a library of small genomic fragments obtained after first randomly or specifically cutting the genomic DNA into large pieces which then, in several subcloning steps, are reduced in size and inserted into vectors like derivatives of Ml 3 or pUC (e.g., Ml 3mpl 8 or M13mpl9) (see FIGURE 1). In a di~rel~lll approach, the fragments inserted in vectors, such as M13, are obtained via subcloning starting with a cDNA library. In yet another approach, the DNA fragments to be sequenced are generated by the polymerase chain reaction (e.g., Higuchi et al., "A General Method of in vitro Pre~ dlion and Mutagenesis of DNA Fragments: Study of Protein and DNA Interactions," Nucleic Acids Res.~ 16, 7351-67 (1988)). As is known in the art, Sanger sequencing can start from one nucleic acid primer (UP) binding to the plus-strand or from another nucleic acid primer binding to the opposite minus-strand. Thus, either the complementary sequence of both strands of a given unknown DNA sequence can be obtained (providing for reduction of ambiguity in the sequence determination) or the length of the sequence information obtainable from one clone can be extended by generating sequence information from both ends of the unknown vector-inserted DNA fragment.
The nucleic acid primer carries, ~ ially at the 5'-end, a linking functionality, L, which can include a spacer of sufficient leng,th and which can interact with a suitable functionality, L', on a solid support to form a reversible linkage such as a photocleavable bond. Since each of the four Sanger sequencing families starts with a nucleic acid primer (L-UP; FIGURE 1) this fragment family can be bound to the solid support by reacting with functional groups, L', on the surface of a solid support and then intensively washed to remove all buffer salts, triphosphates, enzymes, reaction by-products, etc. Furthermore, for mass spectrometric analysis, it can be of importance at this stage to exchange the cation at the phosphate backbone of the DNA fr~gments in order to elimin~te peak bro~(lçnin~ due to a heterogeneity in the cations bound per nucleotide unit.
Since the L-L' linkage is only of a temporary nature with the purpose to capture the nested Sanger DNA or RNA fragments to plopt;lly condition them for mass spectrometric analysis, there are different chemistries which can serve this purpose. In addition to the examples given in which the nested fragments are coupled covalently to the solid support, washed, and cleaved offthe support for mass spectrometric analysis, the temporary O 94/16101 2 1 5 3 3~ ~ PCT/US94/00193 linkage can be such that it is cleaved under the conditions of mass spectrometry, i.e., a photocleavable bond such as a charge transfer complex or a stable organic radical.
Furthermore~ the linkage can be formed with L' being a quaternary arnrnoniu~n group (some examples are given in FIGURE l 9). In this case, preferably, the surface of the solid 5 support carries negative charges which repel the negatively charged nucleic acid backbone and thus facilitates desorption. Desorption will take place either by the heat created by the laser pulse and/or~ depending on L,' by specific absorption of laser energy which is in resonance with the L' chromophore (see, e.g., examples given in FIGURE 19). The functionalities, L and L.' can also forrn a charge transfer complex and thereby forrn the 10 temporary L-L' linkage. Various examples for appropl;ate functionalities with either acceptor or donator properties are depicted without limitation in FIGURES 20A and 20B.
Since in many cases the "charge-transfer band" can be deterrnined by UV/vis speckometry (see e.g. Organic Charge Transfer Complexes by R. Foster, Academic Press, 1969), the laser energy can be tuned to the corresponding energy of the charge-transfer wavelength 15 and, thus, a specific desorption offthe solid support can be initiated. Those skilled in the art will recognize that several combinations can serve this purpose and that the donor functionality can be either on the solid support or coupled to the nested Sanger DNA/RNA
fragments or vice versa.
In yet another approach, the temporary linkage L-L' can be generated by 20 homolytically forming relatively stable radicals as exemplified in FIGURE 21. In example 4 of FIGURE 21, a combination of the approaches using charge-transfer complexes and stable organic radicals is shown. Here, the nested Sanger DNA/RNA fr~gments are captured via the formation of a charge transfer complex. Under the influence of the laser pulse, desorption (as discussed above) as well as ionization will take place at the radical position. In the other 25 examples of FIGURE 21 under the influence of the laser pulse, the L-L' linkage will be cleaved and the nested Sanger DNA/RNA fragments desorbed and subsequently ionized at the radical position formed. Those skilled in the art will recognize that other organic radicals can be selected and that, in relation to the dissociation energies needed to homolytically cleave the bond between them, a corresponding laser wavelength can be selected (see e.g.
30 Reactive Molecules by C. Wentrup. John Wiley & Sons, 1984). In yet another approach, the nested Sanger DNA/RNA fragments are captured via Watson-Crick base pairing to a solid support-bound oligonucleotide complementary to either the sequence of the nucleic acid primer or the tag oligonucleotide sequence (see FIGURE 22). The duplex formed will be cleaved under the influence of the laser pulse and desorption can be initiated. The solid 35 support-bound base sequence can be presented through natural oligoribo- or oligodeoxyribonucleotide as well as analogs (e.g. thio-modified phosphodiester or phosphotriester backbone) or employing oligonucleotide mimetics such as PNA analogs (see e.g. Nielsen et cll., Science~ 254~ 1497 (1991 )) which render the base sequence less susceptible to enzymatic degradation and RECTIFIED SHEET (RULE 91) ISA/EP

-WO 94/16101 ,~ 33~ i -15- PCT/US94/00193 hence increases overall stability of the solid support-bound capture base sequence. With a~propl;ate bonds, L-L', a cleavage can be obtained directly with a laser tuned to the energy necessary for bond cleavage. Thus, the immobilized nested Sanger fragments can be directly ablated during mass spectrometric analysis.
To increase mass spectrometric performance, it may be necessary to modify the phosphodiester backbone prior to MS analysis. This can be accomplished by, for example, using alpha-thio modified nucleotides for chain elongation and termin~tion.
With alkylating agents such as akyliodides, iodoacetamide, ~-iodoethanol, 2,3-epoxy-1-propanol (see FIGURE 10), the monothio phosphodiester bonds of the nested Sangerfragments are transformed into phosphotriester bonds. Multiplexing by mass modification in this case is obtained by mass-modifying the nucleic acid primer (IJP) or the nucleoside triphosphates at the sugar or the base moiety. To those skilled in the art, other modifications of the nested Sanger fr~grnents can be envisioned. In one embodiment of the invention, the linking chemi~try allows one to cleave offthe so-purified nested DNA
enzymatically, chemically or physically. By way of example, the L-L' chemistry can be of a type of disulfide bond (chemically cleavable, for example, by mercaptoethanol or dithioerythrol), a biotin/streptavidin system, a heterobifunctional derivative of a trityl ether group (Koster et al., "A Versatile Acid-Labile Linker for Modification of Synthetic Biomolecules," Tetrahedron Letters 31, 7095 (1990)) which can be cleaved under mildly acidic conditions, a levulinyl group cleavable under almost neutral conditions with a hydræinium/acetate buffer, an arginine-arginine or Iysine-lysine bond cleavable by an endopeptidase enzyme like trypsin or a pyrophosphate bond cleavable by a pyrophosphatase, a photocleavable bond which can be, for example, physically cleaved and the like (see, e.g., FIGURE 23). Optionally, another cation exchange can be ~lrolllled prior to mass spectrometric analysis. In the instance that an enzyme-cleavable bond is utilized to immobilize the nested fragments, the enzyme used to cleave the bond can serve as an internal mass standard during MS analysis.
The purification process and/or ion exchange process can be carried out by a number of other methods instead of, or in conjunction with, immobilization on a solid support. For example, the base-specifically terrnin~ted products can be separated from the re~ct~ntc by dialysis, filtration (including ultrafiltration), and chromatography. Likewise, these techniques can be used to exch~nge the cation of the phosphate backbone with a counter-ion which reduces peak bro~lening.
The base-specifically terrnin~te~l fragment families can be generated by standard Sanger sequencing using the Large Klenow fragment of E. coli DNA polymerase I, by Sequenase, Taq DNA polymerase and other DNA polymerases suitable for this purpose, thus generating nested DNA fragments for the mass spectrometric analysis. It is, however, part of this invention that base-specifically terrnin~ted RNA transcripts of the DNA fragments to be sequenced can also be utilized for mass ~ue~;llollletric sequence 21~387 _ ~ o 94/16101 -16- PCTrJS94/00193 determination. In this case, various RNA polymerases such as the S]~6 or the T7 RNA
polymerase can be used on a~plop~iate vectors co~ g~ for example, the SP6 or the T7 promoters (e.g. Axelrod et al, "Transcription from Bacteriophage T7 and SP6 RNA
Polymerase Promoters in t~e Presence of 3'-Deoxyribonucleoside 5'-triphosphate Chain s Termin~tors," Biochemistry 24,5716-23 (1985)). In this case, the unknown DNAsequence fragments are inserted downstream from such promoters. Transcription can also be initiated by a nucleic acid primer (Pitulle et al., "Initiator Oligonucleotides for the Combination of Chemical and Enzymatic RNA Synthesis," Gene 1 12, l O 1-105 (1992)) which carries, as one embodiment of this invention, allplop,;ate linking functionalities, L, o which allow the immobilization of the nested RNA fragments, as outlined above, prior to mass spectrometric analysis for purification and/or appropl;ate modification and/or conditioning.
For this immobilization process of the DNA/RNA sequencing products for mass spectrometric analysis, various solid ~u~l,o~ls can be used, e.g., beads (silica gel, controlled pore glass, magnetic beads, Sephadex/Sepharose beads, cellulose beads, etc.), capillaries, glass fiber filters, glass snrf~ces, metal surfaces or plastic m~ten~l. Examples of useful plastic m~teri~ls include membranes in filter or microtiter plate formats, the latter allowing the automation of the purification process by employing microtiter plates which, as one embodiment of the invention, carry a permeable membrane in the bottom of the well functionalized with L'. Membranes can be based on polyethylene, polypropylene, polyamide, polyvinyliclene-lifluoride and the like. Examples of suitable metal surfaces include steel, gold, silver, alulllhlulll, and copper. After purification, cation exchange, and/or modification of the phosphodiester backbone of the L-L' bound nested Sanger fragments, they can be cleaved off the solid support chemically, enzymatically or 2s physically. Also, the L-L' bound fr~gment~ can be cleaved from the support when they are subjected to mass spectrometric analysis by using il~ropl;ately chosen L-L' linkages and corresponding laser energies/intensities as described above and in FIGURES 19-23.
The highly purified, four base-specifically termin~tecl DNA or RNA
fragment families are then analyzed with regard to their fragment lengths via determin~tion of their re~eclive molecular weights by MALDI or ES mass spectrometry.
For ES, the sarnples, dissolved in water or in a volatile buffer, are injected either continuously or discontinuously into an atmospheric pressure ionization interface (API) and then mass analyzed by a quadrupole. With the aid of a con~ul~. program, the molecular weight peaks are searched for the known molecular weight of the nucleic acid primer (UP) and determined which of the four chain-termin~ting nucleotides has been added to the UP. This represents the first nucleotide of the unknown sequence. Then, the second, the third, the nth extension product can be identified in a similar manner and, by this, the nucleotide sequence is ~csignP~ The generation of multiple ion peaks which can be obtained using ES mass spectrometry can increase the accuracy of the mass ~ - 17-~ 1 ~33~ ~
determination.
In MALDI mass spectrometry, various mass analyzers can be used, e.g., magnetic sector/magnetic deflection instruments in single or triple quadrupole mode (MS/MS), Fourier transform and time-of-flight (TOF) configurations as is known in the 5 art of mass spectrometry. FIGURES 2A through 6 are given as an example of the data obtainable when sequencing a hypothetical DNA fragment of 50 nucleotides in length (SEQ ID NO:3) and having a molecular weight of 15,344.02 daltons. The molecular weights calculated for the ddT (FIGURES 2A and 2B), ddA (FIGURES 3A and 3B), ddG(FIGURES 4A and 4B) and ddC (FIGURES 5A and 5B) termin~ted products are given 10 (corresponding to fragments of SEQ ID NO:3) and the ide~li7e~1 four MALDI-TOF mass spectra shown. All four spectra are superimposed, and from this, the DNA sequence can be generated. This is shown in the summarizing FIGURE 6, demonstrating how the molecular weights are correlated with the DNA sequence. MALDI-TOF spectra have been generated for the ddT terminated products (FIGURES 16A- 16M) corresponding to 15 those shown in FIGURE 2 and these spectra have been superimposed (FIGURES 17A and 17B). The c(3rrelation of calculated molecular weights of the ddT fragments and their experimentally-verified weights are shown in Table 1. Likewise, if all four chain-termin~ting reactions are combined and then analyzed by mass spectrometry, the molecular weight difference between two adjacent peaks can be used to determine the 20 sequence. For the desorption/ionization process, numerous matrix/laser combinations can be used.
TABLE I
25 Correlation of calculated and experimentally verified molecular weights of the 13 DNA
fragments of FIGURES 2 and 16A- 16M.
Fragment(n-mer)calculatedmassexperimental mass difference 7-mer 2104.45 2119.9 +15.4 10-mer 3011.04 3026.1 +15.1 I l-mer 3315.24 3330.1 +14.9 19-mer 5771.82 5788.0 +16.2 20-mer 6076.02 6093.8 +17.8 24-mer 7311.82 7374.9 +63.1 26-mer 7945.22 7960.9 +15.7 33-mer 10112.63 10125.3 +12.7 37-mer 11348.43 11361.4 +13.0 38-mer 11652.62 11670.2 +17.6 42-mer 12872.42 12888.3 +15.9 46-mer 14108.22 14125.0 +16.8 50-mer 15344.02 15362.6 +18.6 RECTIFIED SHEET (RULE 91) ISA/EP

~o 94/16101 18 2 1 5 3 3 8 7 PCT/US94/00193 In order to increase throughput to a level necessary for high volume genomic and cDNA sequencing projects, a further embodiment of the present invention is to utilize multiplex mass spectrometry to simultaneously determine more than one sequence. This can be achieved by several. albeit different7 methodologies, tbe basic principle being the mass modification of the nucleic acid primer (UP), the chain-elon~ting and/or termin~ting nucleoside triphosphates, or by using mass-differentiated tag probeshybridizable to specific tag sequences. The term "nucleic acid primer" as used herein encompasses primers for both DNA and RNA Sanger sequencing.
By way of example, FIGURE 7A presents a general formula of the nucleic acid primer (UP) and the tag probes (TP). The mass modifying moiety can be attached, for instance, to either the 5'-end of the oligonucleotide (M 1), to the nucleobase (or bases) (M2, M7), to the phosphate backbone (M3), and to the 2'-position of the nucleoside (nucleosides) (M4, M6) or/and to the terminal 3'-position (M5). Primer length can vary between l and 50 nucleotides in length. For the priming of DNA Sanger sequencing, the primer is preferentially in the range of about 15 to 30 nucleotides in length. For artificially priming the transcription in a RNA polymerase-mediated Sanger sequencing reaction, the length of the primer is preferentially in the range of about 2 to 6 nucleotides.
If a tag probe (TP) is to hybridize to the integrated tag sequence of a family chain-terminated fragments, its preferential length is about 20 nucleotides.
The table in FIGURE 7B depicts some examples of mass-modified primer/tag probe configurations for DNA, as well as RNA, Sanger sequencing. This list is, however, not meant to be limiting, since numerous other combinations of mass-modifying functions and positions within the oligonucleotide molecule are possible and are deemed part of the invention. The mass-modifying functionality can be, for example, a halogen, an azido, or of the type, XR, wherein X is a linking group and R is a mass-modifying functionality. The mass-modifying functionality can thus be used to introduce defined mass increments into the oligonucleotide molecule.
In another embodiment, the nucleotides used for chain-elongation and/or termination are mass-modified. Examples of such modified nucleotides are shown in FIGURE 8A and 8B. Here the mass-modifying moiety, M, can be attached either to the nucleobase. M2 (in case of the c7-deazanucleosides also to C-7, M7), to the triphosphate group at the alpha phosphate, M3, or to the 2'-position of the sugar ring of the nucleoside triphosphate M4 and M6. Furthermore, the mass-modifying functionality can be added so as to affect chain termination, such as by attaching it to the 3'-position of the sugar ring in the nucleoside triphosphate, M5. The list in FIGURE 8B represents examples of possible configurations for generating chain-terrnin~ting nucleoside triphosphates for RNA or DNA
Sanger sequencing. For those skilled in the art, however, it is clear that many other combinations can serve the purpose of the invention equally well. In the same way, those skilled in the art will recognize that chain-elongating nucleoside triphosphates can also be RECTI~IED SHEET (RULE 91) IS~/EP

WO 94/l6l01 ~ 533~ -19- PCT/US94/00193 mass-modified in a similar fashion with numerous variations and combinations in functionality and ~tt~rl~ment positions.
Without limiting the scope of the invention, FIGURE 9 gives a more detailed description of particular examples of how the mass-modification, M, can be introduced for s X in XR as well as using oligo-/polyethylene glycol derivatives for R. The mass-modifying increment in this case is 44, i.e. five dirrelelll mass-modified species can be generated by just ch~nging m from 0 to 4 thus adding mass units of 45 (m=0), g9 (m=1), 133 (m=2), 177 (m=3) and 221 (m=4) to the nucleic acid primer (UP), the tag probe (TP) or the nucleoside triphosphates respectively. The oligo/polyethylene glycols can also be monoalkylated by a lower alkyl such as methyl, ethyl, propyl, isopropyl, t-butyl and the like. A selection of linking functionalities, X, are also illustrated. Other chemistries can be used in the mass-modified compounds, as for example, those described recently in Oli~onucleotides and Analo~ues. A Practical Approach~ F. Eckstein, editor, IRL Press, Oxford, 1991.
In yet another embodiment, various mass-modifying functionalities, R, other than oligo/polyethylene glycols, can be selected and attached via appropl;ate linking chemistries, X. Without any limitation, some examples are given in FIGURE 10. A
simple mass-modification can be achieved by substituting H for halogens like F, Cl, Br and/or I, or pseudohalogens such as SCN, NCS, or by using dirrerent alkyl, aryl or aralkyl moieties such as methyl, ethyl, propyl, isopropyl, t-butyl, hexyl, phenyl, substituted phenyl, benzyl, or functional groups such as CH2F, CHF2, CF3, Si(CH3)3, Si(CH3)2(C2H5), Si(CH3)(C2H5)2, Si(C2Hs)3 . Yet another mass-modification can beobtained by ~tt~hing homo- or h~le~opcl~lides through X to the UP, TP or nucleoside triphosphates. One example useful in generating mass-modified species with a mass 2s increment of 57 is the ~ çhment of oligoglycines, e.g., mass-modifications of 74 (r=1, m=O), 131 (r=1, m=2), 188 (r=1, m=3), 245 (r=1, m=4) are achieved. Simple oligoamides also can be used, e.g., mass-modifications of 74 (r=1, m=0), 88 (r=2, m=0), 102 (r=3, m=0), 116 (r=4, m=0), etc. are obtainable. For those skilled in the art, it will be obvious that there are numerous possibilities in addition to those given in FIGURE 10 and the above mentioned reference (Oli~onucleotides and Analo~ues~ F. Eckstein, 1991), for introducing, in a predetermined manner, many different mass-modifying functionalities to UP, TP and nucleoside triphosphates which are acceptable for DNA and RNA Sanger sequencing.
As used herein, the superscript 0-i tie~ign~tes i + 1 mass differenti~te~l 3s nucleotides, primers or tags. In some instances, the ~u~ 0 (e.g., NTP0, UP0) can designate an unmodified species of a particular reactant, and the superscript i (e.g., NTPi, NTP 1, NTP2, etc.) can designate the i-th mass-modified species of that reactant. If, for example, more than one species of nucleic acids (e.g., DNA clones) are to be concul,elllly sequenced by multiplex DNA sequencing, then i + l different mass-modified nucleic acid _~l0 94/16101 21 ~ 3 3 ~ 7 PCT/US94/00193 primers (UPO. UP 1 ...UPi) can be used to distinguish each set of base-specifically terminated fragments, wherein each species of mass-modified upi can be distinguished by mass spectrometry from the rest.
As illustrative embodiments of this invention, three different basic processes for multiplex mass spectrometric DNA sequencing employing the described mass-modified reagents are described below:
A) Multiplexing by the use of mass-modified nucleic acid primers (UP) for Sanger DNA or RNA sequencing (see for example FIGURE 1 1 );
B) Multiplexing by the use of mass-modified nucleoside triphosphates as chain elongators and/or chain terminators for Sanger DNA or RNA sequencing (see for example FIGURE 12); and C) Multiplexing by the use of tag probes which specifically hybridize to tag sequences which are integrated into part of the four Sanger DNA/RNA base-specifically terminated fragment families.
Mass modification here can be achieved as described for FIGURES 7A, 7B, 9 and 10, or alternately, by designing different oligonucleotide sequences having the same or different length with unmodified nucleotides which, in a predetermined way, generate applopfiately differentiated molecular weights (see for example FIGURE 13).
The process of multiplexing by mass-modified nucleic acid primers (UP) is illustrated by way of example in FIGURE 11 for mass analyzing four different DNAclones simultaneously. The first reaction mixture is obtained by standard Sanger DNA
sequencing having unknown DNA fragment 1 (clone 1) integrated in an al)propl;ate vector 25 (e.gM 1 3mpl 8) employing an unmodified nucleic acid primer UPO, and a standard mixture of the four unmodified deoxynucleoside triphosphates, dNTPO, and with 1/lOth of one of the four dideoxynucleoside triphosphates, ddNTPO. A second reaction mixture for DNA fragment 2 (clone 2) is obtained by employing a mass-modified nucleic acid primer UP 1 and. as before. the four unmodified nucleoside triphosphates, dNTPO,containing in each separate Sanger reaction 1/lOth ofthe chain-termin~ting unmodified dideoxynucleoside triphosphates ddNTPO. In the other two experiments, the four Sanger reactions have the following compositions: DNA fragment 3 (clone 3), UP2, dNTPO,ddNTPO and DNA fragment 4 (clone 4), UP3, dNTPO, ddNTPO. For mass spectrometric DNA sequencing. all base-specifically termin~ted reactions of the four clones are pooled and mass analyzed. The various mass peaks belonging to the four dideoxy-terminated (e.g.. ddT-termin~te~) fragment families are assigned to specifically elongated and ddT-terminated fragments by searching (such as by a computer program) for the known molecular ion peaks of UPO. UP I . Up2 and UP3 extended by either one of the four dideoxynucleoside triphosphates, UPO-ddNO, UP1-ddNO, UP2-ddNO and UP3-ddNO. In RECTIFIED SHEET (RULE 91) ISA/EP

WO 94/16101 2~S3'3~ -21- PCT/US94100193 this way, the first nucleotides of the four unknown DNA sequences of clone 1 to 4 are determined. The process is repeated, having memorized the molecular masses of the four specific first extension products, until the four sequences are assigned. Unambiguous mass/sequence ~c~ignment~ are possible even in the worst case scenario in which the four s mass-modified nucleic acid primers are extended by the same dideoxynucleoside triphosphate, the extension products then being, for example, UP0-ddT, UP 1-ddT, UP2-ddT and UP3-ddT, which differ by the known mass increment dirr~ ."i~ting the four nucleic acid primers. In another embodiment of this invention, an analogous technique is employed using different vectors cont~ining, for example, the SP6 and/or T7 promoter o sequences, and performing transcription with the nucleic acid primers UP0, UP1, UP2 and UP3 and either an RNA polymerase (e.g., SP6 or T7 RNA polymerase) with chain-elongating and tçrmin~tin~ unmodified nucleoside triphosphates NTP0 and 3'-dNTP0Here, the DNA sequence is being ~etçrmined by Sanger RNA sequencing.
FIGURE 12 illustrates the process of multiplexing by mass-modified chain-elongating or/and tPrrnin~ting nucleoside triphosphates in which three dirrelent DNA
fragments (3 clones) are mass analyzed simultaneously. The first DNA Sanger sequencing reaction (DNA fragment 1, clone 1) is the standard llliXll.Jlc~ employing unmodified nucleic acid primer UP0, dNTP0 and in each of the four reactions one of the four ddNTP0. The second (DNA fragment 2, clone 2) and the third (DNA fragment 3, clone 3) have the following contents: UP0, dNTP0, ddNTP1 and UP0, dNTP0, ddNTP2, respectively. In a variation of this process, an amplification of the mass increment in mass-modifying the extended DNA fragments can be achieved by either using an equally mass-modified deoxynucleoside triphosphate (i.e., dNTP1, dNTP2) for chain elongation alone or in conjunction with the homologous equally mass-modified dideoxynucleoside triphosphate.
For the three clones depicted above, the contents of the reaction lllixlules can be as follows: either UP0/dNTP0/ddNTP0, UP0/dNTPl/ddNTPO and UP0/dNTP2/ddNTPO or UP0/dNTP0/ddNTP0, UP0/dNTPl/ddNTPl and UP0/dNTP2/ddNTP2. As described above, DNA sequencing can be p~lrolllled by Sanger RNA sequencing employing unmodified nucleic acid primers, UP0, and an appl~liate mixture of chain-elongating and termin~ting nucleoside triphosphates. The mass-modification can be again either in the chain-termin~tin~ nucleoside triphosphate alone or in conjunction with mass-modified chain-elongating nucleoside triphosphates. Multiplexing is achieved by pooling the three base-specifically termin~ted sequencing reactions (e.g., the ddTTP termin~ted products) and simultaneously analyzing the pooled products by mass spe~ llletry. Again, the first 3s extension products of the known nucleic acid primer sequence are assigned, e.g., via a computer program. Mass/sequence ~csignment~ are possible even in the worst case in which the nucleic acid primer is extended/tçrmin~ted by the same nucleotide, e.g., ddT, in all three clones. The following configurations thus obtained can be well differentiated by their different mass-modifications: UP0-ddT0, UP0-ddTl, UP0-ddT2.

_, o 94/16101 2 1 5 3 3 8 7 PCT/US94/00193 In yet another embodiment of this invention. DNA sequencing by multiplex mass spectrometry can be achieved by cloning the DNA fragments to be sequenced in "plex-vectors" containing vector specific "tag sequences" as described (Koster et al., - "Oligonucleotide Synthesis and Multiplex DNA Sequencing Using Chemilurninescent Detection." Nucleic Acids Res. Symposium Ser. No. 24, 318-321 (1991)); then pooling clones from different plex-vectors for DNA ~repaldlion and the four separate Sanger sequencing reactions using standard dNTP0/ddNTP0 and nucleic acid primer UP0;
purifying the four multiplex fragment families via linking to a solid support through the linking group, L, at the 5'-end of UP; washing out all by-products, and cleaving the purified multiplex DNA fragments off the support or using the L-L' bound nested Sanger fragments as such for mass spectrometric analysis as described above; performing de-multiplexing by one-by-one hybridization of specific "tag probes"; and subsequently analyzing by mass spectrometry (see, for example, FIGURE 13). As a reference point, the four base-specifically terminated multiplex DNA fragment families are run by the mass spectrometer and all ddT0-~ ddA0-, ddC0- and ddG0-terrnin~ted molecular ion peaks are respectively detected and memorized. Assignment of, for example, ddT0-terrnin~ted DNA
fragments to a specific fragment family is accomplished by another mass spectrometric analysis after hybridization of the specific tag probe (TP) to the corresponding tag sequence contained in the sequence of this specific fragment farnily. Only thosemolecular ion peaks which are capable of hybridizing to the specific tag probe are shifted to a higher molecular mass by the same kno~vn mass increment (e.g. of the tag probe).
These shifted ion peaks~ by virtue of all hybridizing to a specific tag probe, belong to the same fragment family. For a given fragment family, this is repeated for the rem~ining chain terminated fragment families with the same tag probe to assign the complete DNA
sequence. This process is repeated i-l times corresponding to i clones multiplexed (the i-th clone is identified by default).
The differentiation of the tag probes for the different multiplexed clones can be obtained just by the DNA sequence and its ability to Watson-Crick base pair to the tag sequence. It is well known in the art how to calculate stringency conditions to provide for specific hybridization of a given tag probe with a given tag sequence (see, for exarnple, Molecular Clonino: A laboratorv manual 2ed, ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: NY, 1989, Chapter 11). Furthermore, differentiation can be obtained by designing the tag sequence for each plex-vector to have a sufficient mass difference so as to be unique just by ch~nging the length or base composition or by mass-modifications according to FIGURES 7A, 7B, 9 and 10. In order to keep the duplex between the tag sequence and the tag probe intact during massspectrometric analysis. it is another embodiment of the invention to provide for a covalent attachment mediated by, for example, photoreactive groups such as psoralen and ellipticine and bv other methods known to those skilled in the art (see, for example, RECTlFlED SHEET (RULE 91) ISA/EP

WO 94/16101 ? ~33~'~ 23 - PCTIUS94/00193 Helène ef ul . Nature 344. 358 (1990) and Thuong et al. "Oligonucleotides Attached to Intercalators~ Photoreactive and Cleavage Agents" in F. Eckstein, Oligonucleotides and Analo~ues: A Practical Approach, IRL Press, Oxford 1991, 283-306).
The DNA sequence is unraveled again by searching for the lowest molecular weight molecular ion peak corresponding to the known UP0-tag sequence/tag probe molecular weight plus the first extension product, e.g., ddT0, then the second, the third, etc.
In a combination of the latter approach with the previously described multiplexing processes, a further increase in multiplexing can be achieved by using, in addition to the tag probe/tag sequence interaction, mass-modified nucleic acid primers (FIGURES 7A and 7B) and/or mass-modified deoxynucleoside, dNTP0-i and/or dideoxynucleoside triphosphates, ddNTP0-i. Those skilled in the art will realize that the tag sequence/tag probe multiplexing approach is not limited to Sanger DNA sequencing generating nested DNA fragments with DNA polymerases. The DNA sequence can also be determined by transcribing the unknown DNA sequence from apl)lo~llate promoter-containing vectors (see above) with various RNA polymerases and mixtures of NTP0-i/3'-dNTP0-i, thus generating nested RNA fragments.
In yet another embodiment of this invention, the mass-modifying functionality can be introduced by a two or multiple step process. In this case, the nucleic acid primer. the chain-elongating or termin~ting nucleoside triphosphates and/or the tag probes are, in a first step, modified by a precursor functionality such as azido, -N3, or modified with a functional group in which the R in XR is H (FIGURES 7A, 7B, 9) thus providing temporary functions, e.g.7 but not limited to -OH, -NH2, -NHR, -SH, -NCS, -OCO(CH2)rCOOH (r= 1-20), -NHCO(CH2)rCOOH (r= 1-20), -OSO2OH, -OCO(CH2)rI (r = 1-20), -OP(O-Alkyl)N(Alkyl)2. These less bulky functionalities result in better substrate properties for the enzymatic DNA or RNA synthesis reactions of the DNA sequencing process. The appropriate mass-modifying functionality is then introduced after the generation of the nested base-specifically terrnin~ted DNA or RNA
fragments prior to mass spectrometry. Several examples of compounds which can serve as mass-modifying functionalities are depicted in FIGURES 9 and 10 without limiting the scope of this invention.
Another aspect of this invention concerns kits for sequencing nucleic acids by mass spectrometry which include combinations of the above-described sequencing reactants. For instance, in one embodiment, the kit comprises reactants for multiplex mass spectrometric sequencing of several different species of nucleic acid. The kit can include a solid support having a linking functionality (Ll) for immobilization ofthe base-specifically terminated products: at least one nucleic acid primer having a linking group (L) for reversiblv and temporarily linking the primer and solid support through, for example. a photocleavable bond; a set of chain-elongating nucleotides (e.g., dATP, dCTP.
RECTIFIED SHEET (RULE 91 ) tSA/EP

iO 94/16101 -24- 21~ 3 3 ~ 7 PCT/US94/00193 dGTP and dTTP, or ATP, CTP, GTP and UTP); a set of chain-terrnin~ting nucleotides (such as 2',3'-dideoxynucleotides for DNA synthesis or 3'-deoxynucleotides for RNA
synthesis); and an ~plvpl;ate polymerase for synthesizing complementary nucleotides.
Primers and/or termin~tin~ nucleotides can be mass-modified so that the base-specifically s terrnin~tecl fragments generated from one of the species of nucleic acids to be sequenced can be distinguished by mass spectrometry from all of the others. Alternative to the use of mass-modified synthesis reactants, a set of tag probes (as described above) can be included in the kit. The kit can also include ~l~lul~l;ate buffers as well as instructions for performing multiplex mass spectrometry to concurrently sequence multiple species of lo nucleic acids.
- In another embodiment, a nucleic acid sequencing kit can comprise a solid support as described above, a primer for initiating synthesis of complementary nucleic acid fragments, a set of chain-elongating nucleotides and an appl~pl;ate polymerase. The mass-modified chain-termin~ting nucleotides are selected so that the addition of one of the chain termin~tQrs to a growing complement~ry nucleic acid can be distinguished by mass spectrometry.

Immobilization of primer-extension products of Sanger DNA sequencing reaction for mass spectrometric analysis via disulfide bonds.
As a solid support, Sequelon membranes (Millipore Corp., Bedford, MA) with phenyl isothiocyanate groups are used as a starting material. The membrane disks, with a diameter of 8 mm, are wetted with a solution of N-methylmorpholine/water/2-2s propanol (NMM solution) (2149149 vlvlv), the excess liquid removed with filter paper and placed on a piece of plastic film or alllminl-m foil located on a heating block set to 55C.
A solution of 1 mM 2-mercaptoethylamine (cysteamine) or 2, 2'-dithio-bis(ethylamine) (cystamine) or S-(2-thiopyridyl)-2-thio-ethylamine (10 ul, 10 nmol) in NMM is added per disk and heated at 55C. After 15 min, 10 ul of NMM solution are added per disk and heated for another 5 min. Excess of isothiocyanate groups may be removed by treatment with 10 ul of a 10 mM solution of glycine in NMM solution. For Cyst~mine7 the disks are treated with 10 ul of a solution of l M aqueous dithiothreitol (DTT)12-propanol ( 1:1 v/v) for 15 min at room temperature. Then, the disks are thoroughly washed in a filtration manifold with 5 aliquots of 1 ml each of the NMM solution, then with 5 aliquots of 1 ml 3s acetonitrile/water (1/1 v/v) and subsequently dried. If not used immediately the disks are stored with free thiol groups in a solution of l M aqueous dithiothreitol/2-propanol ( 1:1 v/v) and, before use, DTT is removed by three washings with 1 ml each of the NMM- solution. The primer oligonucleotides with 5'-SH functionality can be prepared by various methods (e.g., B.C.F Chu et al., Nucleic Acids Res. 14, 5591-5603 (1986), Sproat et al., WO 94/16101 ~, i iS ~ 3 ~ 7 -25- PCT/US94/00193 Nucleic Acids Res. 15, 4837-48 (1987) and Oli~onucleotides ~nd Analogues: A Practical Approach (F. Eckstein, editor), IRL Press Oxford, 1991). Sequencing reactions according to the Sanger protocol are performed in a standard way (e.g., H. Swerdlow et al., Nucleic Acids Res. 18, 1415-19 (1990)). In the presence of about 7-10 mM DTT the free s 5'-thiol primer can be used; in other cases, the SH functionality can be protected, e.g., by a trityl group during the Sanger sequencing reactions and removed prior to anchoring to the support in the following way. The four sequencing reactions (150 ul each in an Eppendorf tube) are tçnnin~tecl by a 10 min incubation at 70C to denature the DNA polymerase (such as Klenow fragment, Sequenase) and the reaction mixtures are ethanol precipitated.
The supern~t~nts are removed and the pellets vortexed with 25 ul of an lM aqueous silver nitrate solution, and after one hour at room temperature, 50 ul of an 1 M aqueous solution of DTT is added and mixed by vortexing. After 15 min, the mixtures are centrifuged and the pellets are washed twice with 100 ul ethylacetate by vortexing and centrifugation to remove excess DTT. The primer extension products with free 5'-thiol group are now coupled to the thiolated membrane supports under mild oxidizing conditions. In general, it is sufficient to add the 5'-thiolated primer extension products dissolved in 10 ul 10 mM
de-aerated triethylammonium acetate buffer (TEAA) pH 7.2 to the thiolated membrane supports. Coupling is achieved by drying the samples onto the membrane disks with a cold fan. This process can be repeated by wetting the membrane with 10 ul of 10 mM
TEAA buffer pH 7.2 and drying as before. When using the 2-thiopyridyl derivatized compounds, anchoring can be monitored by the release of pyridine-2-thione spectrophotometrically at 343 nm.
In another variation of this approach, the oligonucleotide primer is functionalized with an amino group at the 5'-end which is introduced by standardprocedures during automated DNA synthesis. After primer extension, during the Sanger sequencing process, the primary amino group is reacted with 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP) and subsequently coupled to the thiolatedsupports and monitored by the release of pyridyl-2-thione as described above. After denaturation of DNA polymerase and ethanol precipitation of the sequencing products, the supern~t~nts are removed and the pellets dissolved in 10 ul 10 mM TEAA buffer pH 7.2 and 10 ul of a 2 mM solution of SPDP in 10 mM TEAA are added. The reaction mixture is vortexed and incubated for 30 min at 25C. Excess SPDP is then removed by three extractions (vortexing, centrifugation) with 50 ul each of ethanol and the resulting pellets are dissolved in 10 ul 10 mM TEAA buffer pH 7.2 and coupled to the thiolated supports 3s (see above).
The primer-extension products are purified by washing the membrane disks three times each with 100 ul NMM solution and three times with 100 ul each of 10 mM
TEAA buffer pH 7.2. The purified primer-extension products are released by threesuccessive treatments with 10 ul of 10 mM 2-mercaptoethanol in 10 mM TEAA buffer pH

O 94116101 -26~1 S 3 3 8 7 PCT/US94/00193 7.2, Iyophilized and analyzed by either ES or MALDI mass spectrometry.
This procedure can also be used for the mass-modified nucleic acid primers upO-i in an analogous and al)p,o~l;ate way, taking into account the chemical plopellies of the mass-modifying functionalities.

EXAMPI,F 2 Immobilization of primer-extension products of Sanger DNA sequencing reaction for mass spectrometric analysis via the levulinyl group o 5-Aminolevulinic acid is protected at the primary amino group with the Fmoc group using 9-fluorenylmethyl N-succinimidyl carbonate and is then transformed into the N-hydroxysuccinimide ester (NHS ester) using N-hydroxysuccinimide and dicyclohexyl carbodiimide under standard conditions. For the Sanger sequencing reactions, nucleic acid primers, UP0-i, are used which are functionalized with a primary amino group at the 5'-end introduced by standard procedures during automated DNAsynthesis with aminolinker phosphoamidites as the final synthetic step. Sanger sequencing is p~.~l..led under standard conditions (see above). The four reaction mixtures (150 ul each in an Eppendorf tube) are heated to 70C for 10 min to inactivate the DNA polymerase, ethanol precipitated, centrifuged and resuspended in 10 ul of 10 mM
TEAA buffer pH 7.2. 10 ul of a 2 mM solution of the Fmoc-5-aminolevulinyl-NHS ester in 10 mM TEAA buffer is added, vortexed and incubated at 25C for 30 min. The excess of the reagent is removed by ethanol precipitation and centrifugation. The Fmoc group is cleaved offby resuspending the pellets in 10 ul of a solution of 20% piperidine in N,N-dimethylformamide/water (1:1 v/v). After 15 min at 25C, piperidine is thoroughly 2s removed by three piecipilalions/centrifugations with 100 ul each of ethanol, the pellets are resuspended in 10 ul of a solution of N-methylmorpholine, 2-propanol and water (2/10/88 vlvlv) and are coupled to the solid support carrying an isothiocyanate group. In the case of the DITC-Sequelon membrane (Millipore Corp., Bedford, MA), the membranes are prepared as described in EXAMPLE 1 and coupling is achieved on a heating block at 55C as described above. RNA extension products are immobilized in an analogous way.
The procedure can be applied to other solid supports with isothiocyanate groups in a similar manner.
The immobilized primer-extension products are extensively washed three times with 100 ul each of NMM solution and three times with 100 ul 10 mM TEAA buffer 3s pH 7.2. The purified primer-extension products are released by three successive treatments with 10 ul of 100 mM hyd~ acetate buffer pH 6.5, lyophilized and analyzed by either ES or MALDI mass spectrometry.

wo 94/1610~ S33~ 27- PCT/US94/00193 Immobilization of primer-extension products of Sanger DNA sequencing reaction for mass spectrometric analysis via a trS psin sensitive linkage Sequelon DITC membrane disks of 8 mm diameter (Millipore Corp., Bedford, MA) are wetted with 10 ul of NMM solution (N-methylmorpholine/propanaol-2/water; 2149149 vlvlv) and a linker arm introduced by reaction with 10 ul of a 10 mM
solution of 1,6-~i~minnhexane in NMM. The excess (li~nnin~ is removed by three washing steps with 100 ul of NMM solution. Using standard peptide synthesis protocols, two L-lysine residues are attached by two successive con-lton~tions with N-Fmoc-N-tBoc-L-lysine pentafluorophenylester, the tçnnin~l Fmoc group is removed with piperidine in NMM and the free a-amino group coupled to 1,4-phenylene diisothiocyanate (DITC).Excess DITC is removed by three washing steps with 100 ul 2-propanol and the N-tBoc groups removed with trifluoroacetic acid according to standard peptide synthesisprocedures. The nucleic acid primer-extension products are prepared from oligonucleotides which carry a primary amino group at the 5'-terminus. The four Sanger DNA sequencing reaction mixtures (150 ul each in Eppendorftubes) are heated for 10 min at 70C to inactivate the DNA polymerase, ethanol precipitated, and the pellets resuspended in 10 ul of a solution of N-methylmorpholine, 2-propanol and water (2/10/88 v/vlv). This solution is transferred to the Lys-Lys-DITC membrane disks and coupled on a heating block set at 55C. After drying, 10 ul of NMM solution is added and the drying process repeated.
The immobilized primer-extension products are extensively washed three times with 100 ul each of NMM solution and three times with 100 ul each of 10 mM TEAA buffer pH 7.2. For mass spectrometric analysis, the bond between the primer-extension products and the solid support is cleaved by treatment with trypsin under standard conditions and the released products analyzed by either ES or MALDI mass spectrometry with trypsin serving as an internal mass standard.

Immobilization of primer-extension products of Sanger DNA sequencing reaction for mass spectrometric analysis via pyrophosphate linkage The DITC Sequelon membrane (disks of 8 mm diameter) are prepared as described in EXAMPLE 3 and 10 ul of a 10 mM solution of 3-aminopyridine ~cleninedinucleotide (APAD) (Sigma) in NMM solution added. The excess APAD is removed bya 10 ul wash of NMM solution and the disks are treated with 10 ul of 10 mM sodium periodate in NMM solution (15 min, 25C). Excess periodate is removed and the primer-extension products of the four Sanger DNA sequencing reactions ( 150 ul each in _ o 94/16101 -28- 215 3 3 ~ 7 PCT/US94/00193 Eppendorf tubes) employing nucleic acid primers with a primary amino group at the 5'-end are ethanol precipitated, dissolved in 10 ul of a solution of N-methylmorpholine/2-propanol/water (2/10/88 vlvlv) and coupled to the 2' 3'-dialdehydo groups of theimmobilized NAD analog.
The primer-extension products are extensively washed with the NMM
solution (3 times with 100 ul each) and 10 mM TEAA buffer pH 7.2 (3 times with 100 ul each) and the purified primer-extension products are released by tre~tm~nt with either NADase or pyrophosphatase in 10 mM TEAA buffer at pH 7.2 at 37C for 15 min, Iyophilized and analyzed by either ES or MALDI mass spectrometry, the enzymes serving o as internal mass standards.

EXAMPLE S

Synthesis of nucleic acid primers mass-modified by glycine residues at the 5'-position of the sugar moiety of the terminal nu~l~c~le Oligonucleotides are synth~si7ecl by standard automated DNA synthesis using 13-cyanoethylphosphoamidites (H. Koster et al., Nucleic Acids Res. 12, 4539 (1984)) and a 5'-amino group is introduced at the end of solid phase DNA synthesis (e.g. Agrawal et al., Nucleic Acids Res. 14, 6227-45 (1986) or Sproat et al., Nucleic Acids Res. 15, 6181-96 (1987)). The total amount of an oligonucleotide synthesis, starting with 0.25 umol CPG-bound nucleoside, is deprotected with concentrated aqueous ammonia, purified via OligoPAKTM Cartridges (Millipore Corp., Bedford, MA) and lyophilized. This material with a 5'-tçrmin~l amino group is dissolved in 100 ul absolute N,N-dimethylformarnide (DMF) and con-lçnsecl with 10 ~lmole N-Fmoc-glycine 2s pell~nuorophenyl ester for 60 min at 25C. After ethanol plecipil~tion and centrifugation, the Fmoc group is cleaved offby a 10 min treatment ~vith 100 ul of a solution of 20% piperidine in N,N-dimethylformamide. Excess piperidine, DMF and the cleavage product from the Fmoc group are removed by ethanol precipitation and the precipitate Iyophilized from 10 mM TEAA buffer pH 7.2. This material is now either used as primer for the Sanger DNA sequencing reactions or one or more glycine residues (or other suitable protected amino acid active esters) are added to create a series of mass-modified primer oligonucleotides suitable for Sanger DNA or RNA sequencing.
Immobilization of these mass-modified nucleic acid primers up-i after primer-extension during the sequencing process can be achieved as described, e.g., in EXAMPLES 1 to 4.
3s wO 94/16101 2 ~- ~ 3 3 ~ ~ -29- PCT/US94/00193 Synthesis of nucleic acid primers mass-modified at C-5 of the heterocyclic base of a pyrimidine nucleoside with glycine residues S Starting material was 5-(3-aminopropynyl-1)-3' 5'-di-p-tolyldeoxyuridine prepared and 3' S'-de-O-acylated according to li~eld~ule procedures (Haralambidis et al., Nucleic Acids Res. 15, 4857-76 (1987)). 0.281 g (1.0 mmole) 5-(3-aminopropyllyl-1)-2'-deoxyuridine were reacted with 0.927 g (2.0 mmole) N-Fmoc-glycine pentafluorophenylester in 5 ml absolute N,N-dimethylformamide in the presence of 0.129 o g (1 mmole; 174 ul) N,N-diisopropylethylamine for 60 min at room temperature. Solvents were removed by rotary evaporation and the product was purified by silica gel chromatography (Kieselgel 60, Merck; column: 2.5x 50 cm, elution with chloroform/methanol mixtures). Yield was 0.44 g (0.78 mmole, 78 %). In order to add another glycine residue, the Fmoc group is removed with a 20 min treatment with 20%
solution of piperidine in DMF, evaporated in vacuo and the rem~ining solid material extracted three times with 20 ml ethyl~cet~te. After having removed the rem~inin~
ethylacetate, N-Fmoc-glycine pentafluo-uphenylester is coupled as described above. 5-(3-(N-Fmoc-glycyl)-amidopropynyl-1)-2'-deoxyuridine is transformed into the S'-O-dimethoxytritylated nucleoside-3'-O-13-cyanoethyl-N,N-diisopropylphosphoamidite and incorporated into automated oligonucleotide synthesis by standard procedures (H. Koster et al., Nucleic Acids Res. 12, 2261 (1984)). This glycine modified thymidine analogue building block for chemical DNA synthesis can be used to substitute one or more of the thymidine/uridine nucleotides in the nucleic acid primer sequence. The Fmoc group is removed at the end of the solid phase synthesis with a 20 min treatment with a 20 %
2s solution of piperidine in DMF at room te~llpe,d~ . DMF is removed by a washing step with acetonitrile and the oligonucleotide deprotected and purified in the standard way.

Synthesis of a nucleic acid primer mass-modified at C-5 of the hct~. uc~clic base of a pyrimidine nucleoside with 13-alanine residues Starting material was the same as in EXAMPLE 6. 0.281 g (1.0 mmole) 5-(3-Aminopropynyl-1)-2'-deoxyuridine was reacted with N-Fmoc-~3-alanine pentafluorophenylester (0.955 g, 2.0 mmole) in 5 ml N,N-dimethylformamide (DMF) in 3s the presence of 0.129 g (174 ul; 1.0 mmole) N,N-disopropylethylamine for 60 min at room telll~eld~llre. Solvents were removed and the product purified by silica gel chromatography as described in EXAMPLE 6. Yield was 0.425 g (0.74 mmole, 74 %).
Another J3-alanine moiety can be added in exactly the same way after removal of the Fmoc group. The preparation of the 5'-O-dimethoxytritylated nucleoside-3'-0-13-cyanoethyl-o 94/16101 ^ 3~ 5 3 3 ~ ~ PCT/US94/00193 N,N-diisopropylphosphoamidite from 5-(3-(N-Fmoc-J3-alanyl)-amidopropynyl-1)-2'-deoxyuridine and incorporation into automated oligonucleotide synthesis is performed under standard conditions. This building block can substitute for any of the thymidine/uridine residues in the nucleic acid primer sequence. In the case of only one 5 incorporated mass-modified nucleotide, the nucleic acid primer molecules prepared according to EXAMPLES 6 and 7 would have a mass difference of 14 daltons.

FX~MPI,F 8 0 Sy~ .is of a nucleic acid primer mass-modified at C-5 of the ;'qCt~l u~,~ cliC base of a pyrimidine nucleQ3i ~ with ethylene glycol monûmethyl ether As a nucleosidic component, 5-(3-aminopropynyl-1)-2'-deoxyuridine was used in this example (see EXAMPLES 6 and 7). The mass-modifying functionality was obtained as follows: 7.61 g (100.0 mmole) freshly distilled ethylene glycol monomethyl ether dissolved in 50 ml absolute pyridine was reacted with lO.01 g (100.0 mmole) recrystAlli7P~l succinic anhydride in the presence of 1.22 g (10.0 mmole) 4-N,N-dimethylaminopyridine overnight at room t~ dLule. The reaction was terrninAted by the addition of water (5.0 ml), the reaction mixture evdpoldledinvacuo~ co-evaporated twice with dry toluene (20 ml each) and the residue redissolved in 100 ml dichloromethane. The solution was extracted successively, twice with 10 % aqueous citric acid (2 x 20 ml) and once with water (20 ml) and the organic phase dried over anhydrous sodium sulfate. The organic phase was evaporated invacuo, the residue redissolved in 50 ml dichloromethane and precipitated into 500 ml pentane and the precipitate dried in vacuo. Yield was 13.12 g (74.0 mmole; 74 %). 8.86 g (50.0 mmole) of succinylated2s ethylene glycol monomethyl ether was dissolved in 100 ml dioxane Co~ g 5% dry pyridine (5 ml) and 6.96 g (50.0 mmole) 4-nitrophenol and 10.32 g (50.0 mmole) dicyclohexylcarbodiimide was added and the reaction run at room te~ c for 4 hours.
Dicyclohexylurea was removed by filtration, the filtrate e~l~oldledin vacuo and the residue redissolved in 50 ml anhydrous DMF. 12.5 ml (about 12.5 mmole 4-nitrophenylester) ofthis solution was used to dissolve 2.81 g (10.0 mmole) 5-(3-aminopropynyl-1)-2'-deoxyuridine. The reaction was performed in the presence of 1.01 g (10.0 mmole; 1.4 ml) triethylamine at room telllp~ldlule overnight. The reaction lllixlule was evaporated in vacuo, co-evaporated with toluene, redissolved in dichloromethane and chromatographed on silicagel (Si60, Merck; column 4x50 cm) with dichloromethane/methanol mixtures. The fractions colltah~ing the desired compound were collected, evaporated, redissolved in 25 ml dichloromethane and precipitated into 250 ml pentane. The dried ~,lccipi~dte of 5-(3-N-(O-succinyl ethylene glycol monomethyl ether)-amidopropynyl-1)-2'-deoxyuridine (yield: 65 %) is 5'-O-dimethoxytritylated and Lldllsro~ ed into the nucleoside-3'-0-13-cyanoethyl-N, N-diisopropylphosphoamidite and wo 94116101 21~ 3 3 ~ 31- PCT/US94/00193 incorporated as a building block in the automated oligonucleotide synthesis according to standard procedures. The mass-modified nucleotide can substitute for one or more of the thymidine/uridine residues in the nucleic acid primer sequence. Deprotection andpurification of the primer oligonucleotide also follows standard procedures.
s FXAMPT,F 9 Synthesis of a nucleic acid primer mass-modified at C-5 of the hete. uc~ clic base of a pyrimidine nucleoside with diethylene glycol monomethyl ether lo Nucleosidic starting m~tçri~l was as in previous examples, 5-(3-aminopropynyl-1)-2'-deoxyuridine. The mass-modifying functionality was obtained similar to EXAMPLE 8. 12.02 g (100.0 mmole) freshly distilled diethylene glycol monomethyl ether dissolved in 50 ml absolute pyridine was reacted with 10.01 g (100.0 mmole) recryst~lli7~d succinic anhydride in the presence of 1.22 g (10.0 mmole) 4-N, N-1S dimethylaminopyridine (DMAP) overnight at room temperature. The work-up was as described in EXAMPLE 8. Yield was 18.35 g (82.3 mmole, 82.3 %). 11.06 g (50.0 mmole) of succinylated diethylene glycol monomethyl ether was transformed into the 4-nitrophenylester and, subsequently, 12.5 mmole was reacted with 2.81 g (10.0 mmole) of 5-(3-aminopropynyl-1)-2'-deoxyuridine as described in EXAMPLE 8. Yield after silica gel column chromatography and precipitation into pentane was 3.34 g (6.9 mmole, 69 %).
After dimethoxytritylation and transformation into the nucleoside-~3-cyanoethylphosphoamidite, the mass-modified building block is incorporated into automated chemical DNA synthesis according to standard procedures. Within the sequence of the nucleic acid primer UP0-i, one or more of the thymidine/uridine residues can be substituted by this mass-modified nucleotide. In the case of only one incorporated mass-modified nucleotide, the nucleic acid primers of EXAMPLES 8 and 9 would have a mass difference of 44.05 daltons.

Synthesis of a nucleic acid primer mass-modified at C-8 of the ~ el~;, oc~clic base of deoxyadenosine with glycine Starting material was N6-benzoyl-8-bromo-5'-0-(4,4'-dimethoxytrityl)-2'-deoxyadenosine prepared according to li~e~ c (Singh et al., Nucleic Acids Res. 18, 3s 3339-45 (1990)). 632.5 mg (1.0 mmole) ofthis 8-bromo-deoxyadenosine derivative was suspended in 5 ml absolute ethanol and reacted with 251.2 mg (2.0 mmole) glycine methyl ester (hydrochloride) in the presence of 241.4 mg (2.1 mmole; 366 ul) N, N-diisopropylethylamine and refluxed until the starting nucleosidic material had disappeared (4-6 hours) as checked by thin layer chromatography (TLC). The solvent was evaporated ~,~o 94/16101 21 5 3;~ 8 7 PCT/US94/00193 and the residue purified by silica gel chromatography (column 2.5x50 cm) using solvent mixtures of chloroform/methanol co~ -g 0.1 % pyridine. The product fractions were combined, the solvent evaporated, the fractions dissolved in 5 ml dichloromethane and precipitated into 100 ml pentane. Yield was 487 mg (0.76 mmole, 76 %). Transformation 5 into the corresponding nucleoside-~3-cyanoethylphosphoamidite and integration into automated chemical DNA synthesis is performed under standard conditions. During final deprotection with aqueous concentrated ammonia, the methyl group is removed from the glycine moiety. The mass-modified building block can substitute one or more deoxyadenosine/adenosine residues in the nucleic acid primer sequence.

Synthesis of a nucleic acid primer mass-modified at C-8 of the heterocyclic base of deoxyadenosine with glycylglycine This derivative was prepared in analogy to the glycine derivative of EXAMPLE 10. 632.5 mg (1.0 mmole) N6-Benzoyl-8-bromo-5'-0-(4,4'-dimethoxytrityl)-2'-deoxyadenosine was suspended in 5 ml absolute ethanol and reacted with 324.3 mg (2.0 mmole) glycyl-glycine methyl ester in the presence of 241.4 mg (2.1 mmole, 366 ~N, N-diisopropylethylamine. The mixture was refluxed and completeness of the reaction checked by TLC. Work-up and purification was similar to that described in EXAMPLE
10. Yield after silica gel column chromatography and pleci~iLalion into pentane was 464 mg (0.65 mmole, 65 %). Transformation into the nucleoside-13-cyanoethylphosphoamidite and into synthetic oligonucleotides is done according to standard procedures. In the case where only one of the deoxyadenosine/adenosine residues in the nucleic acid primer is substituted by this mass-modified nucleotide, the mass difference between the nucleic acid primers of EXAMPLES 10 and 11 is 57.03 daltons.

Synthesis of a nucleic acid primer mass-modified at the C-2' of the sugar moiety of 2'-amino-2'-deoxythymidine with ethylene glycol monomethyl ether residues Starting m~teri~l was 5'-0-(4,4-dimethoxytrityl)-2'-amino-2'-deoxythymidine synthesi7ed according to published procedures (e.g., Verheyden et al., J. Or~. Chem. 36, 250-254 (1971); Sasaki etal., J. Or~. Chem. 41, 3138-3143 (1976); Ima_awaetal.~ J. Or~.
3s Chem. 44, 2039-2041 (1979); Hobbs et al., J. Or~. Chem. 42, 714-719 (1976); Ikehara et al., Chem Ph~rm. Rull. Japan 26, 240-244 (1978); see also PCT Application WO
88/00201). 5'-0-(4,4-Dimethoxytrityl)-2'-amino-2'-deoxylhylllidine (559.62 mg; 1.0 mmole) was reacted with 2.0 mmole of the 4-nitrophenyl ester of succinylated ethylene glycol monomethyl ether (see EXAMPLE 8) in 10 ml dry DMF in the presence of 1.0 wo 94/16101 ~ ~33~t PCT/US94/00193 mmole (140 ~l) triethylamine for 18 hours at room temperature. The reaction mixture was evaporated in vacuo, co-evaporated with toluene, redissolved in dichloromethane and purified by silica gel chromatography (Si60, Merck; column: 2.5x50 cm; eluent:
chloroform/methanol mixtures col-t~it-ing 0.1 % triethylamine). The pro~uct cont~inin~
fractions were combined, evaporated and pl~cipi~ted into pentane. Yield was 524 mg (0.73 mmol; 73 %). Transformation into the nucleoside-13-cyanoethyl-N,N-diisopropylphosphoamidite and incorporation into the automated chemical DNA synthesis protocol is performed by standard procedures. The mass-modified deoxythymidine derivative can substitute for one or more of the thymidine residues in the nucleic acid o primer.
In an analogous way, by employing the 4-nitrophenyl ester of succinylated diethylene glycol monomethyl ether (see EXAMPLE 9) and triethylene glycol monomethyl ether, the corresponding mass-modified oligonucleotides are prepared. In the case of only one incorporated mass-modified nucleoside within the sequence, the mass difference between the ethylene, diethylene and triethylene glycol derivatives is 44.05, 88.1 and 132.15 daltons respectively.

Sy~.lhlsii, of a nucleic acid primer mass-modified in the internucleotidic linkage via alkylation of phosphorothioate groups Phosphorothioate-co.lt;~ g oligonucleotides were prepared according to standard procedures (see e.g. Gait et al., Nucleic Acids Res., 19 1183 (1991)). One, several or all internucleotide linkages can be modified in this way. The (-)-M13 nucleic acid primer sequence (17-mer) S'-dGTAAAACGACGGCCAGT was synth~si7.ecl in 0.25 ~lmole scale on a DNA synth.oci7~r and one phosphorothioate group introduced after the final synthesis cycle (G to T coupling). Sulfurization, deprotection and purification followed standard protocols. Yield was 31.4 nmole (12.6 % overall yield), corresponding to 31.4 nmole phosphorothioate groups. Alkylation was performed by dissolving the residue in 31.4 111 TE buffer (0.01 M Tris pH 8.0, 0.001 M EDTA) and by adding 16 ~l of a solution of 20 rnM solution of 2-iodoethanol (320 nmole; i.e., 10-fold excess with respect to phosphorothioate diesters) in N,N-dimethylformamide (DMF). The alkylated oligonucleotide was purified by standard reversed phase HPLC (RP- 18 Ultraphere,Be~m~n; column: 4.5 x 250 rnm; 100 mM triethylammonium acetate, pH 7.0 and a gradient of 5 to 40 % acetonitrile).
In a variation of this procedure, the nucleic acid primer co.ltS~ g one or more phosphorothioate phosphodiester bond is used in the Sanger sequencing reactions.
The primer-extension products of the four sequencing reactions are purified as exemplified in EXAMPLES 1 - 4, cleaved offthe solid support, lyophilized and dissolved in 4 ~Ll each ~_,o 94/16101 ~34~1 a ~3~7 PCT/US94/00193 of TE buffer pH 8.0 and alkylated by addition of 2 ~ll of a 20 mM solution of 2-iodoethanol in DMF. It is then analyzed by ES and/or MALDI mass spectrometry.
In an analogous way, employing instead of 2-iodoethanol, e.g., 3-iodoplopallol, 4-iodobutanol mass-modified nucleic acid primer are obtained with a mass s difference of 14.03, 28.06 and 42.03 daltons respectively compared to the unmodified phosphorothioate phosphodiester-co~ i,-g oligonucleotide.

EXAMPT,F, 14 0 Synthesis of 2'-amino-2'-deoxyuridine-5'-triphosphate and 3'-amino-2',3'-dideo~ylhy~idine-5'-triphosphate mass-modified at the 2'- or 3'-amino function with glycine or 13-alanine residues Starting material was 2'-azido-2'-deoxyuridine prepared according to literature (Verheyden et al.. J. Or~. Chem. 36, 250 (1971)), which was 4,4-dimethoxytritylated at 5'-OH with 4,4-dimethoxytrityl chloride in pyridine and acetylated at 3'-OH with acetic anhydride in a one-pot reaction using standard reaction conditions.
With 191 mg (0.71 mmole) 2'-azido-2'-deoxyuridine as starting material, 396 mg (0.65 mmol, 90.8 %) 5'-0-(4,4-dimethoxytrityl)-3'-O-acetyl-2'-azido-2'-deoxuridine wasobtained after purification via silica gel chromatography. Reduction of the azido group was performed using published conditions (Barta et al., Tetrahedron 46. 587-594 (1990)).
Yield of 5'-0-(4,4-dimethoxytrityl)-3'-O-acetyl-2'-amino-2'-deoxyuridine after silica gel chromatography was 288 mg (0.49 mmole;
76 %). This protected 2'-amino-2'-deoxyuridine derivative (588 mg, 1.0 mmole) was reacted with 2 equivalents (927 mg, 2.0 mmole) N-Fmoc-glycine pentafluorophenyl ester 2s in 10 ml dry DMF overnight at room te,llpelalul~ in the presence of 1.0 mmole (174 ~l) N,N-diisopropylethylamine. Solvents were removed by evaporation in vacuo and theresidue purified by silica gel chromatography. Yield was 711 mg (0.71 mmole, 82 %).
Detritylation was achieved by a one hour treatment with 80% aqueous acetic acid at room telll~ lure~ The residue was evaporated to dryness, co-evaporated twice with toluene, suspended in 1 ml dry acetonitrile and 5'-phosphorylated with POCl3 according toliterature (Yoshikawa et al., Bull. Chem. Soc. Japan 42, 3505 (1969) and Sowa et al., Bull. Chem. Soc. Japan 48, 2084 (1975)) and directly transformed in a one-pot reaction to the 5'-triphosphate using 3 ml of a 0.5 M solution (1.5 mmole) tetra (tri-n-butylammonium) pyrophosphate in DMF according to lilel~lule (e.g. Seela et al., 3s Helvetica Chimica Acta 74, 1048 (1991)). The Fmoc and the 3'-O-acetyl groups were removed by a one-hour tre~tment with concentrated aqueous ammonia at room telllpel~lule and the reaction mixture evaporated and lyophili7~1 Purification also followed standard procedures by using anion-exchange chromatography on DEAE-Sephadex with a linear gradient of triethylammonium bicarbonate (0.1 M - 1.0 M).

wo 94/16101 2,~ ~33~1 PCT/US94/00193 Triphosphate cont~ining fractions (checked by thin layer chromatography on polyethyleneimine cellulose plates) were collected, evaporated and lyophilized. Yield (by UV-absorbance of the uracil moiety) was 68% (0.48 mmole).
A glycyl-glycine modified 2'-amino-2'-deoxyuridine-5'-triphosphate was s obtained by removing the Fmoc group from 5'-0-(4,4-dimethoxytrityl)-3'-O-acetyl-2'-N-(N-9-fluorenylmethyloxycarbonyl-glycyl)-2'-amino-2'-deoxyuridine by a one-hour treatment with a 20% solution of piperidine in DMF at room temperature, evaporation of solvents, two-fold co-evaporation with toluene and subsequent con-len~tion with N-Fmoc-glycine pentafluorophenyl ester. Starting with 1.0 mmole of the 2'-N-glycyl-2'-lo amino-2'-deoxyuridine derivative and following the procedure described above, 0.72 mmole (72%) of the corresponding 2'-~N-glycyl-glycyl)-2'-amino-2'-deoxyuridine-5'-triphosphate was obtained.
Starting with 5'-0-(4,4-dimethoxytrityl)-3'-O-acetyl-2'-amino-2'-deoxyuridine and coupling with N-Fmoc-~3-alanine pentafluorophenyl ester, the corresponding 2'-(N-~3-alanyl)-2'-amino-2'-deoxyuridine-5'-triphosphate can be synthesized. These modified nucleoside triphosphates are incorporated during the Sanger DNA sequencing process in the primer-extension products. The mass difference between the glycine, ~3-alanine and glycyl-glycine mass-modified nucleosides is, per nucleotide incorporated, 58.06, 72.09 and 115.1 daltons respectively.
When starting with 5'-0-(4,4-dimethoxytrityl)-3'-amino-2',3'-dideoxythymidine (obtained by published procedures, see EXAMPLE 12), the corresponding 3'-(N-glycyl)-3'-amino-/ 3'-(-N-glycyl-glycyl)-3'-amino-/ and 3'-(N-J3-alanyl)-3'-amino-2',3'-dideoxythymidine-5'-triphosphates can be obtained. These mass-modified nucleoside triphosphates serve as a termin~ting nucleotide unit in the Sanger DNA sequencing reactions providing a mass difference per tennin~te~l fragment of 58.06, 72.09 and 115.1 daltons respectively when used in the multiplexing sequencing mode.
The mass-differenti~te~ fragments can then be analyzed by ES and/or MALDI mass spectrometry.

EX~MPLE 15 Synthesis of deoxyuridine-5'-triphosphate mass-modified at C-5 of the heterocyclic base with glycine, glycyl-glycine and ~-alanine residues.
0.281 g (1.0 mmole) 5-(3-Aminopropynyl-1)-2'-deoxyuridine (see EXAMPLE 6) was reacted with either 0.927 g (2.0 mmole) N-Fmoc-glycine pentafluorophenylester or 0.955g (2.0 mmole) N-Fmoc-J3-alanine pentafluorophenyl ester in 5 ml dry DMF in the presence of 0.129 g N, N-diisopropylethylamine (174 ul, 1.0 mmole) overnight at room temperature. Solvents were removed by evaporation in vacuo and the con~lçn~tion products purified by flash chromatography on silica gel (Still et al., _~ o 94/16101 2 1 5 3 3 8 7 PCT/US94/00193 J. Org. Chem. 43, 2923-2925 (1978)). Yields were 476 mg (0.85 mmole: 85%) for the glycine and 436 mg (0.76 mmole; 76%) for the ~3-alanine derivatives. For the synthesis of the glycyl-glycine derivative, the Fmoc group of 1.0 mmole Fmoc-glycine-deoxyuridine derivative was removed by one-hour treatment with 20% piperidine in DMF at room 5 temperature. Solvents were removed by evaporation in vacuo, the residue was co-evaporated twice with toluene and con~çn~ed with 0.927 g (2.0 mmole) N-Fmoc-glycine pentafluorophenyl ester and purified as described above. Yield was 445 mg (0.72 mmole;
72%). The glycyl-, glycyl-glycyl- and B-alanyl-2'-deoxyuridine derivatives, N-protected with the Fmoc group were transformed to the 3'-O-acetyl derivatives by tritylation with 0 4,4-dimethoxytrityl chloride in pyridine and acetylation with acetic anhydride in pyridine in a one-pot reaction and subsequently detritylated by one hour treatment with 80%
aqueous acetic acid according to standard procedures. Solvents were removed, theresidues dissolved in 100 ml chloroform and extracted twice with 50 ml 10% sodium bicarbonate and once with 50 ml water, dried with sodium sulfate, the solvent evaporated and the residues purified by flash chromatography on silica gel. Yields were 361 mg (0.60 mmole; 71%3 for the glycyl-, 351 mg (0.57 mmole; 75%) for the 13-alanyl- and 323 mg (0.49 mmole; 68%) for the glycyl-glycyl-3-O'-acetyl-2'-deoxyuridine derivatives respectively. Phosphorylation at the 5'-OH with POC13, ~ rollllation into the 5'-triphosphate by in-situ reaction with tetra(tri-n-butylammonium) pyrophosphate in DMF, 3'-de-O-acetylation, cleavage of the Fmoc group, and final purification by anion-exchange chromatography on DEAE-Sephadex was ~t;l~l,l,ed as described in EXAMPLE 14.
Yields according to W-absorbance of the uracil moiety were 0.41 mmole 5-(3-(N-glycyl)-amidopropynyl-1)-2'-deoxyuridine-5'-triphosphate (84%), 0.43 mmole 5-(3-(N-13-alanyl)-amidopropynyl-1)-2'-deoxyuridine-5'-triphosphate (75%) and 0.38 mmole 5-(3-(N-~5 glycyl-glycyl)-amidopropyllyl- 1)-2'-deoxyuridine-5'-triphosphate (78%).
These mass-modified nucleoside triphosphates were incorporated during the - Sanger DNA sequencing primer-extension reactions.
When using 5-(3-aminopropynyl-1)-2',3'-dideoxyuridine as starting material and following an analogous reaction sequence the corresponding glycyl-, glycyl-glycyl-and 13-alanyl-2',3'-dideoxyuridine-5'-triphosphates were obtained in yields of 69, 63 and 71% l~e~ ely. These mass-modified nucleoside triphosphates serve as chain-tertnin~ting nucleotides during the Sanger DNA sequencing reactions. The mass-modified sequencing ladders are analyzed by either ES or MALDI mass spectrometry.

3s EXAMP~,F 16 Synthesis of 8-glycyl- and 8-glycyl-glycyl-2'-deoxyadenosine-5'-triphosphate 727 mg (1.0 mmole) of N6-(4-tert-butylphenoxyacetyl)-8-glycyl-5'-(4,4-dimethoxytrityl)-2'- deoxyadenosine or 800 mg (1.0 mmole) N6-(4-tert-wo 94/16101 2 ~j 33~ PCT/US94/00193 butylphenoxyacetyl)-8-glycyl-glycyl-5'-(4,4-dimethoxytrityl)-2'-deoxyadenosine prepared according to EXAMPLES 10 and 11 and literature (Koster et al., Tetr~hedron 37, 362 (1981)) were acetylated with acetic anhydride in pyridine at the 3'-OH, detritylated at the 5'-position with 80% acetic acid in a one-pot reaction and transformed into the 5'-triphosphates via phosphorylation with POC13 and reaction in-situ with tetra(tri-n-butylammonium) pyrophosphate as described in EXAMPLE 14. Deprotection of the N6-tert-butylphenoxyacetyl, the 3'-O-acetyl and the O-methyl group at the glycine residues was achieved with concentrated aqueous ammonia for ninety minutes at room telllp~l~Lu~e. Ammonia was removed by lyophilization and the residue washed with0 dichloromethane, solvent removed by evaporation in vacuo and the rem~ining solid m~t~ri~l purified by anion-exchange chromatography on DEAE-Sephadex using a linear gradient oftriethylammonium bicarbonate from 0.1 to 1.0 M. The nucleoside triphosphate co~ g fractions (checked by TLC on polyethyleneimine cellulose plates) were combined and lyophilli7~tl Yield of the 8-glycyl-2'-deoxyadenosine-5'-triphosphate (~letermined by W-absorbance of the adenine moiety) was 57% (0.57 mmole). The yield for the 8-glycyl-glycyl-2'-deoxyadenosine-5'-triphosphate was 51% (0.51 mmole).
These mass-modified nucleoside triphosphates were incorporated during primer-extension in the Sanger DNA sequencing reactions.
When using the corresponding N6-(4-tert-butylphenoxyacetyl)-8-glycyl- or -glycyl-glycyl-5'-0-(4,4-dimethoxytrityl)-2',3'-dideoxyadenosine derivatives as starting materials prepared according to standard procedures (see, e.g., for the introduction of the 2',3'-function: Seela et al., Helvetica Chimica Acta 74, 1048-1058 (1991)) and using an analogous reaction sequence as described above, the chain-tçrrnin~tinp; mass-modified nucleoside triphosphates 8-glycyl- and 8-glycyl-glycyl-2'.3'-dideoxyadenosine-5'-triphosphates were obtained in 53 and 47% yields respectively. The mass-modifiedsequencing fragment ladders are analyzed by either ES or MALDI mass spectrometry.

F.XAMPl,F 17 Mass-modification of Sanger DNA sequencing fragment ladders by incorporation of chain-elongating 2'-deoxy- and chain-terminating 2',3'-dideoxythymidine-5'-(alpha-S-)-triphosphate and subsequent alkylation with 2-iodoethanol and 3-iodopropanol2',3'-Dideoxythymidine-5'-(alpha-S)-triphosphate was prepared according to published procedures (e.g., for the alpha-S-triphosphate moiety: Eckstein et al., Biochenli~try 15, 1685 (1976) and Accounts Chem. Res. 12, 204 (1978) and for the 2',3'-dideoxy moiety: Seela et al., Helvetica Chimica Acta~ 1~, 1048- 1058 (1991)). Sanger DNA sequencing reactions employing 2'-deoxythymidine-5'-(alpha-S)-triphosphate are performed according to standard protocols (e.g. Eckstein, Ann. Rev. Biochem. 54, 367 (1985)). When using 2',3'-dideoxythymidine-5'-(alpha-S)-triphosphates, this is used _ ,VO 94/16101 21~ 3 3 8 7 PCTrUS94100193 instead of the unmodified 2',3'-dideoxythymidine-5'-triphosphate in standard Sanger DNA
sequencing (see e.g. Swerdlow et al., Nucleic Acids Res. 18, 1415-1419 (1990)). The template (2 pmole) and the nucleic acid M13 sequencing primer (4 pmole) modifiedaccording to EX~PLE 1 are annealed by heating to 65C in 100 ul of 10 mM Tris-HCI
s pH 7.5, 10 mM MgCl2, 50 mM NaCl, 7 mM dithiothreitol (DTT) for 5 min and slowly brought to 37C during a one hour period. The sequencing reaction mixtures contain, as exemplified for the T-specific termination reaction, in a final volume of 150 ul, 200 uM
(final concentration) each of dATP, dCTP, dTTP, 300 uM c7-deaza-dGTP, 5 uM 2',3'-dideoxythymidine-5'-(alpha-S)-triphosphate and 40 units Sequenase (United StatesBiochemicals). Polymerization is performed for 10 min at 37C, the reaction ~ L[e heated to 70C to inactivate the Sequenase, ethanol precipitated and coupled to thiolated Sequelon membrane disks (8 mm diarneter) as described in EXAMPLE 1. Alkylation is performed by treating the disks with 10 ul of 10 mM solution of either 2-iodoethanol or 3-iodoplopanol in NMM (N-methylmorpholine/water/2-propanol, 2149149, vlvlv) (threetimes), washing with 10 ul NMM (three times) and cleaving the alkylated T-tennin~te~l primer-extension products offthe support by tre~tment with DTT as described in EXAMPLE 1. Analysis of the mass-modified fragment families is performed with either ES or MALDI mass spectrometry.

EXAl~IPT,F 18 Analysis of a Mixture of Oligothymidylic Acids Oligothymidylic acid, oligo p(dT)12 18, is commercially available (United States Biochemical, Cleveland, OH). Generally, a matrix solution of 0.5 M in ethanol was 2s prepared. Various matrices were used for this Example and Examples 19- 21 such as 3,5-dihydroxybenzoic acid, sinapinic acid, 3-hydroxypicolinic acid, 2,4,6-trihydroxyacetophenone. Oligonucleotides were lyophilized after purification by HPLC and takerl up in ul~ e water (MilliQ, Millipore) using amounts to obtain a concentration of 10 pmoles/~1 as stock solution. An aliquot (1 ~1) of this concentration or a dilution in ultrapure water was mixed with 1 ~11 of the matrix solution on a flat metal surface serving as the probe tip and dried with a fan using cold air. In some ~c~ lents, cation-ion exchange beads in the acid form were added to the mixture of matrix and sample solution.
MALDI-TOF spectra were obtained for this Exarnple and Examples 19-21 on different commercial instrurnents such as Vision 2000 (Finnigan-MAT), VG TofSpec (Fisons 3s Instruments), LaserTec Research (Vestec). The conditions for this Example were linear negative ion mode with an acceleration voltage of 25 kV. The MALDI-TOF spectrum generated is shown in FIGURE 14. Mass calibration was done externally and generally achieved by using defined peptides of ap~lopl;ate mass range such as insulin, gramicidin S, trypsinogen, bovine serum albumen, and cytochrome C. All spectra were generated by WO 94/16101 2 IS 3 ~ 8 7 PCT/US94/00193 employin~ a nitro~en laser with 5 nsec pulses at a wavelength of 337 nm. Laser energy varied between l o6 and 107 W/cm2. To improve signal-to-noise ratio generally, the intensities of 10 to 30 laser shots were accumulated.

Mass Spectrometric Analysis of a 50-mer and a 99-mer Two lar~e oligonucleotides were analyzed by mass spectrometry. The 50-mer d (TAACGGTCATTACGGCCATTGACTGTAGGACCTGCATTACATGACTAGCT) (SEQ
10 ID NO:3) and dT(pdT)gg were used. The oligodeoxynucleotides were synthesi7~cl using -cyanoethylphosphoamidites and purified using published procedures.(e.g. N.D. Sinha, J.
Biernat, J. McManus and H. Koster, Nucleic Acids Res.. 12, 4539 (1984)) employing commercially available DNA synthesizers from either Millipore (Bedford, MA) or Applied Biosystems (Foster City, CA) and HPLC equipment and RP18 reverse phase columns from Waters (Milford, MA). The samples for mass spectrometric analysis were prepared as described in Example 18. The conditions used for MALDI-MS analysis of each oligonucleotide were 500 fmol of each oligonucleotide~ reflectron positive ion mode with an acceleration of 5 kV and postacceleration of 20 kV. The MALDI-TOF spectra generated were superimposed and are shown in FIGURE 15.

Simulation of the DNA Sequencing Results of FIGURE 2 The 13 DNA sequences representing the nested dT-termin~te~l fragments of the Sanger DNA sequencing for the 50-mer described in Example 19 (SEQ ID NO:3) were synthesized as described in Example 19. The samples were treated and 500 fmol of each fragment was analyzed by MALDI-MS as described in Example 18.. The resulting MALDI-TOF spectra are shown in FIGURES 16A- 16M. The conditions were reflectron positive ion mode with an acceleration of 5 kV and postacceleration of 20 kV. Calculated molecular masses 30 and experimental molecular masses are shown in Table 1.
The MALDI-TOF spectra were superimposed (FIGURES 17A and 17B) to demonstrate that the individual peaks are resolvable even between the 10-mer and 11 -mer (upper panel) and the 37-mer and 38-mer (lower panel). The two panels show two different scales and the spectra analyzed at that scale.

RECTIFIED SHEET (RULE 91) ISA/EP

_ ~o 94/16101 40 2 ~ 5 3 3 ~ 7 PCT/US94100193 MALDI-MS Analysis of a Mass-Modified Oligonucleotide A 17-mer was mass-modified at C-5 of one or two deoxyuridine moieties. 5-[13-s (2-Methoxyethoxyl)-tridecyne-1-yl]-5'-0-(4,4'-dimethoxytrityl)-2'-deoxyuridine-3'-~-cyanoethyl-N, N-diisopropylphosphoamidite was used to synthesi7~ the modified 1 7-mers using the methods described in Example 19.
The modified 1 7-mers were a: d (TAAAACGACGGCCAGUG) (molecular mass: 5454) (SEQ ID NO:4) ~ Xl b: d (UAAAACGACGGCCAGUG) (molecular mass 5634) (SEQ ID NO:5) where X = -C-C-(CH2)1 l-OH
(unmodifiedl7-mer: molecularmass: 5273) The samples were prepared and 500 fmol of each modified 1 7-mer was analyzed using MALDI-MS as described in Example 18. The conditions used were reflectron positive ion mode with an acceleration of 5 kV and postacceleration of 20 kV.
The MALDI-TOF spectra which were generated were superimposed and are shown in FIGURE 18.

All of the above-cited references and publications are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

WO 94/16101 g,~ -41- PCT/US94/00193 SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLIC~NT:
(A) NAME: KOSTER, HUBERT
(B) STREET: 1640 MONU.MENT STREET
(C) CITY: CONCORD
(D) STATE: MASSA~U~ S
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(F) POSTAL CODE (ZIP): 01742 (G) TELEPHONE: (508) 369-9790 (ii) TITLE OF lNv~NlloN: DNA SEQU~N-~lNG BY MASS SPECTROMETRY
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(A) APPLICATION NUMBER: US 08/001,323 (B) FILING DATE: 07-JAN-1993 (C) CLASSIFICATION: 1807 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: DeConti, Giulio A.
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_'O 94/16101 ~ ~ 5 ~ ~ 8 7 PCTrUS94/00193 (2) INFORMATION FOR SEQ ID NO:2:
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(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) HYPOTHETICAL: YES

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(2) INFORMATION FOR SEQ ID NO:3:
(i) S~QU~N~ CHARACTERISTICS:
(A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULE TYPE: other nucleic acid (iii) HYPOTHETICAL: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) HYPOTHETICAL: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
TA~AACGACG GGCCAGXG 17 WO 94/16101 2~33~ -43- PCT/US94/00193 (2) INFORMATION FOR SEQ ID NO:5 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid ~iii) HYPOTHETICAL: YES

(xi) S~Qu~N~ DESCRIPTION: SEQ ID NO:5:
XAA~ACGACG GGCCAGXG 17

Claims (73)

1. A method of sequencing a nucleic acid, comprising the steps of a) synthesizing complementary nucleic acids which are complementary to the nucleic acid to be sequenced, starting from a nucleic acid primer and in the presence of chain-terminating and chain-elongating nucleotides so as to produce four sets of base-specifically terminated complementary nucleic acid fragments;
b) determining the molecular weight value of each nested fragment in each of the four sets of base-specifically terminated fragments by mass spectrometry wherein the molecular weight values of at least two base-specifically terminated fragments are determined concurently; and c) determining the nucleotide sequence by aligning the four sets of molecular weight values according to molecular weight.

2. The method according to claim 1, wherein the four sets of base-specifically terminated fragments are purified before the step of determining the molecular weight values by mass spectrometry.

3. The method according to claim 2, wherein the four sets of base-specifically terminated fragments are purified, comprising the steps of a) immobilizing the complementary nucleic acids on a solid support, and b) washing out all remaining reactants and by-products.

4. The method according to claim 3, further comprising the step of removing the complementary nucleic acids from the solid support.

5. The method according to claim 1, wherein a counter-ion of the phosphate backbone of the complementary nucleic acids is removed or is exchanged with a second counter-ion, the second counter-ion allowing a step of determining the molecular weight values by mass spectrometry.

6. The method according to claim 1, wherein each of the four sets of base-specifically terminated fragments is synthesized in a separate reaction vessel.

7. The method according to claim 6, wherein a step of determining the nucleotidesequence further comprises interpolating the molecular weight values determined for each of the four sets of base-specifically terminated fragments.

8. The method according to claim 1, wherein at least two of the four sets of base-specifically terminated fragments are synthesized concurrently in the same reaction vessel.

9. The method according to claim 8, wherein the chain-terminating nucleotides are chosen such that addition of one species of the chain-terminating nucleotides to the complementary nucleic acid can be distinguished by mass spectrometry from addition of all other species of the chain-terminating nucleotides present in the same reaction vessel.

10. The method according to claim 1, wherein the molecular weight value of each nested fragment are determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).

11. The method according to claim 1 in which the molecular weight value of each nested fragment are determined by electrospray mass spectrometry (ES-MS).

12. The method according to claim 1, wherein the complementary nucleic acid is synthesized using a nucleic acid primer; at least one deoxynucleotide selected from the group consisting of deoxyadenosine triphosphate dATP, deoxythymidine triphosphate dTTP, deoxyguanosine triphosphate dGTP, deoxycytidine triphosphate dCTP, deoxyinosine triphosphate dTTP, a 7-deazadeoxynucleoside triphosphate c7dGTP, a 7-deazadeoxynucleoside triphosphate c7dATP, and a 7-deazadeoxynucleoside triphosphate c7dITP; at least one chain-terminating dideoxynucleotide selected from the groupconsisting of dideoxyadenosine triphosphate ddATP, dideoxythymidine triphosphateddTTP, dideoxyguanosine triphosphate ddGTP, and dideoxycytidine triphosphate ddCTP;
and a DNA polymerase.

13. The method according to claim 1, wherein the complementary nucleic acid is synthesized using a nucleic acid primer; at least one nucleotide selected from the group consisting of adenosine triphosphate ATP, uridine triphosphate UTP, guanosine triphosphate GTP, cytidine triphosphate CTP, inosine triphosphate ITP, a 7-deazanucleoside triphosphate c7ATP, a 7-deazanucleoside triphosphate c7GTP, and a 7-deazanucleoside triphosphate c7ITP; at least one chain-terminating 3'-deoxynucleotide selected from the group consisting of deoxyadenosine triphosphate 3'-dATP, deoxyuridine triphosphate 3'-dUTP, deoxyguanosine triphosphate 3'-dGTP, and deoxycytidine triphosphate 3'-dCTP; and an RNA polymerase.

14. The method according to claim 1, wherein the nucleic acid primer further includes a linking group (L) for reversibly immobilizing the primer on a solid support.

15. The method according to claim 14, wherein the sets of base-specifically terminated fragments are coupled by the linking group (L) to a functionality (L') on the support creating a temporary and cleavable attachment of the complementary nucleic acid to the support.

16. The method according to claim 15, wherein the temporary and cleavable attachment can be cleaved enzymatically, chemically or physically.

17. The method according to claim 16, wherein the temporary and cleavable attachment is selected from the group consisting of a photocleavable bond, a bond based on strong electrostatic interaction, a tritylether bond, .alpha..beta.-benzoylpropionyl group, a levulinyl group, a disulfide bond, an arginine/arginine bond, a lysine/lysine bond, a pyrophosphate bond, and a bond created by Watson-Crick base pairing.

18. The method according to claim 15, wherein the support-bound base-specifically terminated fragments are thoroughly washed to remove all remaining reactants and by-products from the sequencing reaction.

19. The method according to claim 18, wherein the base-specifically terminated fragments are cleaved from the solid support prior to mass spectrometry.

20. The method according to claim 18, wherein the base-specifically terminated fragments are cleaved from the solid support during mass spectrometry.

21. The method according to claim 1, wherein more than one species of nucleic acid are concurrently sequenced by multiplex mass spectrometric nucleic acid sequencing employing tag probes, nucleic acid primers, chain-elongating nucleotides, and chain-terminating nucleotides, wherein one of the sets of base-specifically terminated fragments is unmodified and the other sets of base-specifically terminated fragments are mass modified, and each of the sets of base-specifically terminated fragments has a sufficient mass difference to be distinguished from the others by mass spectrometry.

22. The method according to claim 21, wherein at least one of the sets of mass-modified base-specifically terminated fragments is modified with a mass-modifying functionality (M) at a heterocyclic base of at least one nucleotide.

23. The method according to claim 22, wherein the heterocyclic base-modified nucleotide is selected from the group consisting of a cytosine nucleotide modified at C-5, a thymine nucleotide modified at C-5, a thymine nucleotide modified at the C-5 methyl group, a uracil nucleotide modified at C-5, an adenine nucleotide modified at C-8, a c7-deazadenine modified at C-8, a c7-deazadenine modified at C-7, a guanine nucleotide modified at C-8, a C7-deazaguanine modified at C-8, a c7-deazaguanine modified at C-7, a hypoxanthine modified at C-8, a c7-deazahypoxanthine modified at C-7, and a C7-deazahypoxanthine modified at C-8.

24. The method according to claim 21, wherein at least one of the sets of mass-modified base-specifically terminated fragments is modified with a mass-modifying functionality (M) attached to one or more phosphorus atoms of the internucleotidic linkages of the fragments.

25. The method according to claim 21, wherein at least one of the sets of mass-modified base-specifically terminated fragments is modified with a mass-modifying functionality (M) attached to one or more sugar moieties of nucleotides within the set of mass modified base-specifically terminated fragments at at least one sugar position selected from the group consisting of an internal C-2' position, an external C-2' position, and an external C-5' position.

26. The method according to claim 21, wherein at least one of the sets of mass-modified base-specifically terminated fragments is modified with a mass-modifying functionality (M) attached to the sugar moiety of a 5'-terminal nucleotide and wherein the mass-modifying function (M) is the linking functionality (L).

27. The method according to claim 21, wherein a mass-modifying functionality (M) is attached to a set of base-specifically terminated fragments subsequent to enzymatic synthesis of the base-specifically terminated fragments and prior to determining the molecular weight values for the nested fragments by mass spectrometry.

28. The method according to claim 27, wherein the synthesis of the base-specifically terminated fragments is performed by using at least one reagent selected from the group consisting of a nucleic acid primer, a chain-elongating nucleotide, a chain-terminating nucleotide or a tag probe which has been modified with a precursor of the mass-modifying functionality, M, and a subsequent step comprises modifying the precursor of the mass-modifying functionality, M, to generate the mass-modifying functionality, M, prior to mass spectrometric analysis.

29. The method according to claim 21, wherein mass differentiation of the tag probes is achieved by changing the nucleotide composition of at least one of the tag probes and complementary tag sequence in the species of nucleic acid.

30. The method according to claim 21, wherein the tag probes are covalently bound to the corresponding complementary tag sequence prior to mass spectrometric analysis.

31. The method according to claim 30, wherein binding between the tag probes and the corresponding complementary tag sequences is achieved photochemically via photoactivatable groups.

32. A method of sequencing a nucleic acid, comprising the steps of a) reversibly linking an oligonucleotide primer to a solid support through a linking group;
b) synthesizing complementary nucleic acids which are complementary to the nucleic acid to be sequenced, starting from a nucleic acid primer and in the presence of chain-terminating and chain-elongating nucleotides so as to produce four sets of base-specifically terminated complementary nucleic acid fragments;
c) determining the molecular weight value of each nested fragment in each of thefour sets of base-specifically terminated fragments by matrix assisted laser desorption/ionization mass spectrometry wherein the molecular weight values of at least two base-specifically terminated fragments are determined concurrently and wherein the nested fragments are cleaved from the solid support by a laser during mass spectrometry; and d) determining the nucleotide sequence by aligning the four sets of molecular weight values according to molecular weight.

33. A method of multiplex analysis of nucleic acid sequences, comprising the steps of a) reversibly linking a nucleic acid primer to a solid support through a linkinggroup;
b) synthesizing complementary nucleic acids which are complementary to the nucleic acid to be sequenced, starting from the nucleic acid primer and in the presence of chain-terminating and chain-elongating nucleotides so as to produce four sets of base-specifically terminated complementary nucleic acid fragments;
c) determining the molecular weight value of each nested fragment in each of thefour sets of base-specifically terminated fragments by matrix assisted laser desorption/ionization mass spectrometry wherein the molecular weight values of at least two base-specifically terminated fragments are determined concurrently and wherein the nested fragments are cleaved from the solid support by a laser during mass spectrometry; and d) determining the nucleotide sequence by aligning the four sets of molecular weight values according to molecular weight;
wherein at least one reagent selected from a group consisting of, a nucleic acidprimer, a chain-elongating nucleotide, or a chain-terminating nucleotide is mass-modified, wherein each set of base-specifically terminated fragments has a sufficient mass difference from the other sets of base-specifically terminated fragments so as to be unique, and wherein the molecular weight values of the nested fragments of two or more sets of unseparated base-specifically terminated fragments are determined concurrently.

34. A kit for sequencing one or more species of nucleic acids by multiplex spectrometric nucleic acid sequencing, comprising:
a) a solid support having a linking functionality (L');
b) a set of nucleic acid primers suitable for initiating synthesis of a set of complementary nucleic acids which are complementary to the different species of nucleic acids, the primers each including a linking group (L) able to interact with the linking functionality (L') and reversibly link the primers to the solid support;
c) a set of chain-elongating nucleotides for synthesizing the complementary nucleic acids;
d) a set of chain-terminating nucleotides for terminating synthesis of the complementary nucleic acids and generating sets of base-specific terminated complementary nucleic acid fragments; and e) a polymerase for synthesizing the complementary nucleic acids from the nucleic acid primers, chain-elongating nucleotides and terminating nucleotides, wherein at least one reagent selected from the group consisting of the primers, the chain-elongating nucleotides, and the chain-terminating nucleotides is mass modified to provide distinction between each set of base-specifically terminatednucleotides of each species of nucleic acid by mass spectrometry.

35. A solid support chosen from the group consisting of magnetic beads, cellulose beads, polystyrene beads, Controlled Pore Glass (CPG), silica-gel beads, SEPHAROSE beads, SEPHADEX beads, capillaries, polymeric sheets of polyethylene, polymeric sheets of polypropylene, polymeric sheets of polyamide, polymeric sheets of polyester, polymeric sheets of polyvinylidene-difluoride, glass plates, and metal surfaces, the solid support having a linking functionality, L', which is able to interact with a linking group, L, of a primer, reversibly link the primer to the solid support, and is cleavable enzymatically, chemically or physically.

36. The solid support according to claim 35, wherein the linkage, L-L', is selected from the group consisting of a photocleavable bond, a bond based on strong electrostatic interaction, a tritylether bond, a 13-benzoylpropionyl group, a levulinyl group, a disulfide bond, an arginine/arginine bond, a lysine/lysine bond, a pyrophosphate bond, and a bond created by Watson-Crick base pairing.

37. A solid support comprising a microtiter plate adapted with a functionalized membrane comprising a solid support of claim 33 in each well for reversibly binding a primer.

38. A set of mass-modified nucleic acid primers selected from a group consisting of a collection of mass-modified universal primers for priming DNA synthesis, and a collection of mass-modified initiator oligonucleotides for initiating transcriptional RNA synthesis.

39. The set of mass-modified nucleic acid primers according to claim 38, wherein at least one of the mass-modified primers is modified with a mass modifying functionality (M) at one or more heterocyclic bases within the primers.

40. The set of mass-modified nucleic acid primers according to claim 39, wherein at least one of the mass modified primers comprises at least one heterocyclic base-modified nucleotide selected from the group consisting of a cytosine nucleotide modified at C-5, a thymine nucleotide modified at C-5, a thymine nucleotide modified at the C-5 methyl group, a uracil nucleotide modified at C-5, an adenine nucleotide modified at C-8, a c7-deazadenine modified at C-8, a c7-deazaadenine modified at C-7, a guanine nucleotide modified at C-8, a c7-deazaguanine modified at C-8, a c7-deazaguanine modified at C-7, a hypoxanthine modified at C-8, a c7-deazahypoxanthine modified at C-7, and a c7-deazahypoxanthine modified at C-8.

41. The set of mass-modified nucleic acid primers according to claim 39, wherein at least one of the mass-modified primers is modified with a mass-modifying functionality (M) attached to one or more phosphorus atoms of the internucleotidic linkages within the mass modified primer.

42. The set of mass-modified nucleic acid primers according to claim 39, wherein at least one of the mass-modified primers is modified with a mass-modifying functionality (M) attached to at least one sugar moiety of the nucleotides within the mass-modified primer at at least one sugar position selected from the group consisting of an internal C-2' position, an external C-2' position, and an external C-5' position.

43. The set of mass-modified nucleic acid primers according to claim 39, wherein at least one of the mass-modified primers is modified with a mass-modifying functionality (M) attached to the sugar moiety of a 5'-terminal nucleotide of the primer, and wherein the mass-modifying function (M) is the linking functionality (L).

44. A set of mass-modified nucleotides selected from the group consisting of mass-modified 2'-deoxynucleoside triphosphates suitable for DNA synthesis, mass-modified 2',3'-dideoxynucleoside triphosphates suitable for chain-terminating DNA synthesis, mass-modified nucleoside triphosphates suitable for RNA synthesis, and mass-modified 3'-deoxynucleoside triphosphates suitable for chain-terminating RNA synthesis.

45. The set of mass-modified nucleotides according to claim 44, wherein a mass-modifying functionality (M) is attached to a heterocyclic base of the mass-modified nucleotide.

46. The set of mass-modified nucleotides according to claim 45, wherein the mass-modified nucleotide comprises a modified heterocyclic base selected from the group consisting of a cytosine moiety modified at C-5, a thymine moiety modified at C-5, a thymine moiety modified at the methyl group of C-5, a uracil moiety modified at C-5, an adenine moiety modified at C-8, a c7-deazadenine moiety modified at C-8, a c7-deazadenine moiety modified at C-7, a guanine moiety modified at C-8, a c7-deazaguanine moiety modified at C-8, a c7-deazaguanine moiety modified at C-7, ahypoxanthine moiety modified at C-8, a c7-deazahypoxanthine moiety modified at C-8, and a c7-deazahypoxanthine moiety modified at C-7.

47. The set of mass-modified nucleotides according to claim 44, wherein a mass-modifying functionality (M) is attached to an alpha phosphorus atom of a triphosphate moiety of the mass-modified nucleotide.

48. The set of mass-modified nucleotides according to claim 44, wherein the mass-modified nucleotide comprises a deoxynucleoside triphosphate, and a mass-modifying functionality (M) is attached to a C-2' position of a sugar moiety of the deoxynucleoside triphosphate.

49. The set of mass-modified nucleotides according to claim 44, wherein the mass-modified nucleotide comprises a dideoxynucleoside triphosphate and a mass-modifying functionality (M) is attached to at least one sugar moiety position selected from the group consisting of a C-2' position and a C-3' position.

50. A set of mass-differentiated tag probes complementary, by Watson-Crick base pairing, to tag sequences present within at least one set of base-specifically terminated fragments.

51. The set of mass diferentiated tag probes according to claim 50, wherein mass-differentiation of the tag probe is achieved by attaching a mass-modifying functionality (M) to the tag probe.

52. The set of mass-differentiated tag probes according to claim 51, wherein the mass-modifying functionality (M) is attached to the tag probe at one or more of heterocyclic bases within the tag probe nucleotide sequence.

53. The set of mass-differentiated tag probes according to claim 52, wherein the tag probe comprises at least one mass-modified heterocyclic base selected from the group consisting of a cytosine moiety modified at C-5, a thymine moiety modified at C-5, a thymine moiety modified at the C-5 methyl group, a uracil moiety modified at C-5, an adenine moiety modified at C-8, a c7-deazaadenine moiety modified at C-8, a c7-deazaadenine moiety modified at C-7, a guanine moiety modified at C-8, a c7-deazaguanine moiety modified at C-8, a c7-deazaguanine moiety modified at C-7, a hypoxanthine moiety modified at C-8, a c7-deazahypoxanthine moiety modified at C-8, and a c7-deazahypoxanthine moiety modified at C-7.

54. The set of mass-differentiated tag probes according to claim 52, wherein the mass-modifying functionality (M) is attached to one or more of the phosphorus atoms of an internucleotidic linkage of at least one tag probe.

55. The set of mass-differentiated tag probes according to claim 52, wherein the mass-modifying functionality (M) is attached to at least one tag probe at at least one sugar moiety.

56. The set of mass-differentiated tag probes according to claim 51, wherein the tag probes further include a cross-linking group (CL) which allows for covalent binding to the corresponding and complementary tag sequences.

57. The set of mass-differentiated tag probes according to claim 55, wherein thecrosslinking functionality (CL) is activated photochemically and is derived from at least one photoactivatable group selected from the group consisting of a psoralen and an ellipticine.

58. The set of mass-modified nucleic acid primers according to claim 39, wherein the mass-modifying functionality (M) is selected from a group consisting of F, Cl, Br, I, Si(CH3)3,Si(CH3)2(C2H5),Si(CH3)(C2H5)2,Si(C2H5)3,CH2F,CHF2,and CF3.

59. The set of mass-modified nucleic acid primers according to claim 39, wherein the mass-modifying functionality (M) is generated from a precursor functionality (PF) attached to the mass-modified primers, the precursor (PF) selected from a group consisting of-N3 and XR, wherein R is H and X is selected from a group consisting of-OH,-NH2,-NHR,-SH, -NCS,-OCO(CH2)rCOOH (where r=1-20),-NHCO(CH2)rCOOH (where r=1-20), -OSO2OH,-OCO(CH2)rI(where r= 1-20),and -OP(O-Alkyl)N(Alkyl)2.

60. The set of mass-modified nucleotides according to claim 45, wherein the mass-modifying functionality (M) is selected from a group consisting of F,Cl,Br,I,Si(CH3)3, Si(CH3)2(C2H5),Si(CH3)(C2H5)2,Si(C2H5)3,CH2F,CHF2,and CF3.

61. The set of mass-modified nucleotides according to claim 45, wherein the mass-modifying functionality (M) is generated from a precursor functionality (PF) attached to the mass-modified nucleotides, the precursor (PF) selected from a group consisting of-N3 and XR, wherein R is H and X is selected from a group consisting of -OH, -NH2, -NHR, -SH, -NCS,-OCO(CH2)rCOOH (where r= 1-20),-NHCO(CH2)rCOOH (where r= 1-20), -OSO2OH,-OCO(CH2)rI (where r= 1-20), and -OP(O-Alkyl)N(Alkyl)2.

62. The set of mass-differentiated tag probes according to claim 51, wherein the tag sequence is mass-modified with a mass-modifying functionality (M) selected from a group consisting of XR, F, Cl, Br, I, Si(CH3)3,Si(CH3)2(C2H5),Si(CH3)(C2H5)2,Si(C2H5)3, CH2F,CHF2,and CF3, wherein X is selected from a group consisting of -OH,-NH2,-NHR, -SH,-NCS,-OCO(CH2)rCOOH (where r = 1-20), -NHCO(CH2)rCOOH (where r = 1-20), -OSO2OH,-OCO(CH2)rI (where r = 1-20), and -OP(O-Alkyl)N(Alkyl)2, and R is selected from a group consisting of H, methyl, ethyl, propyl, isopropyl, t-butyl, hexyl, benzyl, benzhydryl, trityl, substituted trityl, aryl, substituted aryl, polyoxymethylene, monoalkylated polyoxymethylene, a polyethylene imine, a polyamide of the general formula (-NH(CH2)rNHCO(CH2)rCO-)m, a polyamide of the general formula (-NH(CH2)rCO-)m, apolyester of the general formula (-O(CH2)rCO-)m, an alkylated silyl compound of the general formula -Si(Y)3, a heterooligo/polyaminoacid of the general formula (-NHCHaaCO-)m, a polyethylene glycol of the general formula -(CH2CH2O)m-CH2CH2OH, and a monoalkylated polyethylene glycol of the general formula -(CH2CH2O)m-CH2CH2O-Y, where m is in the range of 0 to 200, Y is a loweralkyl group selected from a group consisting of methyl, ethyl, propyl, isopropyl, t-butyl, hexyl, r is in the range of 1 to 20, and aa represents the amino acid side chain of a naturally-occurring amino acid.

63. The set of mass-differentiated tag probes according to claim 51, wherein the mass-modifying functionality (M) is generated from a precursor functionality (PF) attached to the mass-differentiated tag probes, the precursor (PF) selected from a group consisting of -N3 and XR, wherein R is H and X is selected from a group consisting of -OH, -NH2, -NHR, -SH, -NCS, -OCO(CH2)rCOOH (where r = 1-20), -NHCO(CH2)rCOOH (where r = 1-20), -OSO2OH, -OCO(CH2)rI (where r = 1-20), and -OP(O-Alkyl)N(Alkyl)2.

64. The set of mass-modified nucleic acid primers according to claim 39, wherein the mass-modifying functionality (M) is given by the general formula XR in which X is selected from a group consisting of-OH, -NH2, -NHR, -SH, -NCS, -OCO(CH2)rCOOH (where r = 1-20), -NHCO(CH2)rCOOH (where r= 1-20), -OSO2OH, -OCO(CH2)rI (where r = 1-20), and-OP(O-Alkyl)N(Alkyl)2, and R is selected from a group concicting of H, methyl, ethyl, propyl, isopropyl, t-butyl, hexyl, benzyl, benzhydryl, trityl, substituted trityl, aryl, substituted aryl, polyoxymethylene, monoalkylated polyoxymethylene, a polyethylene imine, a polyamide of the general formula (-NH(CH2)rNHCO(CH2)rCO-)m, a polyamide of the general formula (-NH(CH2)rCO-)m, a polyester of the general formula (-O(CH2)rCO-)m, an alkylated silyl compound of the general formula -Si(Y)3, a heterooligo/polyaminoacid of the general formula (-NHCHaaCO-)m, a polyethylene glycol of the general formula -(CH2CH2O)m-CH2CH2OH, and a monoalkylated polyethylene glycol of the general formula -(CH2CH2O)m-CH2CH2O-Y, where m is in the range of 0 to 200, Y is a loweralkyl group selected from a group consisting of methyl, ethyl, propyl, isopropyl, t-butyl, hexyl, r is in the range of 1 to 20, and aa represents the amino acid side chain of a naturally-occurring amino acid.

65. The set of mass-modified nucleotides according to claim 45, wherein the mass-modifying functionality (M) is given by the general formula XR in which X is selected from a group consisting of-OH, -NH2, -NHR, -SH, -NCS, -OCO(CH2)rCOOH (where r = 1-20), -NHCO(CH2)rCOOH (where r = 1-20), -OSO2OH, -OCO(CH2)rI (where r = 1-20), and -OP(O-Alkyl)N(Alkyl)2, and R is selected from a group consisting of H, methyl, ethyl, propyl, isopropyl, t-butyl, hexyl, benzyl, benzhydryl, trityl, substituted trityl, aryl, substituted aryl, polyoxymethylene, monoalkylated polyoxymethylene, a polyethylene imine, a polyamide of the general formula (-NH(CH2)rNHCO(CH2)rCO-)m, a polyamide of the general formula (-NH(CH2)rCO-)m, a polyester of the general formula (-O(CH2)rCO-)m, an alkylated silyl compound of the general formula -Si(Y)3, a heterooligo/polyaminoacid of the general formula (-NHCHaaCO-)m, a polyethylene glycol of the general formula -(CH2CH2O)m-CH2CH2OH, and a monoalkylated polyethylene glycol of the general formula -(CH2CH2O)m-CH2CH2O-Y, where m is in the range of 0 to 200, Y is a loweralkyl group selected from a group concisting of methyl, ethyl, propyl, isopropyl, t-butyl, hexyl, r is in the range of 1 to 20, and aa represents the amino acid side chain of a naturally-occurring amino acid.

66. A kit for sequencing nucleic acids by mass spectrometry, comprising:
a) a solid support having a linking functionality (L');
b) a set of nucleic acid primers suitable for initiating synthesis of a set of complementary nucleic acids which are complementary to the different species of nucleic acids, the primers each including a linking group (L) able to interact with the linking functionality (L') and reversibly irnmobilize the primers on the solid support;
c) a set of chain-elongating nucleotides for synthesizing the complementary nucleic acids;
d) a set of chain-terminating nucleotides for terminating synthesis of the complementary nucleic acids and generating sets of base-specific terminated complementary nucleic acid fragments; and e) a polymerase for synthesizing the complementary nucleic acids from the primers, chain-elongating nucleotides and chain-terminating nucleotides, wherein the chain-terminating nucleotides are mass-modified so that addition of one species of the chain-terminating nucleotides to the complementary nucleic acid can be distinguished by mass spectrometry from addition of all other species of chain-terminating nucleotides concurrently analyzed.

67. The method according to claim 32, wherein the base-specifically terminated fragments are cleaved from the solid support prior to mass spectrometry.

68. The method according to claim 32, wherein the base-specifically terminated fragments are cleaved from the solid support during mass spectrometry.

69 The solid support according to claim 36, wherein the photocleavable bond of the linkage, L-L', is selected from the group consisting of a charge transfer complex or a stable organic radical.

70. The method according to claim 32, wherein the reversible linkage is a photocleavable bond.

71. The method according to claim 33, wherein the reversible linkage is a photocleavable bond.

72. The method according to claim 33, wherein the base-specifically terminated fragments are cleaved from the solid support prior to mass spectrometry.

73. The method according to claim 33, wherein the base-specifically terminated fragments are cleaved from the solid support during mass spectrometry.