AU738203B2

AU738203B2 - DNA sequencing by mass spectrometry

Info

Publication number: AU738203B2
Application number: AU91379/98A
Authority: AU
Inventors: Hubert Koster
Original assignee: Sequenom Inc
Current assignee: Sequenom Inc
Priority date: 1993-01-07
Filing date: 1998-11-06
Publication date: 2001-09-13
Anticipated expiration: 2014-01-06
Also published as: AU9137998A

Description

-1-

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

0@ 0 *0* *0 *O 0

S

Name of Applicant: Actual Inventor: SAddress of Service: *see*:

T

5 o Invention Title: SEQUENOM, INC.

Hubert KOSTER ADDRESS FOR SERVICE

ALTERED

BALDWIN SHELSTON AAT Sp r o son 4 Peru3 onz 60 MARgAR -SREET c E SN, B MrTns TouT N SEQUNCW 2900 B MSA STRAeY 0 "DNA SEQUENCING BY MASS SPECTROMETRY" Details of Original Application No. 59929/94 dated 6 January, 1994 The following statement is a full description of this invention, including the best method of performing it known to us:la- DNA SEQUENCING BY MASS SPECTROMETRY Technical Field The present invention relates to mass modified nucleic acid primers and mass modified nucleotides, and their uses in nucleic acid sequencing. The present application is related to AU 694,940, which is incorporated in its entirety herein by reference.

Background of the Invention Since the genetic information is represented by the sequence of the four DNA building blocks deoxyadenosine- (dpA), deoxyguanosine- (dpG), deoxycytidine- (dpC) and deoxythymidine-5'-phosphate (dpT). DNA sequencing is one of the most 10 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 varieties of plants and microorganisms via recombinant DNA technology. In particular, unraveling the DNA sequence helps in understanding human pathological S 15 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, S. in some cases even fatal, consequences. Recently, DNA sequencing has become the core technology of the Human Genome Sequencing Project J.E. Bishop and M.

a Waldholz, 1991, Genome; The Story of the Most Astonishing Scientific Adventure of a 20 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 diseases. To be able to tackle successfully the determination of the approximately 3 billion base pairs of the human genome in a reasonable time frame and in an economical way, rapid, 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 are given by Barrell (The FASEB Journal 5. 40-45 (1991), and Trainor (Anal. Chem. 62.

418-26 (1990)).

Currently, DNA sequencing is performed by either the chemical degradation method of Maxam and Gilbert (Methods in Enzvmologv 65, 499-560 (1980)) or the I 0 S I' lbenzymatic 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.

o 10 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 0 0* a 9 1* 0000 synthesizing a complementary strand by DNA polymerases. such as Klenow fragment of E. coli DNA polymerasc I. a DNA polymerase from Thermus aquaticus. Taq DNA polymerase, or a modified T7 DNA polymerase, Sequenase (Tabor et al.. Proc Na Acad. Sci. UA 84, 4767-4771 (1987)), in the presence of chain-terminating reagents.

Here. the chain-terminating 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-terminating dideoxynucleoside triphosphates, ddATP, ddGTP. ddTTP or ddCTP, respectively, in a limiting small concentration. The four sets of resulting fragments produce, after electrophoresis. four 10 base sptcific ladders from which the DNA sequence can be determined.

A recent modification of the Sanger sequencing stiategy involves the degradation of phosphorothioate-containing DNA fragments obtained by using alpha-thio dNTP instead of the normally used ddNTPs during the primer extension reaction mediated 0:o6: by DNA polymerase (Labeit et al., DA 5. 173-177 (1986): Amersham, PCT-Application 15 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 visualization by radioisotopic 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 treatment (Max-Planck-Gesellschaft. 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 genome, this is a multi-step process involving cloning and subcloning steps using different and appropriate cloning vectors such as YAC, cosmids, plasmids and M 13 vectors (Sambrook et al., Molecular Cloning: A Laboratory 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 M 13 bacteriophage (Vieria and Messing. ene 19. 259 (1982)) and then the double-stranded fonn is transformed 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, Biemat. McManus and K6ster.

Nucleic Acids Res. 12. 4539-57 (1984); U.S. Patent No. 4725677 upstream of the re;triction 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 Annlication. Innis et al..

'I V .3editors, 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 al., Proc. Natl. Acad Sci.

IUSA S, 9436-9440 (1988)). In this case, however, the DNA sequence in the interested region most be known at least to the extent io 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 32 p. 33 p. 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 S* short half-lives which can limit the useful time of the labels. The emission high energy beta radiation, particularly from 32 P, can lead to breakdown of the products via radiolysis so that the sample should be used very quickly after labeling. In addition, high energy 15 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 3 3 P or 35S (see. e.g..

Omstein et al., Biotechniques 1, 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, decontamin.tion and handling 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 be:n 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 321, 674-679 (1986) and EPO Patent No. 87300998.9; Du Pont De Nemours EPO Application No. 0359225; Ansorge et al. J. Biochem. Biophys. Methods 11, 325-32 (1986)) or to the chain-terminating dideoxynucloside triphosphates (Prober et al. 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 by Applied Biosystems (Smith et al., Science 23. G89 (1987); U.S. Patent Nos. 570973 and 689013), Du Pont De Nemours (Prober el al. Science 218, 336-341 (1987); U.S. Patents Nos. 881372 and 57566), Pharmacia-LKB (Ansorge et al. Nucleic Acids Res. 15, 4593- 4602 (1987) and EMBL Patent Application DE P3724442 and P3805808.1) and Hitachi (JP 1-90844 and DE 4011991 Al). A somewhat similar approach was developed b:.

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 usii;g two electrophoretic lanes with two different specific labels per lane is described (PCT Application W092/02635).

A different approach uses fliorescently labeled avidin and biotin labeled primers. Here, the sequencing ladders ending with biotin are reacted during electrophoresis with the 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 ard amplifyable by enzymes have been developed (Beck. O'Keefe, Coull and K6ster, Nucleic Acids Res. 12, 5115-5123 (1989) and Beck and K6ster. Anal. Chem. 62, 2258-2270 (1990)). These labeling methods were combined with multiplex DNA sequencing (Church el al. Scienc 240, 185-188 (1988) to provide for a strategy aimed at high throughput DNA sequencing (Koster el al., Nucleic Acids Res. Symposium Ser. No. 24. 318-321 (1991). University of Utah, PCT S t 1o Application No. WO 90/15883); this strategy still suffers from the disadvantage of being very laborious and difficult to automate.

0* 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 utilizing the 15 strong biotin-avidin/streptavidin interaction (Orion-Yhtyma Oy, U.S. Patent No. 277643; M. Uhlen et al. Nucleic Acids Res. 16, 3025-38 (1988); Cemu Bioteknik, PCT Application No. WO 89/09282 and Medical Research Council, GB, PCT Application No.

,0 WO 92/03575). The primer extension products synthesized on the immobilized template 00: strand are purified of enzymes, other sequencing reagents and by-products by a washing step and then released under denaturing conditions by loosing the hydrogen bonds 0* between the Watson-Crick base pairs and subjected to PAGE separation. In a different appro..ch. the primer extension products (not the template) from a DNA sequencing reaction are bound to a solid support via biotin/avidin (Du Pont De Nemours, PCT 0 Application WO 91/11533). In contrast to the above mentioned methods, here, the intt.action 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. magnetic beads (Dynabeads) and Sepharose beads), filters, capillaries, plastic dipsticks 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 pattern by autoradiorraphy). removing the gel and cleaning the glass plates 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 performed automatically when using the laser-scanning 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 fragments durinz electrophoresis and a possible overlap in the spectral bandwidth of the four specific dyes reducing the discriminating power between neighboring bands, hence, increasing the probability of sequence ambiguities. Artifacts are also produced by basespecific interactions with the polyacrylamide gel matrix (Frank and K6ster. Nucleic Acids Res. 6. 2069 (1979). and by the formation of secondary structures which result in "band compressions" and hence do not allow one to read the sequence. This problem has.

10 in part, been overcome by using 7-deazadeoxyguanosine 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 electrophoresis is capillary zone electrophoresis (CZE) (Jorgenson et al., J. Chromatograph 52, 337 (1986); Gesteland et al.

Nucleic Acids Res. 18, 1415-1419 which, compared to slab gel electrophoresis (PAGE). significantly increases the ;esolution 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 miniaturization 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 proto",l. several methods have been proposed t- do DNA sequencing without an electrophoretic step. One approach calls for hybridization or fragmentation sequencing (Bains, Biotechnology 1Q. 757-58 (1992) and Mirzabekov et FEBS Leers 256. 118-122 (1989)) utilizing the specific hybridization known short oligonucleotides octadeoxynucleotides which gives 65,536 different sequer:ces) 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 by unilaterally degrading one single, immobilized DNA fragment by an exonuclease in a moving flow stream and detecting the cleaved nucleotides by their specific fluorescent tag via laser excitation (Jett et al., J. Biomolecular Structure Dynamics 2. 301-309. (1989); United States Department of Energy, PCT Application No. WO 89/03432). In anothe, system proposed by Hyman (Anal. Biochem. 124. 423-436 (1988)). the pyrophosphate generated when the correct nucleotide is attached to the growing chain on a primertemplate system is used to determine the DNA sequence. The enzymes used and the DNA 11, k -6are held in place by solid phases (DEAE-Sepharose and Sepharose) either by ionic interactions 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 al. (PCT Application No. WO 91/06678). 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 applicatior 0o also describes an apparatus designed 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 volatilization.

Under the iqfluence of combinations of electric and magnetic fields, the ions follow

S

trajec:ories depending on their individual mass and charge In the range of S 15 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 5 determination of the mass of the parent molecular ion. In addition, by arranging collisions of this parent molecular ion with other particles argon atoms), the molecular ion is fragmented forming secondary ions by the so-called collision induced dissociation (CID).

The fragmentation pattern/pathway very often allows the derivatiun 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 Enzvmology.

Vol. 193: "Mass Spectrome.:ry" McCloskey. editor), 1990. Academic Press, New :.:York.

Due to the apparent 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 Spectrometry Reviews 2, 505- 554 (1990). The biggest hurdle to applying mass spectrometry to nucleic acids is the difficulty of volatilizing these very polar biopolymers. Therefore. "sequencing" has been limited to low molecular weight synthetic oligonucleotides hy determining the mass of the parent molecular ion and through this, confirming the already known sequence, or alternatively, confirming the known sequence through the generation of secondary ions (fragment ions) via CID in an MS/MS configuration utilizing, in particular, for the ionization and volatilization, the method of fast atomic bombardment (FAB mass 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 Mas Spectrometrvy 1 1-116(1987)).

Two more recent ionization/desorption techniques are electrospray/ionspray (ES) and matrix-assisted laser desorption/ionization (MALDI). ES mass spectrcmetry has been introduced by Fenn el al. Phvs. Chem. a, 4451-59 (1984); PCT Application No.

WO 90/14148) and current applications are summarized in recent review articles (R.D.

Smith e al., Anal.Chem. 62, 882-89 (1990) and B. Ardrey. Electrospray Mass 10 Spectrometry, Spoctroscopy Europe. 4, 10-18 (1992)). The molecular weights of the tetradecanucleotide d(CATrCCA fGGCATG) (SEQ ID NO: I) (Covey et al. "The Determination of Protein, Oligonucleotide and Peptide Molecular Weights by lonsprav Mass Spectrometry," Rapid Communications in Mass Spectrometry. 2. 249-256 (1988)).

of the 21-mer d(AAATTGTGCACATCCTGCAGC) (SEQ ID NO:2) and without giving 15 details of that of a tRNA with 76 nucleotides (Methods in Enzymology, 19. "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 determination 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 spectromctry, 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 UV-Laser Desorption/lonization: A New Approach to Mass Spectrometry of Large Biomolecules," Biological Mass Spectrometry (Burlingame and McCloskey. editors). Elsevier Science Publishers, Amsterdam, pp. 49-60. 1990.) Since, in most cases, no multiple 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 volatilized (Williams et al., "Volatilization of High Molecular Weight DNA by Pulsed Laser Ablation of Frozen Aqueous Solutions." Science, 246, 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-Fchre el al.. "Matrix-Assisted Laser Desorption Mass Spectrometry of Oligodeoxythymidylic Acids." Rapid Communications in Mass Spectrometry f. 209-13 (1992)) and a double-stranded DNA of 28 base pairs (Williams et al., "Time-of-Flight Mass Spectrometry of Nucleic Acids by Laser Ablation and lonization from a Frozen Aqueous Matrix." Rapid 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.

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 prepared by incorporating NTP's, dNTP's and, as terminating nucleotides, ddNTP's which are substituted at the position of the sugar moiety with one or a combination of the isotopes 12 C, 13 C, 14 C, IH.

2H, 3 H, 160, 17 0 and 180. The polynucleotides obtained are degraded to 3'-nucleotides, cleaved at the N-giycosidic linkage and the isotopically labeled 5'-functionality removed by periodate oxidation and the resulting formaldehyde species determined by mass spectrometry. A specific combination of isotopes serves to discriminate base-specifically between internal nucleotides originating from the incorporation of NTP's and dNTP's and S! to10 terminal nucleotides caused by linking ddNTP's to the end of the polynuclcotide 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 0 ois deduced.

00..

In Japanese Patent No. 59-131909. an instrument is described which detects *o 15 nucleic acid fragments separated either by electrophoresis, liquid chromatography or high speed gel filtration. Mass spectrometric detection is achieved by incorporating into the nucleic acids atoms which normally do not occur in DNA such as S. Br, 1 or Ag. Au. Pt.

Oe 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. Eng. 1206, (New Technol. Cvtom. 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, 34 S, 36S or 3 5 CI. 3 7 CI. 7 9 Br.

81Br to specifically label the chain-terminating 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 sequencing reaction mixtures are separated 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 9000C in an oxygen atmosphere. The SO 2 generated with masses of 64, 65, 66 or 68 is determined on-line by mass spectrometry using, as mass analyzer, a quadrupoie 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., ProcS!E-Int. Soc. Opt. Eng. 1435, (Opt. Methods Ultrasensitive Detect. Anal. Tech.

ApDL). 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 diference is the use of resonance ionization spectroscopy (RIS) in conjunction with a magnetic sector mass analyzer as disclosed in U.S. Patent No.

9 4,442,354 to detect the sulfur isotopes corresponding to the specific nucleotide terminators, and by this, allowing the assignment of the DNA sequence.

EPO Patent Applications No. 0360676 Al and 0360677 Al also describe Sanger sequences using stable isotope substitutions in the ddNTP's such as D, 13 C, 15 N, 170, 180, 32, 33S, 34S 36S, 19F, 35 C1, 79 Br, Br, and 127I or functional groups such as CF 3 or Si(CH 3 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 8000 0 K) and o0 analyzed by a simple mass analyzer. An instrument is proposed which enables one to automate the analysis of the Sanger sequences 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 recently suggested (Bains, "DNA Sequencing by Mass Spectrometry: Outline of a Potential Future Application," Chimicaoggi 9, 13-16 (1991)).

Summary of the Invention Herein disclosed is a set of mass-modified nucleic acid primers selected from a group S consisting of a collection of mass-modified universal primers for priming DNA synthesis, 20 and a collection of mass-modified initiator oligonucleotides for initiating transscriptional RNA synthesis.

Also herein disclosed is 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 25 synthesis, mass-modified nucleoside triphosphates suitable for RNA synthesis and massmodified 3'-deoxynucleoside triphosphates suitable for chain-terminating RNA synthesis.

Also herein disclosed is an ionized mass-modified nucleic acid molecule, comprising at least one mass modified nucleotide from the group consisting of a mass-modified 2'deoxynucleoside triphosphate, a mass modified 2',3'-dideoxy-nucleoside triphosphate, a 30 mass-modified nucleoside triphosphate and a mass-modified 3'-deoxynuclsodie triphosphate.

Also herein disclosed is a set of mass-differentiated tag probes wherein, each tag probe in the set comprises a sequence of nucleotides which is complementary by Watson- Crick base pairing to a tag sequence present within at least one set of base-specifically terminated fragments; the tag sequences to which each tag probe is complementary are different for each tag probe; each tag probe in the set comprises at least one mass-modified nucleotide; and the mass modified nucleotides are not isotopically labelled and have different massmodifications in each tag probe.

Also herein disclosed is an ionized mass-modified nucleic acid molecule comprising A23216 27. JUL. 2001 11:34 SPRUSON AND FERGUSON 61292615486 NO. 1198-P. 7 9a two or more mass modified nucleotides selected from the group consisting of a massmodified 2'-deoxynucleoside triphosphate, a mass-modified 3 '-dideoxynucleosidc triphosphate, a mass-modified nucleoside triphosphate and a mass-modified- 3'deoxynucleoside triphosphate.

Thus according to a first embodiment of the invention, there is provided an ionized and volatilized mass-modified nucleic acid molecule, comprising at least two mass modified nucleotides which are not naturally occurring and which are not isotopically labeled, wherein the molecule is positively charged.

According to a second embodiment of the invention, there is provided an ionized 0 mass-modified nucleic acid molecule, comprising: at least one mass modified nucleotide, wherein the molecule is positively charged, and comprises a member selected from the group consisting of: a mass-modified universal primer and a mass-modified initiator oligonucleotide.

According to a third embodiment of the invention, there is provided a positively i charged ionized mass-modified nucleic acid molecule, comprising: at least one mass-modified nucleotide containing a modified heterocyclic base selected from the group consisting of a cytosine moiety modified at C-5, a thymine moicry 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 c 7 -deazaadenine moiety modified at 20 C-8, a c -deazaadenine moiety modified at C-7, a guanine moiety modified at C-8, a c 7 deazaguanine moiety modified at C-8, a c7-deazaguanine moiety modified at C-7, a hypoxanthine moiety modified at C-8, a ce-deazahypoxanthine moiety modified at C-8, and 7 a c -deazahypoxanthine moiety modified at C-7.

According to a fourth embodiment of the invention, there is provided a positively 25 charged ionized mass-modified nucleic acid molecule, comprising: at least one mass-modified nucleotide containing a mass-modifying functionality

(M)

attached to at least one sugar moiety of the nucleotide.

According to a fifth embodiment of the invention, there is provided a positively *E charged ionized mass-modified nucleic acid molecule, comprising: 30 a mass-modifying functionality attached to at least one sugar moiety of the nucleic acid molecule, wherein the sugar is modified at a position selected from the group consisting of an internal C- 2' position, an external C-2' position, and an external C-5' position.

According to a sixth embodiment of the invention, there is provided an ionized massmodified nucleic acid molecule, comprising at least one mass-modified nucleotide containing a mass-modifying functionality incorporated into the molecule, wherein

(M)

is selected from the group consisting of F, Cl, Br, I, Si(CH3) 3 Si(CH 3 2

(C

2

H),

Si(CH 3

)(C

2 Hs) 2 Si(C 2 H5)3, CH 2 F, CHF2, and CF 3 According to a seventh embodiment of the invention, there is provided a set of massdifferentiated tag probes wherein, each tag probe in the set comprises a sequence of nucleotides which is complementary by Watson-Crick base pairing to a tag sequence present within at least one set ofbase-specifically terminated fragments; AiZI6 9b the tag sequences to which each tag probe is complementary are different for each tag probe; each tag probe in the set comprises at least one mass-modified nucleotide; and the mass-modified nucleotides are not isotopically labelled and have different mass modifications in each tag probe.

According to an eighth embodiment of the invention, there is provided a positively charged ionized mass-modified nucleic acid molecule, comprising: two or more mass modified nucleotides selected from the group consisting of a massmodified 2'-deoxynucleotide, a mass-modified 2',3'-dideoxynucleotide, a mass-modified nucleotide and a mass-modified 3'-deoxynucleotide, wherein the two or more massmodified nucleotides are different from each other.

According to a ninth embodiment of the invention, there is provided an ionized massmodified nucleic acid molecule, comprising at least one mass modified nucleotide selected from the group consisting of a mass-modified 2'-deoxynucleotide, a mass-modified dideoxynucleotide, a mass-modified nucleotide and a mass-modified 3'-deoxynucleotide, wherein the mass modified nucleic acid molecule comprises a modified heterocyclic base selected from a group consisting of a c 7 -deazaadenine moiety modified at C-8, a c 7 -deazaadenine moiety modified at C-7, a c 7 -deazaguanine moiety modified at C-8, a cdeazaguanine moiety modified at C-7, a hypoxanthine moiety modified at C-8, a c 7 20 deazahypoxanthine moiety modified at C-8, and a c 7 -deazahypoxanthine moiety modified at C-7.

Also disclosed herein is an ionized duplex comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein the mass-modified tag probe comprises at least one mass-modified nucleotide.

Thus, according to a tenth embodiment of the invention, there is provided an ionized positively charged duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein the massmodified tag probe comprises at least one mass-modified nucleotide.

According to an eleventh embodiment of the invention, there is provided an ionized duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein the mass-modified tag probe comprises at least one mass-modified nucleotide, wherein at least one of the mass-modified nucleotides comprises a mass-modifying functionality attached to the heterocyclic base.

According to a twelfth embodiment of the invention, there is provided an ionized duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein: the mass-modified tag probe comprises at least one mass-modified nucleotide; and a mass-modifying functionality (M) incorporated into the tag probe is attached to the phosphorus atom forming an Sinternucleotidic linkage of the tag probe.

A23216 27. JUL. 2001 11:34 SPRUSON AND FERGUSON 61292615486 NO, 1198- P. 8- 0 a 9 a a *o *o 9c According to a thirteenth embodiment of the invention, there is provided an ionized duplex, comprising a mass-modi l fed tag probe bound to a tag sequence present within a base-specifically terminated nuclcic acid fragment, wherein: the mass-modified tag probe comprises at least one mass-modified nucleotide; and at least one of the mass-modified S nucleotides comprises a mass-modifying functionality attached to the sugar moiety.

According to a fourteenth embodiment of the invention, there is provided an ionized duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein: the mass-modified tag probe comprises at least one mass-modified nucleotidc; and the tag probe further comprises a cross-linking group (CL) which allows for covalent binding to the tag sequence.

According to a fifteenth embodiment of the invention, there is provided .a ionized mass-modified nucleic acid molecule, comprising at least one mass modified nucleotide wherein a mass-modifying functionality incorporated into the molecule is generated from a precursor functionality (PF) attached to one or more of a nucleic acid primer, a chain-elongating nucleoside triphosphate or a chain-terminating nucleoside triphosphate, and wherein the precursor functionality (PF) is selected from the group consisting of-N 3 -NH2, -SH, -NCS, -OCO(CH 2 ),COOH (where r=1-20), -NHCO(CH2),COOH (where r-l- 20), -OSO2H, -OCO(CH2),I (where r=l-20), -CONH,,

-NH-C(S)-NH

2 OP(O-Alkyl)OH, and O-CO-CH 2

-SH.

According to a sixteenth embodiment of the invention, there is provided an ionized and volatilized mass-modified nucleic acid molecule, comprising at least two mass modified nucleotides which are not naturally occurring and which are not isotopically labeled.

According to a seventeenth embodiment of the invention, there is provided a method 25 of sequencing a nucleic acid, comprising the steps of: a) starting from a nucleic acid primer and in the presence of chain-terminating and chain-elongating nucleotides, synthesizing complementary nucleic acids that are complementary to the nucleic acid to be sequenced, whereby four sets of base-specifically terminated complementary nucleic acid fragments are produced, wherein: the nucleic acid fragments comprise nucleotides with a modification at a base, a sugar or a phosphate of a nucleotide; and the modification improves the separation or resolution of the fragments when analyzed compared to unmodified fragments; b) determining the molecular weight value of each base-specifically terminated fragment simultaneously by mass spectrometry; and c) determining the nucleotide sequence by aligning the base-specifically terminated fragments according to molecular weight.

A23216 27. JUL 2001 11:34 SPRUSON AND FERGUSON 61292615486 NO. 1198-P. 9 9d According to an eighteenth embodiment of the invention, there is provided a method of multiplex sequence analysis of nucleic acid species, comprising the steps of a) reversibly linking nucleic acid primers to a solid support through a linking group; b) synthesizing complementary nucleic acids which are complementary to the nucleic acid species to be sequenced, starting from the nucleic acid primers and in the presence of chain-terminating and chain-elongating nucleotides so as to produce sets of base-specifically terminated complementary nucleic acid fragments for each species; c) determining the molecular weight value of each base-specifically temninated on fragment for all species simultaneously by matrix assisted laser desorption/ionization mass spectrometry wherein the fragments are cleaved from the solid support by a laser during mass spectrometry; and d) determining the nucleotide sequence of the species by aligning the basespecifically terminated fragments according to molecular weight; I s wherein at least one reagent selected from a group consisting of, a nucleic acid pnmer, a chain-elongating nucleotide, or a chain-terminating nucleotide is mass-modified, 9 wherein each set of base-specifically terminated fragments of a species has a sufficient mass difference from the sets of base-specifically terminated fragments of other species to be distinguished by mass spectrometry.

20 According to a nineteenth embodiment of the invention, there is provided a method of sequencing a nucleic acid, comprising the steps of: a) synthesizing complementary nucleic acids that 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, to produce base-specifically terminated 25 complementary nucleic acid fragments; b) exposing the base-specifically terminated complementary nucleic acid fragments to a single laser to produce desorbed/ionized fragments; c) determining the molecular weight value of each desorbed/ionized fragment produced by step by mass spectrometry; and d) determining the nucleotide sequence by aligning the base-specifically terminated nucleic acid fragments according to molecular weight.

According to a twentieth embodiment of the invention, there is provided a method for determining the sequence of a nucleic acid, comprising the steps of: a) generating at least two base-specifically terminated nucleic acid fragments containing deazapurine nucleotides; b) determining the molecular weight value of each base-specifically terminated fragment by mass spectrometry, wherein the molecular weight values of at least two basespecifically terminated fragments are determined simultaneously; and c) determining the sequence of the nucleic acid by aligning the base-specifically terminated nucleic acid fragments according to molecular weight.

According to a twenty-first embodiment of the invention, there is provided a method A4 of sequencing a target nucleic acid, comprising the steps of: A23216 27. JUL. 2001 11:35 SPRUSON AND FERGUSON 61292615486 NO. 1198- 9e a) reversibly linking oligonucleotide primers to a solid support; b) hybridizing to the primers, at least a portion of the target nucleic acid and generating via chain elongation of the primer and subsequent termination, at least two basespecifically terminated nucleic acid fragments containing deazapurine nucleotides; c) determining the molecular weight value of each base-specifically terminated fragment by mass spectrometry wherein the molecular weight values of at least two basespecifically terminated fragments are determined simultaneously and wherein the fragments are cleaved from the solid support during mass spectrometry; and d) detennining the nucleotide Sequence by aligning the base specifically 0t terminated fragments according to molecular weight.

According to a twenty-second embodiment of the invention, there is provided a method for determining the sequence of a nucleic acid comprising the steps of a) generating at least two base-specifically terminated nucleic acid fragments from a nucleic acid to be sequenced, wherein: 5 the fragments are generated under conditions which comprise cation exchange; and the conditions permit sequencing ofoligomers that are 5 0-mers; Sb) determining the molecular weight value of each of the base-specifically terminated fragments by mass spectrometry, wherein the molecular weight values of at least two base-specifically terminated fragments are determined simultaneously; and i: 20 c) determining the sequence of the nucleic acid by aligning the base-specifically terminated nucleic acid fragments according to molecular weight.

According to a twenty-third embodiment of the invention, there is provided a method for determining the sequence of a nucleic acid, comprising the steps of: a) generating at least two base-specifically terminated nucleic acid fragments 25 wherein: at least one of the nucleic acid fragments comprises two different mass modifications b) determining the molecular weight value of each base-specifically terminated fragment by mass spectrometry; and U• c) determining the sequence of the nucleic acid by aligning the one or more sets of base-specifically terminated nucleic acid fragments according to molecular weight.

According to a twenty-fourth embodiment of the invention, there is provided a method of multiplex sequence analysis of nucleic acid species, comprising the steps of a) synthesizing complementary nucleic acids which are complementary to the nucleic acid species to be sequenced, starting from nucleic acid primers and in the presence of chain-terminating and chain-elongating nucleotides so as to produce sets of basespecifically terminated complementary nucleic acid fragments for each species; b) determining the molecular weight value of each base-specifically terminated fragment for all species simultaneously by mass spectrometry; and c) determining the nucleotide sequences of the species by aligning the basespecifically terminated fragments according to molecular weight; wherein at least one reagent selected from a group consisting of, a nucleic acid pnrimer, a chain-elongating nucleotide, or a chain-terminating nucleotide is mass-modified, AJl216 27. JUL 2001 11:35 SPRUSON AND FERGUSON 61292615486 NO., 1198--P. 11 9f wherein each set of base-specifically terminated fragments of a species has a sufficient mass differcnce from sets or base-specifically terminated fragments of other species to be distinguished by mass spectrometry.

According to a twenty-fifth embodiment of the invention, there is provided a kit for sequencing one or more species of nucleic acids by multiplex mass spectrometric nucleic acid sequencing, comprising: a) a solid support having a linking functionality b) a set of nucleic acid primers suitable for initiating synthesis of a set of nucleic acids which are complementary to the different species of nucleic acids, the primers each.

o including a linking group able to interact with the linking functionality and reversibly link the primers to the solid support and optionally, a tag probe; 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 S e) a polymerase for synthesizing the complementary nucleic acids from the nucleic S acid primers, chain-elongating nucleotides and terminating nucleotides, wherein in the absence of a tag probe, at least one reagent selected from the group consisting of the 20 primers, the chain-elongating nucleotides, and the chain-terminating nucleotides is mass modified to provide distinction between each set of base-specifically terminated nucleotides of each species of nucleic acid by mass spectrometry.

According to a twenty-sixth embodiment of the invention, there is provided a kit for sequencing nucleic acids by mass spectrometry, comprising: n a) a solid support having a linking functionality 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 able to interact with the linking functionality and reversibly immobilize the primers on the solid support; c) a set of chain-elongating nucleotides for synthesizing the complementary nucleic acids; d) a set if 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.

A23216 27. JUL. 2001 11:36 SPRUSON AND FERGUSON 61292615486 NO. 1198-P. 12 9g According to a twenty-seventh embodiment of the invention, there is provided a solid support, comprising a linking functionality, L' linked to a primer via a linking group, L, of the primer to form a linkage wherein: the interaction between L and L' is selectively cleavable enzymatically, chemically or physically; and the primer comprises a mass-modifying functionality linked directly to the primer, or the primer comprises an initiated nucleic acid chain that contains a nucleotide with a mass-modifying functionality wherein the linkage is selected from the group consisting of a photocleavable bond, a bond based on a strong electrostatic interaction, a tritylether bond, a P-benzoylpropionyl group, a levulinyl group, an arginine/arginine bond, a lysinc/lysinc bond and a pyrophosphate bond.

According to a twenty-eighth embodiment of the present invention, there is provided a solid support, comprising a linking functionality, L' linked to a primer via a linking group, L, of the primer to form a linkage wherein: the interaction between L and L' is selectively cleavable enzymatically, chemically or physically; and the primer comprises a mass-modifying functionality linked directly to the primer, or the primer comprises an initiated nucleic acid chain that contains a nucleotide with a mass-modifying functionality wherein 20 the linkage is a photocleavable bond or a bond based on a strong electrostatic interaction.

According to a twenty-ninth embodiment of the present invention, there is provided a solid support having a linking functionality, linked to a primer via a linking group, L, *0 forming a photocleavable bond wherein the photocleavable bond is selected to be S 25 selectively cleaved by ultraviolet laser energy.

According to a thirtieth embodiment of the present invention, there is also provided a solid support according to the invention when used for synthesizing nucleic acid molecules, preferably when used in a method according to the invention.

According to a thirty-first embodiment of the present invention, there is provided a 30 microtiter plate adapted with a functionalized membrane, comprising a solid support and a reversibly linked nucleic acid primer in each well.

According to a thirty-second embodiment of the present invention, there is also provided a microtiter plate according to the invention when used for synthesizing nucleic acid molecules, preferably when used in a method according to the invention, Unless the context requires otherwise, throughout the specification, and the claims which follow, the words "comprise", and the like, are to be construed in an inclusive sense, that is as "including, but not limited to".

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 terminated nested fragment family temporarily to a solid support; removing by a washing step all by-products; A2321l r conditioning the nested DNA or RNA fragments by. for example, c.tion-io, exchange or modification reagent and presenting the immobilized nested fragments either directly to mass spectrometric analysis or cleaving the purified fragment familyoff the support and evaporating the cleavage reagent.

FIGURE 2A shows the Sanger sequencing products using ddTTP as terminating deoxynucleoside triphosphate of a hypothetical DNA fragment of nucleotides (SEQ ID NO:3) in length with approximately equally balanced base composition. Th molecular masses of the various chain terminated fragments are given.

FIGURE 2B shows an idealized mass spectrum of such a DNA fragment o 10 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 terminator.

SFIGURES 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).

15 FIGURES 5A 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 DNA 20 sequei.:e (SEQ ID NO:3).

o* FIGURES 7A and 7B illustrate the general structure of mass-modified i 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 25 triphosphates for either Sanger DNA or Sanger RNA sequencing. General formulas of the chain-elongating and the chain-terminating nucleoside triphosphates are demonstrated.

FIGURE 9 outlines various linking chemistries with either polyethylene glycol or terminally monoalkylated polyethylene glycol as an example.

FIGURE 10 illustrates similar linking chemistries as shown in FIGURES 8A and 8B and depicts various mass modifying moieties 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 terminating 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 ofoligothymidylic acids. d(pT) 12-18.

FIGURE 15 shows a superposition of MALDI-TOF spectra of the

I,'

I d(TAACGGTCATTACGGCCATTGACTGTAGGACCTGCATTACATGACTAGCT)

(SEQ

ID NO:3) (500 fmol) and dT(pdT) 9 9 (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 of Figure 2. 500 fmol each. as follows: 16A is a 7-mer; 16B is a 10-mer; 16C is a 1 I-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; 161 is a 37-mer: 16J is a 38-mer: 16K is a 42-mer; 16L is a 46-mer and 16M is a 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 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 Sanalysis of mass-modified oligonucleotides as described in Example 21.

15 FIGURE 19 illustrates various linking chemistries between the solid support and the nucleic acid primer (NA) through a strong electrostatic interaction.

FIGURES 20A and 20B illustrate various linking chemistries between the solid support and the nucleic acid primer (NA) through a charge transfer complex ofa charge transfer acceptor and a charge transfer donor FIGURE 21 illustrates various linking chemistries between the solid support and the nucleic acid primer (NA) through a stable organic radical.

FIGURE 22 illustrates a possible linking chemistry between the solid support and the nucleic acid primer (NA) through Watson-Crick base pairing.

FIGURE 23 illustrates linking the solid support 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 spcctrometry such as matrix-assisted laser desorption/ionization (MALDI) or electrospray (ES) mass spectrometry to analyze the Sanger sequencing reaction mixtures.

In Sanger sequencing. four families of chain-terminated 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 interpolation) of the molecular weight peaks of the four individual experiments. In another embodiment, the molecular weights of the four specifically terminated fragment families can be determined simultaneously by MS. either 11.1 by mixing the products of all four reactions run in at least two separate reaction vessels all run separately, or two together, or three together) or by running one reaction having all four chain-terminating r.ucleotides a reaction mixture comprising d"TP.

ddTTP. dATP. ddATP. dCTP. ddCTP. dGTP. ddGTP) in one reaction vessel. By simultaneously analyzing all four base-specifically terminated reaction products, the *o o *i: *oo oooo il o molecular weight values have been, in effect, interpolated. Comparison of the mass difference measured between fragments with the known masses of each chain-terminating nucleotide allows the assignment of sequence to be carried out. In some instances, it may be desirable to mass modify, as discussed below, the :hain-terminating nucleotides so as to expand the difference in molecular weight between each nucleotide. It will be apparent to those skilled in the art when mass-modification of the chain-terminating nucleotides is desirable and can depend, for instance, on the resolving ability of the particular spectrometer employed. By way of example, it may be desirable to produce four chainterminating nucleotides, ddTTP, ddCTP 1 ddATP 2 and ddGTP 3 where ddCTpl. ddATP 2 and ddGTP 3 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-terminating nucleotides are well known in the art. For DNA, chain-elongating nucleotides include 2'-deoxyribonucleotides and chain-terrinating nucleotides include 3'-dideoxyribonucleotides. For RNA, chain-elongating nucleotides inc!ude ribonucelotides and chain-terminating 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.

20 Since mass spectrometry is a serial method, in contrast to currently 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 massmodified nucleic acid sequencing primers or chain-elongating and/or terminating nucleoside triphosphates so as to shift the molecular weight of the base-specifically terminated fragments of a particular DNA or RNA species being sequenced in a predetermined manner. For thr first lime. 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 terminated fragments are determined concurrently using mass spectrometry. The molecular weight valtes of preferably at least five and more preferably at least ten base-specifically terminated fragments are determined by mass spectrometry.

-13- Also included in the invention are determinations of the molecular weight values of at least base-specifically terminated fragments and at least 30 base-specifically terminated fragments. Further, the nested base-specifically terminated 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 terminated fragments is analyzed concurrently and the molecular weight values are determined. At least two base-specifically terminated fragments are analyzed concurrently by mass spectrometry when the fragments are contained in the same sample.

In general, the overall mass spectrometric DNA sequencing process will start 0o 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 M13 or pUC MI 3mpl 8 or M13mpl9) (see FIGURE In a different approach, the fragments inserted in vectors.

such as Ml 3. 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 Higuchi et al.. "A General Method of in vitro Preparation and Mutagenesis of DNA Fragments: Study of Protein and DNA Interactions," Nucleic Acids Res.. 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 20 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 ir, 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, preferentially at the 5'-end. a linking functionality, L, which can include a spacer of sufficient length and which can ;nteract with a suitable functionality, on a solid support to form a reversible linkage such as a photocleivable 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 33 support by reacting with functional groups, on the surface of a solid support and then intensively washed to remove all buffer salts, triphosphates. enzymes, reaction byproducts. etc. Furthermore, for mass spectrometric analysis. it can be of importance at this stage to exchange the cation at the phosphate bacl.bone of the DN.\ fragments in order to eliminate peak broadening 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 properly condition them for mass spectrometnc 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 off the support for mass spectrometric analysis. the temporary 14linkage car be such that it is cleaved under the conditions of mass spectrometry. 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 ammonium group (some examples are given in FIGURE 19). In this case, preferably. the surface of the *-olid support carries negative charges which repel the negatively charged nucleic acid backbone and thus facilitates desorption. Desorption will take p.ace either by the heat created by the laser pulse and/or, depending on by specific absorption of laser energy which is in resonance with the L' chromophore (see. examples given in FIGURE 19). The functionalities. L and can also form a charge transfer complex and thereby form the temporary L-L' linkage. Various examples for appropriate functionalities with either acceptor or donator properties are depicted without limitation in FIGURES 20A and Since in many cases the "charge-transfer band" can be determined by UV/vis spectrometry (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 off the 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. I. 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 fragments 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 DNAIRNA 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.

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 support-bound base sequence can be presented through natural oligoribo- or oligodeoxvribonucleotide as well as analogs thio-modified phosphodiester or phosphotriester backbone) or employing oligonucleotide mimetics such as PNA analogs (see c.g. Nielsen et ul.. Science. 254. 1497 (1991)) which render the base sequence less susceptible to enzymatic degradation and hence increases overall stability of the solid support-bound capture base sequence. With appropriate bonds. 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 termination.

With alkylating agents such as akyliodides, iodoacetamide, P-iodoethanol. 2,3-epor.y-1propanol (see FIGURE 10), the monothio phosphodiester bonds of the nested Sanger fragments are transformed into phosphotriester bonds. Multiplexing by mass modification in this case is obtained by mass-modifying the nucleic acid primer (UP) or nucleoside triphosphates at, the sugar or the base moiety. To those skilled in the art, other modifications of the nested Sanger fragments can be envisioned. In one embodiment of the invention, the linking chemistry allows one to cleave off the so-purified nested DNA 15 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 (Kster 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 hydrazinium/acetate buffer, an arginine-arginine or lysine-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, FIGURE 23). Optionally, another cation exchange can be performed 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 terminated products can be separated from the reactants by dialysis, filtration (including ultrafiltration), and chromatography. Likewise.

these techniques can be used to exchange the cation of the phosphate backbone with a counter-ion which reduces peak broadening.

The base-specifically terminated fragment families can be generated by standard Sanger sequencing using the Large Klenow fragment of E. coli DNA polymerase 1, by Sequenase, Taq DNA polymerase and other DN 4 polymerases suitable for this purpose, thus generating nested DNA fragments for the mass spectrom.tric analysis. It is.

however, part of this invention that base-specifically terminated RNA transcripts of the DNA fragments to be sequenced can also be utilized for mass spectrometric sequence -16determination. In this case, various RNA polymerases such as the SP6 or the T7 RNA polymerase can be used on appropriate vectors containing, for example, the SP6 or the T7 promoters Axelrod et al, "Transcription from Bacteriophage T7 and SP6 RNA Polymerase Promoters in the Presence of 3'-Deoxyribonucleoside 5'-triphosphate Chain Terminators." Biochemistr 24. 5716-23 (1985)). In this case, the unknown DNA sequence fragments are inserted downstream from such promoters. Transcription can also be initiated by a nucle', acid primer (Pitulle et al.. "Initiator Oligonucleotides for the Combination of Chemical and Enzymatic RNA Synthesis," GQne 12,. 101-105 (1992)) which carries, as one embodiment of this invention, appropriate linking functionalities.

L.

which allow the immobilization of the nested RNA fragments, as outlined above, prior to mass spectrometric analysis for purification and/or appropriate modification and/or conditioning.

0 *For this immobilization process of the DNA/RNA sequencing products for mass spectrometric analysis, various solid supports can be used, beads (silica gel.

15 controlled pore glass, magnetic beads, Sephadex/Sephar- .e beads, cellulose beads, etc.).

capillaries, glass fiber filters, glass surfaces, metal surfaces or plastic material. Examples of useful plastic materials 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 20 well functionalized with Membranes can be based on polyethylene, polypropylene.

polyamide, polyvinylidenedifluoride and the like. Examples of suitable metal surfaces include steel, gold, silver, aluminum, and copper. After purification. cation exchange.

S 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 25 physically. Also, the L-L' bound fragments can be cleaved from the support when they are subjected to mass spectrometric analysis by using appropriately chosen L-L' linkages and corresponding laser energies/intensities as described above and in FIGURES 19-23.

The highly purified, four base-specifically terminated DNA or RNA fragment families are then analyzed with regard to their fragment lengths via determination of their respective molecular weights by MALDI or ES mass spectrometry.

For ES, the samples, 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 computer 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-terminating nucleotides has been added to the UP. This represents the first nucleotide of the unknown sequence. Then. tre second, the third, the nth extension product can be identified in a similar manner and. by this, the nucleotide sequence is assigned. The generation of multiple ion peaks which can be obtained using ES mass spectrome'ry can increase the accuracy of the mass 17determination.

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 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 SA and 5B) terminated products are given (corresponding to fragments of SEQ ID NO:3) and the idealized 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 correlation of calculated molecular weights of the ddT fragments and their experimentally-verified weights je shown in Table 1. Likewise, if all four chainterminating 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 Correlation of calculated and experimentally verified molecular weights of the 13 DNA fragments of FIGURES 2 and 16A-16M.

Fragment (n-mer) calculated mass experimental mass difference 7-mer 2104.45 2119.9 +15.4 3011.04 3026.1 +15.1 11-mer 3315.24 3330.! +14.9 19-mer 5771.82 5788.0 +16.2 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-mcr 14108.22 14125.0 +16.8 15344.02 15362.6 +18.6 18- 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 different, methodologies, the basic principle being the mass modification of the nucleic acid primer the chain-elongating and/or terminating nucleoside triphosphates. or by using mass-differentiated tag probes hybridizable 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 The mass modifying moiety can be attached, for instance, to either the 5'-end of the oligonucleotide (M to the nucleobase (or bases)

(M

2

M

7 to the phosphate backbone and to the 2'-position of the nucleoside (nucleosides) (M 4

M

6 or/and to the terminal 3'-position (M 5 Primer length can vary between I and 50 nucleotides in length. For the priming of DNA Sanger sequencing, the 15 primer is preferentially in the range of about 15 to 30 nucleotides in length. For artificially priming the transcription in a RNA polyvmerase-rr.diated 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 chainterminated fragments. its preferential length is about 20 nucleotides.

20 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 massmodifying functions and positions within the oligonucleotide molecule are possible and S* are deemed part of the invention. The mass-modifying functionality can be. for example.

25 a halogen. an azido. or of the type. XR. wherein X is a linking group and R is a mass- 0. modifying functionality. The mass-modifying functionality can thus be used to introduce defined mass increments into the oligonucleotide molecule.

In another embodiment. the r.ucleotides 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. M 2 (in case of the c 7 -deazanucleosides also to C-7. M 7 to the triphosphate group at the alpha phosphate. M 3 or to the 2'-position of the sugar ring of the nucleoside triphosphate. M 4 and M 6 Furthermore, the mass-modifying functionality can be added so as to affect chain termination, such as by attaching it to the 3'-positicn of the sugar ring in the nucleoside triphosphate. M 5 The list in FIGURE 8B represents examples of possible configurations for generating chain-terminating 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 mass-modified .n a similar fashion with numerous variations and combinations in fu nctionality and attachment 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 X in XR as well as using oligo-/polyethylene glycol derivatives for R. The massmodifying increment in this case is 44, i.e. five different mass-modified species can be generated by just changing m from 0 to 4 thus adding mass units of 45 89 1).

133 177 and 221 to the nucleic acid primer the tag probe (TP) or the nucleoside triphncphates 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 Oligonucleotides and Analogues. A Practical Approach, F. Eckstein, editor. IRL Press, Oxford, 1991.

15 In yet another embodiment, various mass-modifying functionalities, R, other than oligo/polyethylene glycols, can be selected and attached via appropriate 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, CI, Br and/or I, or pseudohalogens such as SCN, NCS. or by using different alkyl. aryl or aralkyl 20 moieties such as methyl, ethyl, propyl, isopropy!, t-butyl, hexyl, phenyl, substituted phenyl, benzyl, or functional groups such as CH 2 F, CHF 2

CF

3 Si(CH 3 3 Si(CH 3 2 (C2H 5 Si(CH 3

)(C

2

H

5 2 Si(C-H 5 3 Yet another mass-modification can be S obtained by attaching homo- or heteropeptides through X to the UP, TP or nucleoside Striphosphates. One example useful in generating mass-modified species with a mass 25 increment of 57 is the attachment of oligoglycines, mass-modifications of 74 (r=l.

131 188 245 m=4) are achieved. Simple oligoamides e' also can be used, mass-modifications of 74 88 102 (r=3, 116 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 (Oligonucleotides and Analogues, 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 designates i I mass differentiated nucleotides, primers or tags. In some instances, the superscript 0 NTPO, UP

O

can designate an unmodified species of a particular reactant, and the superscript i NTP i NTPI, NTP 2 etc.) can designate the i-th mass-modified species of that reactant. If. for example, more than one species of nucleic acids DNA clones) are to be concurrently sequenced by multiplex DNA sequencing, then i 1 different mass-modified nucleic acid primers (UP 0 UP I UPi) can be used to distinguish each set of base-specifically terminated fragments. wherein each speci.s 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 empi- the described massmodified 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 11): 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 i hybridize to tag sequences which are integrated into part of the four 15 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 appropriately 20 differentiated molecular weights (see for example FIGURE 13).

5 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 DNA clones simultaneously. The first reaction mixture is obtained by standard Sanger DNA sequencing having unknown DNA fragment I (clone I) integrated in an appropriate vector 25 M 13mpl8). employing an unmodified nucleic acid primer UPO and a standard mixture of the four unmodified deoxynucleoside triphosphates, dNTPO, and with 1/10th of one of the four dideoxynucleoside triphosphates. ddNTP 0 A second reaction mixture for DNA fragment 2 (clone 2) is obtained by employing a mass-modified nucleic acid primer UP I and. as before, the four unmodified nucleoside triphosphates, dNTPO.

containing in each separate Sanger reaction 1/10th of the chain-terminating unmodified dideoxynucleoside triphosphates ddNTP 0 In the other two experiments, the four Sanger reactions have the following compositions: DNA fragment 3 (clone UP 2 dNTPO, ddNTPO and DNA fragment 4 (clone UP 3 dNTPO. ddNTP 0 For mass spectrometric DNA sequencing, all base-specifically terminated reactions of the four clones are pooled and mass analyzed. The various mass peaks belonging to the four dideoxy-terminated ddT-terminated) fragment families are assigned to specifically elongated and ddTterminated fragments by searching (such as by a computer program) for the known molecular ion peaks of UP 0

UP

I

UP

2 and UP 3 extended by either one of the four dideoxynucleoside triphosphates. UPO-ddNO, UPl-ddN 0

UP

2 -ddN 0 and UP 3 -ddN 0 In this way, the first nucleotides of the four unknown DNA sequences of clone I 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 assignments are possible even in the worst case scenario in which the four mass-modified nucleic acid primers are extended by the same dideoxynucleoside triphosphate, the extension products then being, for example, UP 0 -ddT, UPl-ddT, UP 2 ddT and UP 3 -ddT, which differ by the known mass increment differentiating the four nucleic acid primers. In another embodiment of this invention, an analogous technique is employed using different vector; containing, for example, the SP6 and/or T7 promoter i10 sequences, and performing transcription with the nucleic acid primers UPO, UP 1

UP

2 and

UP

3 and either an RNA polymerase SP6 or T7 RNA polymerase) with chainelongating and terminating unmodified nucleoside triphosphates NTPO and 3'-dNTP o Here, the QNA sequence is being determined by Sanger RNA sequencing.

FIGURE 12 illustrates the process of multiplexing by mass-modified chain- 15 elongating or/and terminating nucleoside triphosphates in which three different DNA fragments (3 clones) are mass analyzed simultaneously. The first DNA Sanger sequencing reaction (DNA fragment 1, clone 1) is the standard mixture employing unmodified nucleic "acid primer UPO, dNTPO and in each of the four reactions one nf the four ddNTP 0 The second (DNA fragment 2, clone 2) and the third (DNA fragment 3, clone 3) have the following contents: Up0, dNTPO, ddNTP and UP 0 dNTPO, ddNTP 2 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-modif d deoxynucleoside triphosphate dNTP 1 dNTP 2 for chain elongation alone or in conjunction with the homologous equally mass-modified dideoxynucleoside triphosphate.

25 For the three clones depicted above, the contents of the reaction mixtures can be as follows: either UPO/dNTPO/ddNTPO, UP 0 /dNTP 1 /ddNTPO and UP 0 /dNTP2!ddNTP 0 or

UP

0 /dNTP 0 /ddNTPO, UP 0 /dNTpl/ddNTP 1 and UP 0 /dNTP 2 iddNTP 2 As described above, DNA sequencing can be performed by Sanger RNA sequencing employing unmodified nucleic acid primers, UPO. and an appropriate mixture of chain-elongating and terminating nucleoside triphosphates. The mass-modification can be again either in the chain-tenninating nucleoside triphosphate alone or in conjunction with mass-modified chain-elongating nucleoside triphosphates. Multiplexing is achieved by pooling the three base-specifically terminated sequencing reactions tle ddTTP terminated products) and simultaneously analyzing the pooled products by mass spectrometry. Again, the first extension products of the known nucleic acid primer sequence are assigned, via a computer program. Mass/sequence assignments are possible even in the worst case in which the nucleic acid primer is extended/terminated by the same nucleotide. ddT. in all three clones. The following configurations thus obtained can be well differentiated by their different mass-modifications: UPO-ddT

O

UP

0 -ddT

I

UPO-ddT 2 -22- 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 Chemiluminescent Detection." Nucleic Acids Res. Symposium Ser. No. 24. 318-321 (1991)); then pooling clones from different plex-vectors for DNA preparation and the four separate Sanger sequencing reactions using standard dNTP 0 /ddNTPO and nucleic acid primer UPO; 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 10 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 demultiplexing by one-by-one hybridization of specific "tag probes": and subsequently analyzing by rfiass spectrometry (see. for example. FIGURE 13). As a reference point, the four base-specifically terminated multiplex DNA fragment families are run by the mass *0 15 spectrometer and all ddTO-. ddAO-. ddC 0 and ddG 0 -terminated molecular ion peaks are respectively detected and memorized. Assignment of. for example. ddTO-terminated 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 family. Only those molecular ion peaks which arc capable of hybridizing to the specific tag probe are shifted to a higher molecular mass by the same known mass increment 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 remaining chain terminated fragment families with the same tag probe to assign the complete DNA sequence. This process is repeated i-I 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 example.

Molecular Cloning: A laboratory manual 2ed. ed. by Sambrook. Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: NY. 1989. Chapter 11). Furthermore, differet: ation 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 changing 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 mass spectrometric 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 by other methods known to those skilled in the art (sec. for example.

-23- Helene ce ul.. ature 344. 358 (1990) and Thuong et u. "Oligonucl-otides Attached to Intercalators. Photoreactive and Cleavage Agents" in F. Eckstein. Cligonucleotides and Analogues: 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 UPO-tag sequence/tag probe molecular weight plus the first extension product, ddT

O

then the second, the third.

etc.

In a combination of the latter approach with the previously described multiplexing processes, a further increase in multiplexing c;,n be achieved by using, in 10 addition to the tag probe/tag sequence interaction, mass-modified nucleic acid primers (FIGURES 7A and 7B) and/or mass-modified deoxynucleoside. dNTPO-i- and/or dideoxynucleoside triphosphates. .dNTPO-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 15 be determined by transcribing ,he unknown DNA sequence from appropriate promotercontaining vectors (see above) with various RNA polymerases and mixtures of NTPO-i/3'dNTPO-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 20 acid primer, the chain-elongating or terminating nucleoside triphosphates and/or the tag probes are. in a first step. modified by a precursor functionality such as azido, -N 3 or Smodified with a functional group in which the R in XR is H (FIGURES 7A, 7B, 9) thus providing temporary functions, but not limited to -OH. -NH 2 -NHR. -SH, -NCS.

-OCO(CH2)rCOOH (r 1-20). -NHCO(CH2)rCOOH (r 1-20). 25 -OCO(CH2)rl (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 terminated 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 (L1) for immobilization of the basespecifically terminated products: at least one nucleic acid primer having a linking group for reversibly and temporarily linking the primer and solid support through, for example. a photocleavable bond: a set of chain-elongating nucleotides dATP. dCTP.

-24dGTP and dTTP, or ATP, CTP, GTP and UTP); a set of chain-terminating nucleotides (such as 2',3'-dideoxynucotides for DNA synthesis or 3'-deoxynucleotides for RNA synthesis); and an appropriate polymerase for synthesizing complementary nucleotides.

Primers and/or terminating nucleotides can be mass-modified so that the base-specifically terminated 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 appropriate buffers as well as instructions for performing multiplex mass spectrometry to concurrently sequence multiple species of 10 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 appropriate polymerase. The mass-modified chain-terminating nucleotides are selected so that the addition of one of the chain terminators to a growing complementary nucleic acid can be distinguished by mass spectrometry.

EXAMPLE 1 Immobilization of primer-extension products of Sanger DNA sequencing reaction for S* 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/2propanol (NMM solution) (2/49/49 the excess liquid removed with filter paper and placed on a piece of plastic film or aluminum foil located on a heating block set to 550C.

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 550C. 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 cystamine, the disks are treated with 10 ul of a solution of 1M aqueous dithiothreitol (DTT)/2-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 I ml 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 I M aqueous dithiothreitol/2-propanol (1:1 v/v) and, before use, DTT is removed by three washings with I ml each of the NMM solution. The primer oligonucleotides with 5'-SH functionality can be prepared by various methods B.C.F Chu et al., Nucleic Acids Res. 14, 5591-5603 (1986), Sproat ct a..

,I I, Nucleic Acids Res. 1i, 4837-48 (1987) and Olipgonucleotides and Analogues: A Practical Approach Eckstein, editor), IRL Press Oxford. 19?1). Sequencing reactions according to the Sanger protocol are performed in a standard way H. Swerdlow et al., Nucleic Acids Res. 18, 1415-19 (1990)). In the presence of about 7-10 mM DTT the free 5'-thiol primer can be used; in other cases, the SH functionality can be protected. 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 terminated by a 10 min incubation at 70 0 C to denature the DNA polymerase (such as Klenow fragment, Sequerase) and the reaction mixtures are ethanol precipitated.

10 The supematants are removed and the pellets vo-r.xed with 25 ul of an IM 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 vortex..,g and centrifugation to remove excess DTT. The primer extension products with free 5'-thiol group are now 15 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 ffunctionalized with an amino group at the 5'-end which is introduced by standard 25 procedures 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 thiolated supports and monitored by the release ofpyridyl-2-thione as described above After denaturation of DNA polymerase and ethanol precipitation of the sequencing products, the supematants 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 250C. Excess SPDP is then removed by three extractions (vortexing, centrifugation) with 50 ul each of ethanol and the resulting pellets art dissolved in 10 ul 10 mM TEAA buffer pH 7.2 and coupled to the thiolated supports (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 three successive treatments with 10 ul of 10 mM 2-mercaptoethanol in 10 mM I EAA buffer pH -26- 7.2, lyophilized and analyzed by either ES or MALDI mass spectrometry.

This procedure can also be used for the mass-modified nucleic acid primers

UP

0 i in an analogous and appropriate way, taking into acc,'jun the chemical properties of the mass-modifying functionalities.

EXAMPLE 2 Immobilization of primer-extension products of Sanger DNA sequencing reaction for mass spectrometric analysis via the levulinyl group S* 10 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 dicyclohexyPcarbodiimide under standard conditions. For the Sanger sequencing reactions, nucleic acid primers, UPOi are used which are functionalized with a primary 15 amino group at the 5'-end introduced by standard procedures during automated DNA synthesis with aminolinker phosphoamidites as the final synthetic step. Sanger sequencing is performed under standard conditions (see above). The four reaction mixtures (150 ul each in an Eppendorf tube) are heated to 70 0 C 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 0* o in 10 mM TEAA buffer is added, vortexed and incubated at 25oC for 30 min. The excess of the reagent is removed by ethanol precipitation and centrifugatio.. The Fmoc group is cleaved off by resuspending the pellets in 10 ul of a solution of 20% piperidine in N.Ndimethylformamide/water (1:1 After 15 min at 250C, piperidine is thoroughly removed by three precipitations/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 v/vv) 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 cn a heating block at 55 0 C 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 pH 7.2. The purified primer-extension products are released by three successive treatments with 10 ul of 100 mM hydrazinium acetate buiffer pH 6.5, lyophilized and analyzed by either ES or MALDI mass spectrometry.

EXAMPLE 3 Immobilization of primer-extension products of Sanger DNA sequencing reaction for mass spectrometric analysis via a trypsin 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; 2.'49/49 v/v/v) and a linker arm introduced by reaction with 10 ul of a 10 mM solution of 1,6-diaminohexane in NMM. The excess diamine is removed by three washing steps with 100 ul of NMM solution. Using standard peptide synthesis protocols, 10 two L-lysine residues are attached by two successive condensations with N-Fmoc-N-tBoc- L-lysine pentafluorophenylester, the terminal Fmoc group is removed with piperidine in g* i 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 synthesis 15 procedures. The nucleic acid pr.ner-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 Eppendorf tubes) are heated for 10 min .'at 70 0 C 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 This solution is transferred to the Lys-Lys-DITC membrane disks and coupled on heating block set at 55 0 C. 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 typsin serving as an internal mass standard.

EXAMPLE 4 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 adenine dinucleotide (APAD) (Sigma) in NMM solution added. The excess APAD is removed by a 10 ul wash of NMM solution and the disks are treated with 10 ul of 10 mM sodium periodate in NMM solution (15 min, 25oC). Excess periodate is removed and the primerextension products of the four Sanger DNA sequencing reactions (150 ul each in -28- Eppendorf tubes) employing nucleic acid primers with a primary amino group at the end are ethanol precipitated, dissolved in 10 ul of a solution of N-methylmorpholine/2propanol/water (2/10/88 v/v/v) and coupled to the 2' 3'-dialdehydo groups of the immobilized 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 treatment with either NADase or pyrophosphatase in 10 mM TEAA buffer at pH 7.2 at 37 0 C for 15 min, Ig; lyophilized and analyzed by either ES or MALDI mass spectrometry, the enzymes serving 10 as internal mass standards.

SEXAMPLE 0 Synthesis of nucleic acid primers mass-modified by glycine residues at the of the sugar moiety of the terminal nucleoside Oligonucleotides are synthesized by standard automated DNA synthesis using B-cyanoethylphosphoamidites K6ster et al., Nucleic Acids Res. 12, 4539 (1984)) and a 5'-amino group is introduced at the end of solid phase DNA synthesis Agrawal et al., Nucleic Acids Res 14, 6227-45 (1986) or Sproat et al., Nucleic Acids Res. 20 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'-terminal amino group is dissolved in 100 ul absolute N,Ndimethylformamide (DMF) and condensed with 10 pmole N-Fmoc-glycine pentafluorophenyl ester for 60 min at 250C. After ethanol precipitation and centrifugation, the Fmoc group is cleaved off by a 10 min treatment with 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 lyophilized 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 massmodified primer oligonucleotides suitable for Sanger DNA or RNA sequencing.

Immobilization of these mass-modified nucleic acid primers UP 0 i after primer-extension during the sequencing process can be achieved as described, in EXAMPLES I to 4.

1 -29- EXAMPLE 6 Synthesis of nucleic acid primers mass-modified at C-5 of the beterocyclic base of a pyrimidine nucleoside with glycine residues Starting material was 5-(3-aminopropynyl- prepared and 3' 5'-de-O-acylated according to literature procedures (Haralambidis et al., Nucleic Acids Res. 4857-76 (1987)). 0.281 g (1.0 mmole) 5-(3-aminopropynyl-l)-2'deoxyuridine were reacted with 0.927 g (2.0 mmole) N-Fmoc-glycine pentafluorophenylester in 5 ml absolute N.N-dimeth:.lformamide in the presence of 0.129 S 10 g (I 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 Schloroform/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 15 solution of piperidine in DMF, evaporated in vacuo and the remaining solid material extracted three times with 20 ml ethylacetate. After having removed the remaining ethylacetate, N-Fmoc-glycine pentafluorophenylester is coupled as described above. 5-(3- (N-Fmoc-glycyl)-amidopropynyl-l)-2'-deoxyuridine is transformed into the dimethoxytritylated nucleoside-3'-O-B-cyanoethyl-N,N-diisopropylphosphoamidite and incorporated into automated oligonucleotide synthesis by standard procedures K6ster el 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 *i 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 25 solution of piperidine in DMF at room temperature. DMF is removed by a washing step with acetonitrile and the oligonucleotide dcprotected and purified in the standard way.

EXAMPLE 7 Synthesis of a nucleic acid primer mass-modified at C-5 of the heterocyclic base of a pyrimidine nucleoside with i-alanine residues Starting material was the same as in EXAMPLE 6. 0.281 g (1.0 mmole) 5-(3-Aminopropynyl- )-2'-deoxyuridine was reacted with N-Fmoc-B-alanine pentafluorophenylester (0.955 g. 2.0 mmole) in 5 ml N.N-dimethylformamide (DMF) in the presence of 0.129 g (174 ul; 1.0 mmole) N.N-disopropylethylamine for 60 min at room temperature. 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 B-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'-O-B-cyanoethyl- N,N-diisopropylphosphoamidite from 5-(3-(N-Fmoc-B-alanyl)-amidopropynyl-1 deoxyuridine and inccrporation into automated oligonccleotide 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 incorporated mass-modified nqcleotide, the nucleic acid primer molecules prepared according to EXAMPLES 6 and 7 would have a mass difference of 14 daltons.

EXAMPLE 8 S* o 10 Synthesis of a nucleic acid primer mass-modified at C-5 of the heterocyclic base of a pyrimidine nucleoside with ethylene glycol monomethyl ether As a nucleosidic component, 5-(3-aminopropynyl-1)-2'-deoxyuridine was used in this exarhple (see EXAMPLES 6 and The mass-modifying functionality was obtained as follows: 7.61 g (100.0 mmole) freshly distilled ethylene glycol monomethyl 15 ether dissolved in 50 ml absolute pyridine was reacted with 10.01 g (100.0 mmole) recrystallized succinic anhydride in the presence of 1.22 g (10.0 mmole) 4-N.Ndimethylaminopyridine overnight at room temperature. The reaction was terminated by the addition of water (5.0 ml), the reaction mixture evaporated in vacuo, 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 in vacuo, the residue redissolved in 80040* 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 succinylated 25 ethylene glycol monomethyl ether was dissolved in 100 ml dioxane containing 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 temperature for 4 hours.

Dicyclohexylurea was removed by filtration, the filtrate evaporated in vacuo and the residue redissolved in 50 ml anhydrous DMF. 12.5 ml (about 12.5 mmole 4nitrophenylester) of this solution was used to dissolve 2.81 g (10.0 mmole) 5-(3aminopropvnyl-l)-2'-deoxyuridine. The reaction was performed in the presence of 1.01 g (10.0 mmole; 1.4 ml) triethylamine at room temperature overnight. The reaction mixture 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 containing the desired compound were collected, evaporated, redissolved in 25 ml dichloromethane and precipitated into 250 ml pentane. The dried precipitate of 5-(3-N-(O-succinyl ethylene glycol monomethy! ether)amidopropynyl-l)-2'-deoxyuridine (yield: 65 is 5'-O-dimethoxytritylated and transformed into the nucleoside-3'-O-B-cyanoethyl-N. N-diisopropylphosphoamidite and -31incorporated 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 and purification of the primer oligonucleotide also follows standard procedures.

EXAMPLE 9 Synthesis of a nucleic acid primer mass-modified at C-5 of the heterocyclic base of a 0 pyrimidine nucleoside with diethylene glycol monomethyl ether Nucleosidic starting material was as in previous examples, 5-(3aminopropynyl- )-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) recrystallized succinic anhydride in the presence of 1.22 g (10.0 mmole) 4-N, N- 15 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 S mmole) of succinylated diethylene glycol monomethyl ether was transformed into the 4nitrophenylester and, subsequently, 12.5 mmole was reacted with 2.81 g (10.0 mmole) of 5-(3-aminopropynyl-l)-2'-deoxyuridine as described in EXAMPLE 8. Yield after silica 20 gel column chromatography and precipitation into pentane was 3.34 g (6.9 mmole, 69%).

After dimethoxytritylation and transformation into the nucleoside-Bcyanoethylphosphoamidite, the mass-modified building block is incorporated into automated chemical DNA synthesis according to standard procedures. Within the sequence of the nucleic acid primer UPO-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.

EXAMPLE Synthesis of a nucleic acid primer mass-modified at C-8 of the beterocyclic base of deoxyadenosine with glycine Starting material was N6-benzoyi-8-bromo-5'-O-(4,4'-dimethoxytrityl)- 2 deoxyadenosine prepared according to literature (Singh et al., Nucleic Acids Res. 18.

3339-45 (1990)). 632.5 mg (1.0 mmole) of this 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. Ndiisopropylethylamine and refluxed until the starting nucleosidic material had disappeared (4-6 hours) as checked by thin layer chromatography (TLC). The solvent was evaporated -32and the residue purified by silica gel chromatography (column 2.5x50 cm) using solvent mixtures of chloroform/methanol containing 0. 1 pyridine. The product fractions were combined, the solvent evaporated, the fractions dissolved in 5 mlJ dichioromethane and precipitated into 100 ml pentane. Yield was 487 mg (0.76 mmole, 76 Transformation into the corresponding nucleoside-B-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.

EXAMPLE II 0 Synthesis of a nucleic acid primer mass-rnodifked at C-8 of the heterocyclic base of deoxyadenosine with glycylglycine This derivative was prepared in analogy to the glycine derivative of *0EXAMPLE 10. 632.5 mg (1.0 mzr. N 6 -Benzoyl-8-bromo-5'-O-(4.4'-dimethoxytrityl)- :2'-deoxyadenosine was suspended in 5 ml absolute ethanol and reacted with 324.3 mg mmole) glycyl-glycine methyl ester in the presence of 241.4 mng (2.1 mmnole, 366 Al) N. N-diisopropylethylamine. The mixture was refluxed and completeness of the reaction 20 checked by TLC. Work-up and piification was similar to that described in EXAMPLE Yield after silica gel column chromatography and precipitation into pentane was 464 0: 0 mg (0.65 mmole, 65 Transformation into the nuclcoside-r-cyanoethylphiosphoamidite 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 I11 is 57.03 daltons.

EXAMPLEJ

Synthesis of a nucleic acid primer mass-modified at the C-2' of the sugar moiety of 2'-amino-2'-deoxythymidine with ethylene glycol monoinethyl ether residues Starting material was 5'-O-(4,4-dimethoxytrityl)-2'-arnino-2'-deoxythymidinc synthesized according to published procedures Verheyden et al., J. rg, Chen. 3.6.

250-254 (1971); Sasaki et al,L.QOg..Chem. 41, 3138-3143 (1976); Imazawa e al.J. Org.

Chemn. -44, 2039-2041 (1979); Hobbs el aL., J. rg- C em.!42.714-719 (1976); Ikehara et Chem. Pharm. Bull. Jap a, 6 240-244 (1978); see also PCT Application WO 88/00201). 5'-O-(4,4-Dirnethoxytrityl)-2'-amino-2'-deoxythymidine (559.62 mg. mmole) was reacted with 2.0 m-mole of the 4-nitrophenyl ester of succinylated ethylene glycol monomethyl ether (see EXAMPLE 8) in 10 ml dry DMF in the presence of

(I

Y

U II -33mmole (140 pl) 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 containing 0.1 triethylamine). The product containing fractions were combined, evaporated and precipitated into pentane. Yield was 524 mg (0.73 mmnol; 73 Transformation into the nucleoside-B-cyanoethyl-N,Ndiisopropy;phosphoamidite 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 acio 10 primer.

In an analogous way, by employing the 4-nitrophenyl es(er of succinylated S* diethylene glycol monomethyl ether (see EXAMPLE 9) ar.d triethylene glycol mor.omethyl ether, the corresponding mass-modified oligonucleotides are prepared. In the case of only one incorporated mass-modified nucleoside within the sequence, the mass difference bc-.vecn the ethylene, diethylene and triethylene glycol derivatives is 44.05, 88.1 and 132.15 daltons respectively.

EXAMPLE 13 Synthesis of a nucleic acid primer mass-modified in the internucleotidic linkage via alkylation of phosphorothioate groups Phosphorothioate-containing oligonucleotides were prepared according to standard procedures (see e.g. Gait et al., Nucleic Acids Res., 19 1183 (1991)). One, several or all interucleotide linkages can be modified in this way. The nucleic acid primer sequence (17-mer) 5'-dGTAAAACGACGGCCAGT was synthesized in 0.25 gmole scale on a DNA synthesizer 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 pl TE buffer (0.01 M Tris pH 8.0, 0.001 M EDTA) and by adding 16 1l of a solution of 20 mM solution of 2-iodoethanol (320 nmole; 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, Beckman; column: 4.5 x 250 mm; 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 containing 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 off the solid support, lyophilized and dissolved in 4 pl each .1 .1 -34of TE buffer pH 8.0 and alkylated by addition of 2 p1 of a 20 mM solution of 2iodoethanol in DMF. It is then analyzed by ES and/or MALDI mass spectrometry.

In an analogous way, employing instead of 2-iodoethaniol, 3iodopropanol, 4-iodobutanol mass-modified nucleic acid primer are obtained with a mass difference of 14.03, 28.06 and 42.03 daltons respectively compared to the unmodified phosphorothioate phosphodiester-containing oligonucleotide.

EXAMPTS 14 Synthesis of 2'-amino-2'-deoxyuridine-5'-triphosphate and 3'-amino-2',3'dideoxythymidine-S'-tripho3phate mass-modified at the or 3'-amino function with glycine or 11-alanine residues Starting material was 2'-azido-2'-deoxyuridine prepared according to literature (Verheyden el aL, J. r.Chem~. U. 250 (197 which was 4,4is 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.

000 With 191 mg (0.71 romole) 2'-azido-2'-deoxyuridine as starting material, 396 mg (0.65 mmol, 90.8 5'-O-(4,4-dirnethoxytrityl)-3'-O-acetyl-2-azido-2'-deoxuridine was O :obtained after purification via silica gel chromatography. Reduction of the azido group was performed using published conditions (Barta et aL, Terhdo 6 587-594 (1990)).

000000Yield of 5'-O-(4,4-diniethoxytrityl)-3'-O-acetyl-2'-anino-2'-deoxyuxidine after silica gel chromatography was 288 mg (0.49 mmole; 76 This protected 2'-axnino-2'-deoxyuridine derivative (588 mg, 1.0 mniole) was o reacted with 2 equivalents (927 mg, 2.0 mnmole) N-Fmoc-glycine pentafluorophenyl ester 25 in 10 ml dry DMF overnight at room temperature in the presence of 1.0 mmole (174 jil) N,N-diisopropylethylamine. Solvents were rem oved by evaporation in Vacuo and the residue purified by silica gel chromatography. Yield was 711 mg (0.71 mrnole, 82 Detritylation was achieved by a one hour tretment with 800/ aqueous acetic acid at room temperature. The residue was evaporated to dryness, co-evaporated twice with toluene.

suspended in I ml dry acetonitrile and 5'-phosphorylatcd with POC1 3 according to literature (Yoshikawa etl Bull, Chem, Soc. Japa -42. 3505 (1969) and Sowa elat.

Bull. Chem. Soc. Japan 41, 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 (trnbutylammonium) pyrophosphate in DMF according to literature Seela et at., Helvetica Chimica Acta 7-4. 1048 (1991)). The Fmoc and the Y-O-acetyl groups were removed by a one-hour treatment with concentrated aqueous ammonia at room temperature and the reaction mixture evaporated and lyophilized. Purification also followed standard procedures by using anion-exchange chromatography on DEAE- Sephadex with a linear gradient of triethylamoniuni bicarbonate 1 M 1.0 M).

Triphosphate containing 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 mmnole).

A glycyl-glycine modified 2'-aniino-2'-deoxyuridine-5'-triphosphate was sobtained by removing the Fmoc group from 5'-O-(4,4-dimethoxytrityl)-3'-Q-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 condensation with N- S Fmoc-glycine pentafluorophenyl ester. Starting with 1.0 rnrole of the 2'-N-glycyl-2'to 1 amino-2'-deoxyuridine derivative and following the procedure described above, 0.72 *mxnole of the corresponding 2'-(N-glycyl-glycyl)-2-amino-2'-deoxyunidine-5'-; L' -osphate was obtained.

5 Sarting with 4 ,4-dimethoxytrityl)-3'-O-acetyl-2'-amino-2'deoxyuridine and coupling with N-Fmoc-B-alanine pentafluorophenyl ester, the corresponding 2'-(N-B3-alanyl)-2'-aznino-2'-deoxyuridine-5.-tri phosphate 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, 8-alanine and glycyl-glycine mass-modified nucleosides is, per nucleotide incorporated, 58.06, 72.09 and 115.1 daltons respectively.

20 When starting with 5'-O-(4,4.-dimethoxytrityl)-3-amnino-2',3'dideoxythymnidine (obtained by published procedures, see EXAMPLE 12), the corresponding 3'-(N-glycyl)-3'-amino-/ 3'-(-N-glycyl-glycyl)-3'-arnino-/ and alanyl)-3'-arnino -2,3'-dideoxythymidine-5'-triphosphates can be obtained. These massmodified nucleoside triphosphates serve as a terminating nucleotide unit in the Sanger DNA sequencing reactions providing a mass difference per terminated fragment of 58.06, 72.09 and 115.1 daltons; respectively when used in the multiplexing sequencing mode.

The mass-differentiated fragments can then be analyzed by ES and/or M.ALDI mass spectrometry.

Synthesis of deoxyuridine-5'-triphosphate mass--nodiied at C-5 of the heterocyclic base with glycine, glycyl-glycine and B-alanine residues.

0.281 g (1.0 mxnole) 5-(3-Aminopropynyl- 1 )-2'-deoxyu-idine (see EXAMPLE 6) was reacted with either 0.927 g (2.0 mmole) N-Fmoc-glycine pentafluorophenylester or 0.955g (2.0 nole) N-Fmoc-3-alanine pentafluorophenyl ester in 5 ml dry DMF in the presence of 0. 129 g N, N-diisopropylethylamine (174 ul, nunole) overnight at room temperature. Solvents were removed by evaporation in vacuo and the condensation products purified by flash chromatography on silica gel (Still et al..

J. rs. Chem. 2923-2925 (1978)). Yields were 476 mg (0.85 mniole: 85%) for the glycine -ind 436 mg (0.76 nunole; 76%) for the 11-alanine derivatives. For the synthesis of the glycyl-glycine derivative. the Fmoc group Of 1 .0 mrnole Fmoc-glvcine-deoxyuridine derivative was removed by one-hour treatment with 20% piperidine in DMF at room temperature. Solvents were removed by evaporation in vacuo, the residue was coevaporated twice with toluene and condensed with 0.927 g (2.0 mmole) N-Fmoc-glycine pentafluorophenyl ester and purified as described above. Yield was 445 mg (0.72 mmole; The glycyl-, glycyl-glycyl- and B3-alanyl-2'-deoxyuridine derivatives. N-protected I:;:with the Fmoc group were transformed to the 3'-O-acetyl derivatives by tritylation with io 4.4-dimethoxytrityl chloride in pyridine and acety lation with acetic anhydride in pyridine in a one-pot reaction and subsequently detritylated by one hour treatment with aqucous acetic acid according to standard procedures. Solvents were removed, the gg~g *residues dissolved in 100 ml chloroform and extracted twice with 50 ml 10/* sodium C 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 *g~gg mmole; 71 for the glycyl-, 351 mg (0.57 mmnole; 75%) for the 3-alanyl- and 323 mg (0.49 mmrole; 68%) for the glycyl-glycyl-3-O'-acetyl-2'-deoxyuridine derivatives respectively. Phosphorylation at the 5'-QH %%ith POC1 3 transformation into the triphosphate by in-situ reaction with tetra(tri-n-butylamnmonium) pyrophosphate in DMF.

20 3'-de-O-acetylation, cleavage of the Fmoc: group, and final purification by anion-exchange chromatography on DEAE-Sephadex was performed as described in EXAMPLE 14.

Yields according to UV-absorbance of the uracil moiety were 0.41 mmole e glycyl)-amidopropynyl-l1)-2'-deoxyuridine-S'-triphosphate 0.43 mmole al any l)-amidopropynyl -l)-2'-deoxyuridine- 5'-ri phosphate and 0.38 mmolc glycyl-glycyl)-antidopropynyl- I)-2'-deoxyuridine-5'-triphosphate These -mass-modified nucleoside triphosphates were incorporated during the Sanger DNA sequencing primer-extension reactions.

When using 5-(3-aininopropynyl-l )-2',3'-dideoxyuridine as starting material and following an analogous reaction sequence the corresponding glycyl-. glycyl-glycyland B-alanyl-2,.3'-dideoxyuridine-5'-triphosphates were obtained in yields of 69. 63 and 7 1% respectively. These mass-modified nucleoside triphosphates serve as chainterminating nucleotides during the Sanger DNA sequencing reactions. The mass-modified sequencing ladders are analyzed by either ES or M1ALDI mass spectrometry.

EXAMPLIF 1 Synthesis Of 8-glycyl- and 8-glycyl-glycyl-2'-deoxvadenoine-5'-tripbospbate 727 mg (1.0 rnmole) o, N 6 -(4-tert-butylphenoxyacctyl)-8-glycvl-5'-(4.4dimethoxytrirl)-2'- deoxyadenosine or 800 mg (1 .0 mmole) N 6 -(4-tertbutlphnoxaccyl-8-]Ycl-gycl.5-(44-dmetoxtriyl)2'-eoyadenosine prepared according to EXAMPLES 10 and I11 and literature (K6ster el at. Ixmh 31. 362 (1981)) were acetylated with acetic anhydride in pyridine at the 3-O11, detritylated at the with 801/ acetic acid in a one-pot reaction and transformed into the triphosphates via phosphorylation with POC1 3 and reaction in-situ with. tetra(trj-nbutylamnnonium) pyrophosphate as described in EXAMPLE 14. Deprotection of the N 6 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 S temperature. Ammonia was removed by lyophilization and the residue washed with 10 dichloromethane, solvent removed by evaporation in vacua and the remaining solid material purified by anion-exchange chromatography on DEAE-Sephadex using a linear gradient of triethylammoniuxn bicarbonate from 0. 1 to 1.0 M. The nucleoside triphosphate S containing fracp~ons (checked by LC on polyethyleneimine cellulose plates) were 33*combined and lyophillized. Yield of the 8 (determined by UV-absorbance of the adenine moiety) was 57% (0.57 rnnole). The yield for the 8-lclgyy-'doydnsn-'tihsht was 51% (0.51 mmole).

33. These mass-modified nucleoside triphosphates were incorporated during :~.primer-extension in the Sanger DNA sequencing reactions.

When using the corresponding

N

6 4 -tert-butylphenoxyacetyl)8glycyl. or 20 glclgyy-'O(,-ichxtiy)2.'ddoydnsn derivatives as starting materials prepared according to standard procedures (see, for the introduction of the 2',3'-function: Secla et at., Helvetica Chiica Acta 14, 1048-1058 (1991)) and using an :0 analogous reaction sequence as described above, the chain-terminating mass-modified nucleoside triphosphates 8-glycyl- and 8 -glycyl-giycyl-2'.3'-dideoxyadenosine-5.

triphosphates were obtained in 53 and 47% yields respectively. The mass-modified sequencing fragment ladders are analyzed by either ES or MALDI mass spectrometry.

EXAM LEJ17 Mass-Modification of Sanger DNA sequencing fragment ladders by incorporation of chain-elongating 2'-deoxy- and chain-terminating 29 S-)-triphosphate and subsequent alkylation with 2-iodoethanoj and 3-iodopropanol 2 3 -Dideoxythymidine5'-(alphaS)tiphsphate was prepared according to published procedures for the alpha-S-tri phosphate moiety: Eckstein et at., Bachcm~.is 1685 (1976) and Accounts Chem. Res- 12, 204 (1978) and for the dideoxy moiety: Seela et at., Helvetica Chimica Acta, 7A, 1048-1058 (199 Sanger DNA sequencing reactions employing 2 '-deoxythymidine-5'-(alpha..S)-tiphosphate are performed according to standard protocols Eckstein, Ann Rv. iochem. 5A. 367 (1985)). When using 2 3 '-dideoxythymidines'-(alpha-S)uiphosphatcs. this is used instead of the unmodified 2',3'-dideoxythymidine-5'-triphosphate in standard Sanger DNA sequencing (see e.g. Swerdlow et al., Nucleic Acids Res. 1, 1415-1419 (1990)). The template (2 pmole) and the nucleic acid M 13 sequencing primer (4 pmole) modified according to EXAMPLE 1 are annealed by heating to 65 0 C in 100 ul of 10 mM Tris-HCI pH 7.5, 10 mM MgCl 2 50 mM NaCI, 7 mM dithiothreitol (DTT) for 5 min and slowly brought to 37°C 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 ofdATP, dCTP, dTTP, 300 uM c7-deaza-dGTP, 5 uM *dideoxythymidine-5'-(alpha-S)-triphosphate and 40 units Sequenase (United States 10 Biochemicals). Polymerization is performed for 10 min at 370C, the reaction mixture heated to 70°C to inactivate the Sequenase, ethanol precipitated and coupled to thiolatcd Sequelon membrane disks (8 mm diameter) as described in EXAMPLE 1. Alkylation is *performed by treating the disks with 10 ul of 10 mM solution of either 2-iodoethanol or 3iodopropanol in NMM (N-methylmorpholine/water/2-propanol, 2/49/49. v/v/v) (three times), washing with 10 ul NMM (three times) and cleaving the alkylated T-terminated primer-extension products off the support by treatment with DTT as described in EXAMPLE 1. Analysis of the mass-modified fragment families is performed with either "ES or MALDI mass spectrometry.

20 EXAMPLE 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 prepared. Various matrices were used for this Example and Examples 19- 21 such as dihydroxybenzoic acid, sinapinic acid, 3-hydroxypicolinic acid, 2,4,6trihydroxyacetophenone. Oligonucleotides were lyophilized after purification by HPLC and taken up in ultrapure water (MilliQ, Millipore) using amounts to obtain a concentration of pmoles/gl as stock solution. An aliquot (1 l) of this concentration or a dilution in ultrapure water was mixed with I l of the matrix solution on a flat metal surface serving as the probe tip and dried with a fan using cold air. In some experiments, 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 Example and Examples 19-21 on different commercial instruments such as Vision 2000 (Finnigan-MAT), VG TofSpec (Fisons 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 appropriate mass range such as insulin, gramicidin S.

trypsinogen, bovine serum albumen, and cytochrome C. All spectra were generated by -39employing a nitrogen laser with 5 nsec pulses at a wavelength of 337 rnm. Laser energy varied between 106 and 107 W/cm 2 To improve signal-to-noise ratio generally, the intensities of 10 to 30 laser shots were accumulated.

EXAMPLE19 Mass Spectrometric Analysis of a 50-mer and a 99-mer Two large oligonucleotides were analyzed by mass spectrometry. The d (TAACGGTCATTACGGCCATTGACTGTAGGACCTGCATTACATGACTAGCT) (SEQ S. 10 ID NO:3) and dT(pdT) 9 9 were used. The oligodeoxynucleotides were synthesized using 0 -cyanoethylphosphoamidites and purified using published procedures.(e.g. N.D. Sinha. J.

Biernat. J. McManus and H. K6ster. 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 a*so ffmol 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 EXAMPLE Simulation of the DNA Sequencing Results of FIGURE 2 The 13 DNA sequences representing the nested dT-terminated fragn..nis of tin 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 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 1I -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.

EXAMPLE 21 MALDI-MS Analysis of a Mass-Modified Oligonucleotide A 17-iner was mass-modified at C-5 of one or two deoxywidine moieties. 5-[13- (2-Methoxyethoxyl)-tridecyne- I -yl]-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyuridine-3Y45-cyanoethyl- N, N-diisopropylphosphoamidite was used to synthesize the modified I 7-mers using the methods described in Example 19.

V.a The modified 17-mers were 1 W a: d (TAAAACGACGGCCAGUG) (molecular mass: 5454) (SEQ ID NO:4) X 0 b: d (UAAAACGACGGCCAGUG) (molecular mass 5634) (SEQ ID where X I I-OH (unmodified I 7-mer. molecular mass: 5273) a~aa.~ yzed The samples were prepared and 500 fmol of each modified I 7-mer was anlzdusing MALDI-MS as described in Example 18. The conditions used were 25 reflectron positive ion mode with an accceration of 5 kV and postacceleration of 20 MV t loe The MALDI-TOF spectra which were generated were superimposed and arc show.%n in' FIGURE 18.

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

EQUIVALENTIS

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 arc covered by the following claims.

SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: KOSTE;, HUBERT STREET: 1640 MONUMENT STREET CITY: CONCORD STATE: MASSACHUSETTS COUNTRY: USA POSTAL CODE (ZIP): 01742 TELEPHONE: (508) 369-9790 6 15 (ii) TITLE OF INVENTION: DNA SEQUENCING BY MASS SPECTROMETRY Sb (iii) NUMBER OF SEQUENCES: COMPUTER READABLE FORM: 20 MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: ASCII (text) 25 (vi) CURRENT APPL.CATION

DATA:

s-o APPLICATION

NUMBER:

FILING DATE: 06-JAN-1994

CLASSIFICATION:

30 (vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: US 08/001.323 FILING DATE: 07-JAN-1993 CLASSIFICATION: 1807 0e S' 35 (viii) ATTORNEY/AGENT

INFORMATION:

NAME: DeConti, Giulio A.

REGISTRATION NUMBER: 31,503 REFERENCE/DOCKET NUMBER: HKI-003CP (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (617) 227-7400 TELEFAX: (617) 227-5941 INFORMATION FOR SEQ ID NO:l: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) HYPOTHETICAL:

YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: -42- CATGCCATGG CATG 14 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) HYPOTHETICAL:

YES

fes*: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 00 20 AAATTGTGCA LPATCCTGCAG C 21 INFORMATION FOR SEQ ID NO:3: cc CiW SEQ~UENCE CHARACTEISTICS: se 25 LENGTH: So base pairs CB) TYPE: nucleic acid Ce STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid C Ciii) HYPOTIETICAL: YES 0060 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TAACGGTCAT TACGGCCATT GACTGTAG.GA CCTGCATTAC ATGACTAGCT so INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single CD) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: TAAAACGACG GGCCAGXG 17 -43- INFORMATION FOR SEQ, ID NO:S SEQUENCE CHARACTER.ISTICS: LsNGT: 17 base pairs TYPE: nucleic acid CC) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) HYPOTHETICAL: YES is (xi) SEQUENCE DESCRIPTION: SEQ ID NO:S: XAAAACGACG GGCCAZXG S S *5 S 0 *5

S

0 5005@5

S

0 0

S

SS

0 0 *0 S S SS S 0 0

S

Se

S

0@5@

S

Claims

1. An ionized and volatilized mass-modified nucleic acid molecule, comprising at least two mass modified nucleotides which are not naturally occurring and which are not isotopically labeled, wherein the molecule is positively charged.

2. An ionized mass-modified nucleic acid molecule, comprising: at least one mass modified nucleotide, wherein the molecule is positively charged, and comprises a member selected from the group consisting of: a mass-modified universal primer and a mass-modified initiator oligonucleotide.

3. An ionized mass-modified nucleic acid molecule of claim 1, comprising at least io one mass-modified nucleotide containing a mass-modifying functionality attached to a hcterocyclic base of the nucleotide.

4. A positively charged ionized mass-modified nucleic acid molecule, comprising: at least one mass-modified nucleotide containing a modified heterocyclic base selected from the group consisting of a cytosine moiety modified at C-5, a thymine moiety 5 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 c -deazaadenine moiety modified at C-8, a c'-deazaadeninc moiety modified at C-7, a guanine moiety modified at C-8, a c 7 deazaguanine moiety modified at C-8, a c 7 -deazaguanine moiety modified at C-7, a hypoxanthine moiety modified at C-8, a c 7 -deazahypoxanthine moiety modified at C-8, and 20 a c -deazahypoxanthine moiety modified at C-7. An ionized mass-modified nucleic acid molecule of claim 1, comprising at least one mass-modified nucleotide containing a mass-modifying functionality attached to at least one phosphorus of the nucleotide.

6. A positively charged ionized mass-modified nucleic acid molecule, comprising: 25 at least one mass-modified nucleotide containing a mass-modifying functionality (M) attached to at least one sugar moiety of the nucleotide.

7. A positively charged ionized mass-modified nucleic acid molecule, comprising: a mass-modifying functionality attached to at least one sugar moiety of the nucleic acid molecule, wherein the sugar is modified at a position selected from the group consisting of an internal C- 2' position, an external C-2' position, and an external C-5' position.

8. A mass-modified nucleic acid molecule of claim 2, wherein a mass-modifying functionality is attached to at least one sugar moiety of a 5'-terminal nucleotide of the primer, and wherein the mass-modifying function is a linking functionality

9. An ionized mass-modified nucleic acid molecule, comprising at least one mass- modified nucleotide containing a mass-modifying functionality incorporated into the molecule, wherein is selected from the group consisting of F, Cl, Br, I, Si(CH) 3 Si(CH3) 2 (C 2 Hs), Si(CH 3 )(C2H5) 2 Si(C 2 H 5 3 CH 2 F, CHF 2 and CF 3 A set of mass-differentiated tag probes wherein, each tag probe in the set comprises a sequence of nucleotides which is complementary by Watson-Crick base pairing to a tag sequence present within at least one 7 set of base-specifically terminated fragments; A24216 the tag sequences to which each tag probe is complementary are different for each tag probe; each tag probe in the set comprises at least one mass-modified nucleotide; and the mass-modified nucleotides are not isotopically labelled and have different mass modifications in each tag probe.

11. The set of mass-differentiated tag probes of claim 10, wherein at least one of the mass-modified nucleotides comprises a mass-modifying functionality attached to the heterocyclic base.

12. The set of mass-differentiated tag probes of claim 11, wherein the mass- o0 modified heterocyclic base is 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, a uracil moiety modified at C-5, an adenine moiety modified at C-8, a 7-deazaadenine moiety modified at C-8, a, a c7deazaadenine moiety modified at C-7, a guanine moiety modified at C-8, a c -deazaguanine moiety modified at C-8, a guanine moiety modified at C-8, a c 7 -deazaguanine moiety modified at C-8, a c 7 -deazaguanine moiety modified at C-7, a hypoxanthine moiety modified at C-8, a c 7 deazahypoxanthine moiety modified at C-8, and a c 7 -deazahypoxanthine moiety modified at C-7.

13. The set of mass-differentiated tag probes of claim 10, wherein at least one of the mass-modified nucleotides comprises a mass-modifying functionality attached to the phosphorus atom forming an intemucleotidic linkage of the tag probe.

14. The set of mass-differentiated tag probes of claim 10, wherein at least one of Sthe mass-modified nucleotides comprises a mass-modifying functionality attached to the sugar moiety. The set of mass-differentiated tag probes of claim 10, wherein at least one of S 25 the tag probes further comprises a cross-linking group (CL) which allows for covalent binding to the corresponding and complementary tag sequences.

16. The set of mass-differentiated tag probes of claim 15, wherein the cross-linking group (CL) is activated photochemically and is derived from at least one photoactivatable group selected from the group consisting ofpsoralen and an ellipticine. 30 17. The set of mass-differentiated tag probes of claim 10, wherein at least one of the tag probes is mass-modified with a mass-modifying functionality selected from the group consisting of XR, F, Cl, Br, I, Si(CH 3 3 Si(CH 3 2 (C 2 H 5 Si(CH 3 )(C 2 Hs) 2 Si(C 2 H 5 3 CH 2 F, CHF 2 and CF 3 wherein X is selected from the group consisting of -OCO(CH 2 )rCOO- (where r=1-20), -NHCO(CH 2 )rCOO- (where r=l-20), OS0 2 and R is selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, t-butyl, hexyl, benzyl, benzhydryl, halogen, trityl, substituted trityl, aryl, substituted aryl, polyoxymethylene, monoalkylated polyoxymethylene, a polyethylene imine, -(NH(CH2)rNHCO(CH 2 )rCO-)m-NH-(CH 2 )r-NH-CO-(CH 2 )r-COOH, -(NH(CH 2 )rCO-)m- NH-(CH 2 )r-COOH, -(O(CH 2 )rCO-)m-O-(CH 2 )r-COOH, -Si(Y) 3 -(NHCHaaCOOH), -(CH 2 CH20)m-CH 2 CH 2 0H, and -(CH 2 CH 2 0),-CH 2 CH 2 0-Y, where m is in the range of 0 to 200, Y is a lower alkyl group selected from a group consisting of methyl, ethyl, propyl, A23216 Q 46 isopropyl, t-butyl and hexyl, r is in the range of 1 to 20, and aa represents the amino acid side chain of a naturally occurring amino acid.

18. The set of mass-differentiated tag probes of claim 10, wherein one or more mass-modifying functionalities incorporated into the probes are generated from one or more precursor functionalities (PF) attached to the mass-differentiated tag probes, and wherein the precursor functionalities (PF) are selected from the group consisting of -N 3 -NH 2 -SH, -NCS, -OCO(CH 2 )rCOOH (where r=l-20), -NHCO(CH 2 )rCOOH (where r=l- -OSO 2 0H, -OCO(CH 2 )rI (where r=l-20), and -OP(O-Alkyl)N(Alkyl) 2

19. A positively charged ionized mass-modified nucleic acid molecule, comprising: two or more mass modified nucleotides selected from the group consisting of a mass- modified 2'-deoxynucleotide, a mass-modified 2',3'-dideoxynucleotide, a mass-modified nucleotide and a mass-modified 3'-deoxynucleotide, wherein the two or more mass- modified nucleotides are different from each other. An ionized mass-modified nucleic acid molecule, comprising at least one mass modified nucleotide selected from the group consisting of a mass-modified 2'- deoxynucleotide, a mass-modified 2',3'-dideoxynucleotide, a mass-modified nucleotide and a mass-modified 3'-deoxynucleotide, wherein the mass modified nucleic acid molecule comprises a modified heterocyclic base selected from a group consisting of a c 7 deazaadenine moity modified at a cdeazaadenine moiety modified at C-7, a 20 deazaguanine moiety modified at C-8, a c-deazaguanine moiety modified at C-7, a deazaguanine moiety modified at C-8, a c -deazaguanine moiety modified at C-7, a hypoxanthine moiety modified at C-8, a c 7 -deazahypoxanthine moiety modified at C-8, and S a c -deazahypoxanthine moiety modified at C-7.

21. An ionized positively charged duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein the mass-modified tag probe comprises at least one mass-modified nucleotide.

22. An ionized duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein the S mass-modified tag probe comprises at least one mass-modified nucleotide, wherein at least 30 one of the mass-modified nucleotides comprises a mass-modifying functionality (M) attached to the heterocyclic base.

23. An ionized duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein: the mass-modified tag probe comprises at least one mass-modified nucleotide; and a mass- modifying functionality incorporated into the tag probe is attached to the phosphorus atom forming an internucleotidic linkage of the tag probe.

24. An ionized duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein: the mass-modified tag probe comprises at least one mass-modified nucleotide; and at least one of the mass-modified nucleotides comprises a mass-modifying functionality attached to Sthe sugar moiety. A23216 47 An ionized duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein: the mass-modified tag probe comprises at least one mass-modified nucleotide; and the tag probe further comprises a cross-linking group (CL) which allows for covalent binding to the tag sequence.

26. An ionized mass-modified nucleic acid molecule, comprising at least one mass modified nucleotide wherein a mass-modifying functionality incorporated into the molecule is generated from a precursor functionality (PF) attached to one or more of a nucleic acid primer, a chain-elongating nucleoside triphosphate or a chain-terminating nucleoside triphosphate, and wherein the precursor functionality (PF) is selected from the group consisting of -N 3 -NH 2 -SH, -NCS, -OCO(CH 2 )rCOOH (where r=l-20), NHCO(CH 2 )rCOOH (where r=l-20), -OSO20H, -OCO(CH 2 )rI (where r=l-20), -CONH 2 NH-C(S)-NH 2 OP(O-Alkyl)OH, and O-CO-CH 2 -SH.

27. The set of mass-differentiated tag probes of claim 10, wherein one or more mass-modifying functionalities incorporated into the probes are generated from a precursor functionality (PF) attached to the mass-differentiated tag probes, and wherein the precursor functionalities are selected from the group consisting of -N 3 -NH 2 -SH, -NCS, -OCO(CH 2 )rCOOH (where r-1-20), -NHCO(CH 2 )rCOOH (where r=l-20), -OCO(CH 2 )rI (where r=1-20), -CONH 2 -NH-C(S)-NH 2 OP(O-Alkyl)OH, and O-CO-CH 2 S* 20 SH.

28. The set of mass-differentiated tag probes of claim 10, wherein the tag probes are mass-modified with a mass-modifying functionality selected from the group consisting ofXR, F, C1, Br, I, Si(CH 3 3 Si(CH 3 2 (C 2 Hs), Si(CH 3 )(C 2 H 5 2 Si(C 2 Hs) 3 CH 2 F, CHF 2 and CF 3 wherein X is selected from the group consisting of -OCO(CH 2 )rCOO- (where r=l-20), -NHCO(CH 2 )rCOO- (where r=l-20), and -OP(O-Alkyl)O- and R is selected from the group consisting of H, N 3 methyl, ethyl, propyl, isopropyl, t-butyl, hexyl, benzyl, benzhydryl, halogen, trityl, substituted trityl, aryl, substituted aryl, (-NH(CH2)rNHCO(CH 2 )rCO-)m-NH-(CH 2 )r-NH-CO-(CH 2 )r-COOH, (-NH(CH 2 )rCO-)m-NH-(CH 2 )r-COOH, (-O(CH 2 )rCO-)m-O-(CH 2 )r-COOH, -Si(Y) 3 30 -(NHCHaaCO-)m-NHCHaaCOOH, -(CH 2 CH 2 0)m-CH 2 CH 2 0H, and -(CH 2 CH 2 0)m-CH 2 CH 2 0-Y, where m is in the range of 0 to 200, Y is a lower alkyl group selected from a group consisting of methyl, ethyl, propyl, isopropyl, t-butyl and hexyl, r is in the range of 1 to 20, and aa represents the amino acid side chain of a naturally occurring amino acid.

29. The set of mass-differentiated tag probes of claim 10, wherein the tag probes are mass-modified with a mass-modifying functionality selected from the group consisting of XR, F, Cl, Br, I, Si(CH 3 3 Si(CH 3 2 (C 2 H 5 Si(CH 3 )(C 2 Hs) 2 Si(C 2 H 5 3 CH 2 F, CHF 2 and CF 3 wherein X is selected from the group consisting of -OCO(CH 2 )rCOO- (where r=1-20), -NHC(O), -CONH-, -NH-C(S)-NH-, -NHCO(CH 2 )rCOO- (where r=l-20), -OS0 2 -O-CO-CH 2 and -OP(O-Alkyl)O- and R S is selected from the group consisting of H, N 3 methyl, ethyl, propyl, isopropyl, t-butyl, hexyl, benzyl, benzhydryl, halogen, trityl, substituted trityl, aryl, substituted aryl, (CH 2 )m- A23216 27 JUL. 2001 11:36 SPRUSON AND FERGUSON 61292615486 NO, 1198 P. 14 48 CH 2 -OH, (CH2)m-CH2-O-Y, (CH2CH2NH),-CH2-CH2-NH2, -(NH(CH 2 ),NHCO(CH 2 ),CO- )m-NH-(CH2),-NH-CO-(CH 2 )r-COOH, -(NH(CH2),CO-)n-NH-(CH2)r-COOH, -(NH-CHY- CO)m-NH-CHY-COOH, (-O(CH2)rCO-)m-O-(CH 2 ),-COOH, -Si(Y) 3 -(NHCHaaCO-),- NHCHaaCOOH, CH 2 F, CHF2, CF3, -(CH2CH 2 0)m-CHH 2 C OH, and -(CH2CH0)m-CH 2 CH20-Y, where m is in the range of 0 to 200, Y is a lower alkyl group selected from a group consisting of methyl, ethyl, propyl, isopropyl, t-butyl and hexyl, r is in the range of I to 20, and aa represents the amino acid side chain of a naturally occurring amino acid. An ionized and volatilized mass-modified nucleic acid molecule, comrising at least two mass modified nucleotides which are not naturally occurring and which are not isotopically labeled.

31. A method of sequencing a nucleic acid, comprising the steps of: a) starting from a nucleic acid primer and in the presence of chain-terminating and chain-elongating nucleotides, synthesizing complementary nucleic acids that are comple- mentary to the nucleic acid to be sequenced, whereby four sets of base-specifically terminated complementary nucleic acid fragments are produced, wherein: the nucleic acid fragments comprise nucleotides with a modification at a base, a sugar or a phosphate of a nucleotide; and the modification improves the separation or resolution of the fragments when 20 analyzed compared to unmodified fragments; b) determining the molecular weight value of each base-specifically terminated fragment simultaneously by mass spectrometry; and c) determining the nucleotide sequence by aligning the base-specifically Sterminated fragments according to molecular weight. 2s

32. A method of multiplex sequence analysis of nucleic acid species, comprising the steps of a) reversibly linking nucleic acid primers to a solid support through a linking group; b) synthesizing complementary nucleic acids which are complementary to the nucleic acid species to be sequenced, starting from the nucleic acid primers and in the presence of chain-terminating and chain-elongating nucleotides so as to produce sets of base-specifically terminated complementary nucleic acid fragments for each species; c) determining the molecular weight value of each base-specifically terminated fragment for all species simultaneously by matrix assisted laser desorption/ionization mass spectrometry wherein the fragments are cleaved from the solid support by a laser during mass spectrometry; and d) determining the nu c leotide sequence of the species by aligning the base- specifically terminated fragments according to molecular weight; wherein at least one reagent selected from a group consisting of, a nucleic acid priner, a chain-elongating nucleotide, or a chain-terminating nucleotide is mass-modified, wherein each set of base-specifically terminated fragments of a species has a sufficient A2316 27. JUL. 2001 11:37 SPRUSON AND FERGUSON 61292615486 NO, 1198-'P. 49 mass difference from the sets of base-specifically terminated fragments of other species to be distinguished by mass spectrometry.

33. A method of sequencing a nucleic acid, comprising the steps of: a) synthesizing complementary nucleic acids that 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, to produce base-specifically terminated complementary nucleic acid fragments; b) exposing the base-specifically terminated complementary nucleic acid fragments to a single laser to produce desorbed/ionized fragments; c) determining the molecular weight value of each desorbed/ionized fragment produced by step by mass spectrometry; and d) determining the nucleotide sequence by aligning the base-specifically terminated nucleic acid fragments according to molecular weight.

34. The method of claim 33, wherein the molecular weight value of each fragment S is is determined by matrix-assisted laser desorption/ionization (MALDI-MS). S.

35. The method of claim 33, wherein the molecular weight value of each fragment is determined by electrospray mass spectrometry (ES-MS).

36. The method of claim 33, wherein more than one species of nucleic acid are simultaneously sequenced by multiplex mass spectrometric nucleic acid sequencing 20 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. 25

37. The method of claim 33, wherein the chain-elongating nucleotides and/or the chain-terminating nucleotides and/or the primer include modified nucleotides.

38. A method for determining the sequence of a nucleic acid, comprising the steps of: a) generating at least two base-specifically terminated nucleic acid fragments containing deazapurine nucleotides; b) determining the molecular weight value of each base-specifically terminated fragment by mass spectrometry, wherein the molecular weight values of at least two base- specifically terminated fragments are determined simultaneously; and c) determining the sequence of the nucleic acid by aligning the base-specifically terminated nucleic acid fragments according to molecular weight.

39. The method of claim 38, wherein the deazapurine moieties are selected from the group consisting of: C -deazaguaine, C'-deazaadenine and C 7 -deazainosine triphosphate. A method of claim 38, wherein the mass spectrometer is selected from the group consisting of: Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI- TOF), Electrospray (ES) and Fourier Transform and combinations thereof

41. The method of claim 38, wherein more than one species of nucleic acid are simultaneously sequenced by multiplex mass spectrometric nucleic acid sequencing A2216i 27. JUL. 2001 11:37 SPRUSON AND FERGUSON 61292615486 NO. 1198 P. 16 employing tag probes, nucleic acid primers, chain-elongating nucleotides, and chain- terminating nucleotides, wherein one or the sets of base-specifically terminated fragments is unmodified and the other sets of base-specifically terminated nucleic acid fragments are mass modified, and each of the sets of base-specifically terminated nucleic acid fragments has a sufficient mass difference to be distinguished from the others by mass spectrometry.

42. A method of sequencing a target nucleic acid, comprising the steps of: a) reversibly linking oligonucleotide primers to a solid support; b) hybridizing to the primers, at least a portion of the target nucleic acid and generating via chain elongation of the primer and subsequent termination, at least two base- specifically terminated nucleic acid fragments containing deazapurine nuclcotides; c) determining the molecular weight value of each base-specifically terminated fragment by mass spectrometry wherein the molecular weight values of at least two base- specifically terminated fragments are determined simultaneously and wherein the fragments are cleaved from the solid support during mass spectrometry; and 1 5 d) determining the nucleotide sequence by aligning the base specifically two terminated fragments according to molecular weight.

43. The method of claim 42, wherein the deazapurine moieties are selected from the group consisting of: C'-deazaguanine, C 7 -deazaadenine and C 7 -deazainosine triphosphate

44. The method of claim 42, wherein the mass spectrometer is selected from the group consisting of: Matrix-Assisted Laser Desorption/lonization Time-of-Flight (MALDI-TOF), Electrospray (ES) and Fourier Transform and combinations thereof.

45. The method of claim 42, wherein more than one species of nucleic acid are simultaneously sequenced by multiplex mass spectrometric nucleic acid sequencing employing tag probes, nucleic acid primers, chain-elongating nucleotides, and chain- 25 terminating nucleotides, wherein one of the sets of base-specifically terminated fragments is unmodified and the other sets ofbase-specifically terminated nucleic acid fragments are mass modified, and each of the sets of base-specifically terminated nucleic acid fragments has a sufficient mass difference to be distinguished from the others by mass spectrometry.

46. A method for determining the sequence of a nucleic acid comprising the steps of: a) generating at least two base-specifically terminated nucleic acid fragments from a nucleic acid to be sequenced, wherein: the fragments are generated under conditions which comprise cation exchange; and the conditions permit sequencing of oligomers.that are b) determining the molecular weight value of each of the base-specifically terminated fragments by mass spectrometry, wherein the molecular weight values of at least two base-specifically terminated fragments are determined simultaneously; and c) determining the sequence of the nucleic acid by aligning the base-specifically terminated nucleic acid fragments according to molecular weight,

47. A method for determining the sequence of a nucleic acid, comprising the steps A23216 27. JUL. 2001 11:38 SPRUSON AND FERGUSON 61292615486 NO, 1198 P. 17 51 a) generating at least two base-specifically terminated nucleic acid fragments wherein: at least one of the nucleic acid fragments comprises two different mass modifications b) determining the molecular weight value of each base-specifically terminated fragment by mass spectrometry; and c) determining the sequence of the nucleic acid by aligning the one or more sets of base-specifically terminated nucleic acid fragments according to molecular weight.

48. The method of claim 32, wherein four sets of base-specifically terminated fragments are produced for each species of nucleic acid. zo 49. A method of multiplex sequence analysis of nucleic acid species, comprising thile steps of a) synthesizing complementary nucleic acids which are complementary to the nucleic acid species to be sequenced, starting from nucleic acid primers and in the presence of chain-terminating and chain-elongating nucleotides so as to produce sets of base- specifically terminated complementary nucleic acid fragments for each species; b) determining the molecular weight value of each base-specifically terminated fragment for all species simultaneously by mass spectrometry; and c) determining the nucleotide sequences of the species by aligning the base- Sspecifically terminated fragments according to molecular weight; wherein at least one reagent selected from a group consisting of, a nucleic acid primer, a chain-elongating nucleotide, or a chain-terminating nucleotide is mass-modified, wherein each set of base-specifically terminated fragments of a species has a sufficient mass difference from sets of base-specifically terminated fragments of other species to be s distinguished by mass spectrometry. 25 50. The method of claim 49, wherein mass modification comprises employing tag probes- pr 51. A kit for sequencing one or more species of nucleic acids by multiplex mass spectrometric nucleic acid sequencing, comprising: a) a solid support having a linking functionality b) a set of nucleic acid primers suitable for initiating synthesis of a set of nucleic acids which are complementary to the different species of nucleic acids, the primers each including a linking group able to interact with the linking functionality and reversibly link the primers to the solid support and optionally, a tag probe; 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 in the absence of a tag probe, at least one reagent selected from the group consisting of the Sprimers, the chain-elongating nucleotides, and the chain-terminating nucleotides is mass AUz216 27. JUL. 2001 11:38 SPRUSON AND FERGUSON 61292615486 NO. 1198- P. 18 52 modified to provide distinction between each set of base-specifically terminated nucleotides of each species of nucleic acid by mass spectrometry.

52. A kit for sequencing nucleic acids by mass spectrometry, comprising: a) a solid support having a linking functionality 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 able to interact with the linking functionality and reversibly immobilize the primers on the solid support; c) a set of chain-elongating nucleotides for synthesizing the complementary nucleic acids; d) a set of chain-tenninating nucleotides for tenninating synthesis of the complementary nucleic acids and generating sets of base-specific tenninated complementary nucleic acid fragments; and a polymerase for synthesizing the complementary nucleic acids from the s primers, chain-elongating nucleotides and chain-terminating nucleotides, wherein the chain-terminating nucleotides are mass-modified so that addition of one species of the :ee: 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. 20

53. A solid support, comprising a linking functionality, L' linked to a primer via a linking group, L, of the primer to form a linkage wherein: the interaction between L and L' is selectively cleavable enzymatically, chemically or physically; and the primer comprises a mass-modifying functionality linked directly to the 25 primer, or the primer comprises an initiated nucleic acid chain that contains a nucleotide with a mass-modifying functionality wherein S: the linkage is selected from the group consisting of a photocleavable bond, a bond based on a strong electrostatic interaction, a tritylether bond, a P-benzoylpropionyl group, a levulinyl group, an arginine/arginine bond, a lysine/lysine bond and a pyrophosphate bond.

54. The solid support of claim 53, wherein the photocleavable bond of linkage L-L', is selected from the group consisting of a charge transfer complex and a moiety, which forms a stable orgaric radical upon cleavage. A solid support of claim 53, wherein the solid support is selected from the group consisting of: a bead, capillary, polymeric sheet, glass plate, and metal surface.

56. A solid support, comprising a linking functionality, L' linked to a primer via a linking group, L, of the primer to form a linkage wherein: the interaction between L and L' is selectively cleavable enzymatically, chemically or physically; and the primer comprises a mass-modifying functionality linked directly to the prnmer, or the primer comprises an initiated nucleic acid chain that contains a nucleotide with a mass-modifying functionality wherein A2 16 27, JUL. 2001 11:38 SPRUSON AND FERGUSON 61292615486 NO. 1198 19 53 the linkage is a photocleavable bond or a bond based on a strong electrostatic interaction.

57. A microtiter plate adapted with a functionalized membrane, comprising a solid support and a reversibly linked nucleic acid primer in each well.

58. A solid support having a linking functionality, linked to a primer via a linking group, L, forming a photocleavable bond wherein the photocleavable bond is selected to be selectively cleaved by ultraviolet laser energy.

59. The solid support of claim 53, wherein: the mass-modification is a modification of a sugar moi c ty, base moiety or phosphate backbone; and is a modification of a nucleobase or bases in the chain or in the primer, to the Sphosphate backbone in the chain or in the primer or to a 2 '-position of the nuclcoside or nucleosides in the chain or in the primer.

60. An ionized mass-modified nucleic acid molecule, substantially as hereinbefore S s5 described, with reference to any one of the examples.

61. An ionized mass-modified nucleic acid molecule, substantially as hereinbefore o *described, with reference to the accompanying drawings.

62. An ionized and volatilized mass-modified nucleic acid molecule, substantially as hereinbefore described, with reference to any one of the examples.

63. An ionized and volatilized mass-modified nucleic acid molecule, substantially as hereinbefore described, with reference tothe accompanying drawings.

64. A positively charged ionized mass-modified nucleic acid molecule, Ssubstantially as hereinbefore described, with reference to any one of the examples. A positively charged ionized mass-modified nucleic acid molecule, 25 substantially as hereinbefore described, with reference to the accompanying drawings.

66. A process for preparing a set of mass-differentiated tag probes, substantially as hereinbefore described, with reference to any one of the examples.

67. A process for preparing a set of mass-differentiated tag probes, substantially as hereinbefore described, with reference to the accompanying drawings.

68. A set of mass-differentiated tag probes prepared by a process according to claim 66 or claim 67.

69. A set of mass-differentiated tag probes, substantially as hereinbefore described, with reference to any one of the examples. A set of mass-differentiated tag probes, substantially as hereinbefore described, with reference to the accompanying drawings.

71. An ionized duplex, substantially as hereinbefore described, with reference to any one of the examples.

72. An ionized duplex, substantially as hereinbefore described, with reference to the accompanying drawings.

73. A method according to any one of claims 31 to 49, wherein one or more of the nucleic acid fragment(s) upon which mass-spectrometric molecular weight determination is performed is/are ionized mass-modified nucleic acid molecule(s) according to any one of claims 1 to 9, 19, 20, 26, or A2321 27. JUL. 2001 11:39 SPRUSON AND FERGUSON 61292615486 NO. 1198 P. 54

74. A method according to claim 47 or claim 73, wherein one or more of the nucleic acid fragment(s) upon which mass-spectrometric molecular weight determination is performed is/are ionized mass-modified nucleic acid molecule(s) according to any one of claims 1, 19 or 30.

75. A method according to any one of claims 31 to 50, wherein one or more of the nucleic acid fragment(s) upon which molecular weight determination is performed is/are hybridised to a complementary sequence, and the species subjected to mass-spectrometric analysis is/are ionized duplcx(es) according to any one of claims 21 to

76. A method according to any one of claims 36, 41, 45, 50 or 75, wherein mass to modification of the nucleotide sequences for mass-spectrometric molecular weight determination comprises employing a set of mass-differentiated tag probes according to any one of claims 10 to 18, 27 to 29, or 68 to

77. A method of sequencing a nucleic acid as defined in any one of claims 31, 33, 38, 42, 46, or 47, substantially as hereinbefore described, with reference to any one of the S 15 examples. S78. A method of sequencing a nucleic acid as defined in any e ne of claims 31, 33, 38, 42, 46, or 47, substantially as hereinbefore described, with reference to the accompanying drawings.

79. A method of multiplex sequence analysis of nucleic acid species as defined in S 20 claim 32 or claim 49, substantially as hereinbefore described, with reference to any one of the examples.

80. A method of multiplex sequence analysis of nucleic acid species as defined in S claim 32 or claim 49, substantially as hereinbefore described, with reference to the accompanying drawings. 25

81. A process for preparing a solid support comprising a linking functionality, substantially as hereinbefore described, with reference to any one of the examples.

82. A process for preparing a solid support comprising a linking functionality, substantially as hereinbefore described, with reference to the accompanying drawings. S"83. A solid support comprising a linking functionality, prepared by a process according to claim 81 or claim 82.

84. A solid support comprising a linking functionality, substantially as hereinbefore described, with reference to any one of the examples. A solid support comprising a linking functionality, substantially as hereinbefore described, with reference to the accompanying drawings.

86. A microtiter plate according to claim 57, wherein said solid support is a support according to any one of claims 53 to 56, 58, 59, or 83 to

87. A microtiter plate adapted with a functionalized membrane, substantially as hereinbefore described.

88. A kit according to claim 51, wherein said tag probe is a member of a set.of mass-differentiated tag probes according to any one of claims 10 to 18, 27 to 29, or 68 to

89. A kit according to any one of claims 51, 52 or 88, wherein said solid support is a support according to any one of claims 53 to 56, 58, 59, or 83 to A1316 27. JUL 2001 11:39 SPRUSON AND FERGUSON 61292615486 NO, 1198-P. 21-- A kit according to any one of claims 51, 52, 88 or 89, wherein said solid support is located in a microtiter plate according to any one of claims 57, 86 or 87

91. A kit for sequencing nucleic acids by mass spectrometry, substantially as hereinbefore described.

92. A kit for sequencing one or more species of nucleic acids by multiplex mass spectrometric nucleic acid scquencing, substantially as hereinbefore described

93. A kit according to any one of claims 51, 52 or 88 to 92, when used in a method according to any one of claims 31 to

94. A solid support according to any one of claims 53 to 56, 58, 59, or 83 to when used for synthesizing nucleic acid molecules. A solid support when used according to claim 94, wherein said use is in a method according to any one of claims 31 to

96. A solid support when used according to claim 94 or claim 95, wherein said nucleic acid molecules are mass-modified. S 15

97. A solid support, when used for synthesizing nucleic acid molecules, substantially as hereinbefore described, with reference to any one of the examples.

98. A solid support, when used for synthesizing nucleic acid molecules, i substantially as hereinbefore described, with reference to the accompanying drawings. 2

99. A solid support, when used for determining/analysing the sequence of one or more nucleic acid molecules, substantially as hereinbefore described, with reference to any one of the examples.

100. A solid support, when used for determining/analysing the sequence of one or more nucleic acid molecules, substantially as hereinbefore described, with reference to the accompanying drawings. 25

101. A microtiter plate according to any one of claims 57, 86 or 87, when used for synthesizing nucleic acid molecules.

102. A microtiter plate when used according to claim 101, wherein said use is in a method according to any one of claims 31 to

103. A microtiter plate when used according to claim 101 or claim 102, wherein said nucleic acid molecules are mass-modified.

104. A microtiter plate, when used for synthesizing nucleic acid molecules, substantially as hereinbefore described, with reference to any one of the examples.

105. A set of mass-differentiated tag probes, when used for determining/analysing the sequence of one or more nucleic acid molecules, substantially as hereinbefore described, with reference to any one of the examples. Dated 26 July, 2001 Sequenom, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON A23216