OLIGONUCLEOTIDE SIZING USING CLEAVABLE PRIMERS

Abstract: The present invention provides modified oligonucleotide primers designed to incorporate a cleavable moiety so that a (3') portion of the primer (linked to an extension product) can be released from an upstream (5') portion of the primer. Upon selective cleavage of the cleavable site, primer extension products that contain about five or fewer base pairs of the primer sequence are released, to provide more useful sizing and sequence information per fragment than extension products containing the entire primer.

Description: OLIGONUCLEOTIDE SIZING USING CLEAVABLE PRIMERS FIELD OF THE INVENTION The present invention relates to oligonucleotide compositions containing cleavable primers and diagnostic and analytical methods employing such primers. REFERENCES Agrawal, S., and Goodchild, J., Tetrahedron Lett. 28:3539-3542 (1987). Ausubel, F.M., et al., in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY. John Wiley and Sons, Inc., Media PA (1988). Bannwarth, W., et al, DNA 5:413 (1986). Bannwarth, W., Chimia 4.1:302 (1987). Bannwarth, W., Helvetica Chimica Ada 71:1517-1527 (1988). Bischoff, R., et al, Analytical Biochemistry 164:336-344 (1987). Collins, S.J., et al., Science 225:72 (1984). Cormier, J., et al., Nucleic Acids Res. 16:4583-4594 (1988). Corey, E.J., and Snider, B.B., J. Am. Chem. Soc. 24:2549-2550 (1972). Cosstick, R. et al., J. Chem. Soc, Chem. Comm 992 (1988). Cosstick, R., et al., Tetrahedron Letters 30(35):4693-4696 (1989). Daley, G.Q., et al, Proc. Natl. Acad. Sci. USA 85:9312-9316 (1988). Dattagupta, N., U.S. Patent No. 4,818,681 (1989). Erlich, H.A., in PCR TECHNOLOGY. Stockton, New York (1989). Fodor, S.P.A., et al., Science 251:767-773 (1991). Gale, R.P., et al, Proc. Natl. Acad. Sci. USA 81:5648 (1984). Ghosh, S., et al. , Nucleic Acids Research 15:5353-5372 (1987). Gingeras, T., et al., Nucleic Acids Research 15:5373-5390 (1987). Glazer, A., et al., Nature 359:859-861 (1992). Goldkorn, T., et al, Nucleic Acids Research, _14(22):9171-9191 (1986). Green, N.M., in ADVANCES IN PROTEIN CHEMISTRY (Avidin, Ed.) Academic Press, New York, NY, U.S.A., p. 29, 85-133 (1975). Gromova, E.S., Bioorg. Khim 13:269 (1987). Gyllensten, U.B., BioTechniques 1H00 (1989). Hasa, T., et al, Chem Lett 601 (1976). Hegner, M., et al, FEBS Letters 336:452-456 (1993a). Hegner, M., et al, Surface Sci. 291:39-46 (1993b) Hillenkamp, R., Adv. Mass Spectrometry HA:354-361 (1988). Hobbs, J.B., Oranophosphorous Chem. 21:201-321 (1990). Innis, M.A., et al., in PCR PROTOCOLS. Academic Press, San Diego (1990). Khrapko, K., et al, DNA Sequence, 1:375-388 (1991). Koole, L.H., et al, Proc. K. Ned. Akad. Wet. jB91:205-209 (1988). Kremsky, J., et al. , Nucleic Acids Research 15:2891-2909 (1987). Kusukawa, N., et al., BioTechniques, 9_ι66 (1990). Longo, M.C., et al, Gene, 93:125 (1990). Mag, M., et al., Nucleic Acids Research 19(7): 1437-1441 (1991). Maniatis, T., et al., in MOLECULAR CLONING: A LABORATORY MANUAL. Cold Spring Harbor Laboratory (1982). Maskos, U., et al, Nucleic Acids Research 20(7): 1679-1684 (1992). McBride, L.J., et al., Tetrahedron Lett. 24:245 (1983). Miller, P.S., et al, J. Am. Chem. Soc. 23:6657-6665 (1971). Moody, H.M., et al, Nucleic Acids Research 12:4769-4782 (1989). Mullis, K.B., et al, U.S. Patent No. 4,683,195, issued 28 July 1987. Mullis, K.B., U.S. Patent No. 4,683,202, issued 28 July 1987. Nadji, S., et al, J. Am. Chem. Soc. 114:9299-9269 (1992). Nakamaye, K.I., et al, Nucleic Acids Research 16:9947-9959 (1988). Nelson, R.W., et al, Science, 246:1585-1587 (1989). Ogilvie, K., et al., Tetrahedron Lett. 26:4159-4162 (1986). Olsen, D., et al, Methods in Enzymology. 218:79-92 (1993). Olsen, G.J., et al., Nucleic Acids Res. 20:2199 (1992). Primings, et al, Methods in Enzymology 65:561-580 (1980). Saha, A., et al, J. Org. Chem, 58:7827-7831 (1993). Sanger, F., J. of Molecular Biology 94:441-448 (1975). Saiki, R.K., et al, Proc. Natl Acad. Set, 86:6230-6234 (1989). Schmidt, T.M., et al., Methods in Enzymology 235:205-222 (1994). Seliger, H., et al, Nucleosides Nucleotides 6:483-484 (1987). Southern, et al, Genomics 13:1008-1017 (1992). Sproat, B.S., et al, Nucleic Acids Res. 15:4837 (1987). Szczylik, C, et al, Science 253:562-565 (1991). Szybalski, W., Gene 40:169-173 (1985). Tomasz, J., et al, Tetrahedron Lett. 22:3905-3908 (1981). Townsend, L.B., et al, Eds., in NUCLEIC ACID CHEMISTRY: IMPROVED AND NEW SYNTHETIC PROCEDURES. METHODS, AND TECHNIQUES. John Wiley and Sons, New York, NY, pp. 337 (1986). Van Ness, J. et al, Nucleic Acids Res. 12:3345-3350 (1991). Wu, K.J., et al, Rapid Communications in Mass Spectrometry 7:142-146 (1993). Watson, J.D., et al, in RECOMBINANT DNA Second Edition, Scientific American, Inc., Chapter 27 (1992). Yamamoto, I., et al, J. Chem. Soc, Perldn Trans. 1 1:306 (1980). Youngquist, R.S., et al, Rapid Communications in Mass Spectrometry j8:77 '-81 (1994). Zhang, Y., et al. , Nucleic Acids Res. 12:3929-3933 (1991). BACKGROUND OF THE INVENTION DNA, the primary genetic material, is a complex molecule consisting of two intertwined polynucleotide chains, each nucleotide containing a deoxyribose unit, a phosphate group and a nitrogenous heterocyclic base. The two polynucleotide strands are held together via hydrogen bonding interactions between complementary base pairs. A normal human being possesses 23 pairs of chromosomes containing a total of about 100,000 genes. The length of DNA contained within the human chromosomes totals about 3.3 billion base pairs, with a typical gene containing about 30,000 base pairs. Due to the vast amount of genetic information yet to be gathered in both human and non- human genomes, intense efforts are underway to develop new and faster methods of DNA detection, sizing, quantification, sequencing, and gene identification including the mapping of human disease genes. Although the efficiency of these processes has been improved by automation, faster and cheaper methods must still be developed to efficiently carry out genomic- scale DNA analyses. Oligonucleotide sizing and sequence analysis is typically carried out by first utilizing either the enzymatic method developed by Sanger and Coulson, or by chemical degradation, developed by Maxam and Gilbert. The Sanger method uses enzymatic chain extension coupled with chain terminating dideoxy precursors to produce randomly terminated DNA fragments. The Maxam and Gilbert technique involves four different base-specific reactions carried out on portions of the DNA target to produce four sets of radiolabeled fragments. Both techniques utilize gel electrophoresis to separate resultant DNA fragments of differing lengths. In conventional DNA analysis, the DNA fragments are labeled with radioisotopes. After separation on sequencing gels, the fragments are visualized by the image they generate upon a piece of film applied to the gel. Other methods of DNA analysis have been described which eliminate the use of radioisotopes. One example of such a method uses fluorophores or fluorescent tags. In general, four different fluorophores, each having a different absorption and emission spectrum, are attached to the DNA primers using chemical DNA synthesis techniques. Primers with different fluorescent labels are used in each of the four enzymatic sequencing reactions. In an alternate approach to four dye fluorescence-based detection, a dye is chemically attached to a chain-terminating base analog after enzymatic extension. In this approach, synthesis of the different dye-primers is avoided. Mono and polyfunctional intercalator compounds have also been developed as reagents for high-sensitivity fluorescence detection (Glazer, et al., 1992). These planar aromatic fluorophores (e.g., ethidium homodimer, thiazole orange homodimer, oxazole yellow homodimer) insert between adjacent base pairs of double stranded DNA. Efforts to analyze DNA have been greatly aided by the development of a process for in vitro amplification of DNA, namely, the polymerase chain reaction (PCR). PCR provides the ability to amplify and obtain direct sequence information from as little as one copy of a target DNA sequence. Typically, PCR amplification is carried out by placing a mixture of target double stranded DNA, a mixture of deoxynucleotide triphosphates, buffer, two primers (one phosphate- labeled) and DNA polymerase (e.g., heat stable Taq olymerase) in a thermocycler which cycles between temperatures for denaturation, annealing, and synthesis. The selection of primers defines the region to be amplified. In the first stage of the cycle, the temperature is raised to separate the double-stranded DNA strands to form the single-stranded templates for amplification. The temperature is then lowered to generate the primed templates for DNA polymerase. In a third stage, the temperature is raised to promote Tα^-promoted DNA synthesis, and the cycle of strand separation, annealing of primers, and synthesis is repeated for about as many as 30-60 cycles. Standard detection, sizing, and sequencing methods as described above, while providing useful information, are often tedious and costly. Many of the commonly employed techniques involve multiple handling steps. Further, the most common method of fragment analysis, gel electrophoresis, is a relatively time-consuming process. SUMMARY OF THE INVENTION The present invention provides an oligonucleotide composition containing a modified primer having a 5' end and a 3' end and containing at least one selectively cleavable site. Preferably, the cleavable site is located at or within about five nucleotides from the 3' end of the primer. The modified oligonucleotide primer, which has a 5' end and a 3' end, is composed of two separate nucleotide regions. The first region contains the 5' end, while the second region contains the 3' end of the primer, where the 3' end is capable of serving as a priming site for enzymatic extension, typically by a polymerase or ligase. The second region also contains a cleavable site which connects the first and second primer regions. In a preferred embodiment, the first region of the primer contains at least three nucleotides. The first primer region may optionally contain one or more secondary cleavable sites, located between the 5' end of the primer and the second region cleavable site (e.g., to breakdown the first region into smaller fragments). For a modified primer containing a secondary cleavable site, the furthest downstream cleavable site should ideally be cleaved with near 100% efficiency, to avoid the formation of secondary or shadow products with additional bases. In one embodiment of the invention, the cleavable site is located at or within about five nucleotides from the 3' end of the primer. In an alternate embodiment, the second primer region consists of a single nucleotide that also contains the cleavable site, such as a ribonucleotide. The second region may alternatively be composed of only the cleavable site. Cleavable sites contained within the modified primer composition include chemically cleavable groups such as dialkoxysilane, 3'-(S)-phosphorothioate, 5'-(S)-phosphorothioate, 3'- (N)-phosphoramidate, 5'-(N)-phosphoramidate, and ribose. Additional cleavable sites include nucleotides cleavable by an enzyme such as a nuclease. In one embodiment, the cleavable site within the modified primer composition is a single uracil incorporated to replace a thymine, where the uracil is cut site-specifically by treatment with uracil DNA-glycosylase, followed by alkali treatment. In another embodiment, the cleavable site is a restriction endonuclease cleavable site, where the recognition sequence is located in the first primer region (i.e., upstream of the cleavage site). In a preferred embodiment, the restriction endonuclease cleavable site is located at or within about five nucleotides from the 3' end of the primer. It is also possible, such as with class IIs restriction enzymes, to position the cleavable site or cut site within the extension product. Restriction endonucleases for use in cleaving the modified primers of the invention include class IIs restriction endonucleases such as Bpml, Bsgl, BseJU, -9jw.FI, and Fokl. A modified primer including a Bpml or Bsgl recognition site contains (i) a first region containing the recognition site, 5'-CTGGAG-3'or 5'-GTGCAG-3', respectively, and (ii) located 16 bases downstream from the last nucleotide of the recognition sequence, in the second primer region, is the cleavable site. A modified primer containing a BseRI or BsmFl recognition site, e.g., 5'-GAGGAG-3', or 5'-GGGAC-3, respectively, contains a cleavable site located 10 bases downstream from the last nucleotide of the recognition sequence, while a primer containing a Fokl recognition sequence (e.g., 5'-GGATG-3') possesses a cleavable site 9 bases downstream from the last nucleotide of the recognition sequence. In a yet another embodiment, the cleavable site is a restriction endonuclease cleavable site, where the recognition sequence is located in the first primer region (i.e., upstream of the cleavage site), and the first primer region contains a 5' hairpin-type (self-complementary double stranded) domain. The 5' hairpin domain includes the double stranded recognition site for the restriction enzyme. The second (single stranded) primer region contains (i) the cleavable site (i.e., restriction endonuclease cut site), and (ii) is composed of nucleotides complementary to a single stranded target, thus serving as a priming site for enzymatic extension. Following enzymatic extension of the primer, the product is cleaved by treatment with a suitable class US restriction endonuclease, followed by denaturation to release the single stranded extension segment. In another embodiment, the cleavable site is a nucleotide or series of nucleotides capable of blocking or terminating 5' to 3' enzyme-promoted digestion by an enzyme having 5' to 3' exonuclease activity, such as T7 Gene 6 Exonuclease. Blocking nucleotides include peptide nucleic acids and nucleotides containing a phosphorothioate or borano-phosphate group. In a primer extension reaction utilizing a modified primer containing a blocking nucleotide as the cleavable site, following a primer extension reaction, the resulting product, composed of (i) a modified primer containing a blocking nucleotide, and (ii) an extension segment, is treated with a nuclease such as an exonuclease having a 5' to 3' exonuclease activity. Nuclease treatment typically results in digestion of the first region of the primer to generate an extension segment composed of nucleotides downstream (i.e., 3') of the cleavable site. Preferably, the blocking group does not inhibit enzymatic extension of the primer. The modified primer may further include an immobilization attachment site for binding to a solid support. The immobilization attachment site may be located either upstream (i.e., 5' to) or downstream (i.e., 3' to) of the cleavable site. In one embodiment, the immobilization attachment site is located at the 5' end or 5' relative to the cleavable site (i.e., upstream of the cleavable site) of the modified primer. In another embodiment, the immobilization attachment site is located at the 3' end or 3' relative to the cleavable site (i.e., downstream of the cleavable site). Alternatively, the immobilization attachment site may be contained within the extension segment resulting from an enzymatic extension reaction, or, may be contained within a target nucleic acid. For modified primers including an immobilization attachment site, the primer is attachable to a solid support by either a covalent or non-covalent linkage between the solid support and the primer immobilization attachment site to provide an immobilized modified oligonucleotide composition. Solid supports for use in the present invention include glass, silicon, polystyrene, cellulose, teflon, polystyrene divinyl benzene, aluminum, steel, iron, copper, nickel, silver and gold. In one embodiment, the primer is attachable to a solid support via an intervening spacer arm, with a typical spacer being six or more atoms in length. In another embodiment, the modified primer contains an immobilization attachment site in the first primer region composed of a series of bases complementary to an intermediary oligonucleotide. The modified primer is immobilizable by specific hybridization of the immobilization attachment site to the intermediary oligonucleotide, which is bound to a solid support. The intermediary oligonucleotide can be complementary to all or a portion of the sequence of the modified primer. The intermediary nucleotide is typically composed of 6 or more bases, and preferably more than 8 bases. Additionally, the intermediary oligonucleotide may also be homologous to a region within a target nucleic acid (template) molecule. In one embodiment, the modified primers of the present invention are oligonucleotides, such as DNA or RNA, having phosphodiester internucleotide linkages. In another embodiment, the modified primers are oligonucleotide analogues composed of alternative backbone structures containing internucleotide linkages such as methylphosphonate, phosphotriester, phosphorothioate, peptide, and the like. Primers for use in the invention should be capable of hydrogen bonding in a sequence-specific manner to a target sequence. The invention also provides a method for determining the size of a primer extension product using the modified primers of the present invention. In employing the method, oligonucleotide size analysis is carried out by first contacting a modified primer of the present invention with a target nucleic acid molecule (e.g., DNA or RNA) to effect hybridization of the primer with the single stranded target. The modified primer is complementary to the target and contains a first region containing the 5' end of the primer, and a second region containing the 3' end of the primer, where the 3' end is capable of serving as a priming site for enzymatic extension. The second region of the primer also contains a cleavable site. In determining the size of a primer extension product, the primer is extended by enzymatic means, typically by action of a polymerase or ligase, to generate a mixture containing a product composed of the primer and one or more extension segments. The resulting product is cleaved at the cleavable site and the resulting extension segment is then sized by any of a number of suitable analytical techniques, preferably mass spectrometry. In accordance with the invention, the mass of the extension segment is decreased and the read length of the extension segment is increased relative to the read length of the product composed of the modified primer and the extension segment. In a preferred embodiment for determining the size of an extension product, the modified primer (first or second region) or template is immobilized by attachment to a solid support. Immobilization may be via a covalent or non-covalent linkage. Exemplary non-covalent linkages include ligand-protein interactions and base-specific hydrogen bonding. The extension segment from an enzymatic extension reaction can also be immobilized by attachment to a solid support, and is released from the solid support prior to sizing or sequence determination. In the latter embodiment, enzymatic extension is typically carried out in the presence of a nucleotide containing (i) an immobilization attachment site and (ii) a releasable site, such as the exemplary nucleotide, biotinylated-disulfide-dideoxynucleotide. Following enzymatic extension, the extension product is immobilized, denatured, and cleaved at the cleavable site, leaving the extension segment affixed to the solid support. Released primer (the portion upstream of the cleavable site), template and additional mixture components are typically removed by washing, and the immobilized extension segment is then cleaved at the releasable site, to release the extension segment for sizing. In one embodiment of the method, the modified primer is immobilized via specific hybridization of the immobilization attachment site to an intermediary oligonucleotide which is bound to a solid support (solid phase bound intermediary oligonucleotide, SPBIO). In one particular embodiment, the immobilization attachment site is located in the first primer region and is composed of a series of bases complementary to the intermediary oligonucleotide. Alternatively, if a portion of the sequence of the extension product is known, the immobilization attachment sight may be contained within a region of the extension segment. In yet another embodiment, the SPBIO is homologous to a target nucleic acid molecule, resulting in a competition between the SPBIO and the target for hybridizing to the modified primer. Following enzymatic extension, the product (composed of the primer and an extension segment) is attached to a solid support via hybridization to the SPBIO. To promote hybridization of the SPBIO to the product, the amount of target molecule may be reduced either by (i) carrying out a target-selective digestion which leaves the primer and extension product intact, or (ii) reducing the amount of target molecule. Template specific digestion may be chemical or enzymatic. In a related embodiment of the method, the modified primer is immobilized via hybridization to a SPBIO, where the first primer region contains a first portion which is complementary to the SPBIO, and downstream or 3' of this portion of the first region is a second portion of the first primer region which is complementary to the target molecule but is not complementary to the SPBIO. The first portion of the first region of the modified primer will typically be composed of at least six or more nucleotides which are complementary to the SPBIO. Alternatively, indirect immobilization of the modified primer can be accomplished by carrying out an enzymatic extension reaction with a target nucleic acid which is attached to a solid support. The target molecule can be immobilized either prior to or subsequent to primer extension. Optionally, the reaction mixture containing immobilized and non-immobilized species is washed prior to cleavage at the cleavable site, allowing ready separation of immobilized versus non-immobilized species prior to sizing of the extension segments. The primer can be immobilized at the immobilization attachment site either prior to or after enzymatic extension, depending upon the nature of immobilization. Generally, when the immobilization attachment site is contained within the first primer region, the immobilization attachment remains intact under the selected cleavage conditions to retain a significant portion of nucleotides from the modified primer (e.g., those comprising the first primer region) in immobilized form. In accordance with the present method, the read length of the extension segment resulting from cleavage at the cleavable site is increased relative to the read length of the product composed of the primer and the extension segment. In one embodiment of the invention, the extension segments, typically containing no more than about five nucleotides derived from the modified oligonucleotide primer, are sized using mass spectrometry. Such sizing may utilize matrix-assisted laser desorption ionization mass spectrometry, and more particularly, may be accomplished using time-of-flight mass spectrometry,. The sizing method may also be coupled with amplification of a target nucleic acid. In one embodiment of this aspect of the invention, first and second primers are combined with a target nucleic acid under conditions effective to promote the hybridization of the primers to the nucleic acid to generate primer /nucleic acid complexes. One of the primers (e.g. , the first primer) is complementary to the target nucleic acid and has a first region containing the 5' end of the primer and an immobilization attachment site. The first primer further contains a second region containing the 3' end of the primer, where the 3' end is capable of serving as a priming site for enzymatic extension. The second region of the first primer further contains a cleavable site. The second primer is homologous to the target nucleic acid. The primer/nucleic acid complexes are converted to double-strand fragments in the presence of a suitable polymerase and all four deoxynucleotide triphosphates (dNTPs) or modified versions thereof. The number of primer-containing fragments is amplified by successively repeating the steps of (i) denaturing the double-strand fragments to produce single- strand fragments, (ii) hybridizing the single strands with the primers to form strand/primer complexes, (iii) generating double-strand fragments from the strand/primer complexes in the presence of a polymerase and all four dNTPs, and (iv) repeating steps (i) to (iii) until a desired degree of amplification has been achieved. The amplified fragments are then denatured to generate a mixture including a product composed of the first primer and an extension segment. In one embodiment of this aspect of the invention, the amplified fragments containing the first primer are immobilized at the immobilization attachment site and the non-immobilized amplified fragments are removed, typically by washing. The first primer is then cleaved from the immobilized product at the cleavable site, causing the release of the extension segment from the support. In an alternate embodiment, the amplified fragments may be immobilized prior to denaturing. Generally, the amplified fragments are immobilized prior to cleaving at the cleavable site, to enable release and subsequent analysis of the extension segments resulting from such cleavage, in the absence of other species (e.g., primers, reactants, excess dNTPs). The extension segments are then sized by mass spectrometry. The read length of the extension segment is increased relative to the read length of the product composed of the first primer and the extension segment. Another embodiment of the sizing method provides first and second primers, where one of the primers, i.e., the first primer, contains a cleavable site, and another primer, i.e., the second primer, contains an immobilization attachment site for binding to a solid support. The second primer is composed of a 5' end and a 3' end, is homologous to the target nucleic acid, and includes a first segment containing the 3' end of the second primer, and a second segment containing the 5' end of the primer and an immobilization attachment site. These first and second primers are combined with a target nucleic acid to generate primer/nucleic acid complexes and converted to double-stranded fragments in the presence of a polymerase and deoxynucleoside-triphosphates. The sizing method can be carried out using a high concentration of target nucleic acid to generate substantial amounts of primer extension product, or alternatively, may be coupled with various rounds of amplification. Upon achieving a desired amount of product, extension products containing the second primer are immobilized by attachment at the immobilization attachment site. The extension product is then cleaved at the cleavable site to generate a mixture which includes a double-stranded product. Non- immobilized cleaved fragments are removed, preferably by washing, and the double-stranded product is denatured to release the extension segment, which is sized by mass spectrometry, where the mass of the extension segment is decreased and the read length of the extension segment is increased relative to the read length of the primer/nucleic double stranded fragments. Immobilization of the extension product may occur either before or after cleavage at the cleavable site. As will be appreciated, the cleavable site of the first primer and the immobilization attachment site of the second primer include those of the type described above. In one embodiment, the first primer contains a class us restriction enzyme recognition site in the first primer region, and a cleavable site in the second primer region, and the second primer contains an immobilization attachment site for attachment to a solid support. Cleavage at the cleavable site is carried out by addition of a restriction endonuclease selective for the recognition site contained in the first primer region to provide (i) released fragments containing the first region of the first primer and (ii) a double-stranded product, which is immobilized prior to denaturing to release the desired extension segment. In a related aspect, a method of sequencing is provided that utilizes the modified primers of the invention for determining the sequence of a target molecule by mass spectrometry. In one embodiment of this aspect of the invention, the sequence of a target nucleic acid is determined by hybridizing a modified immobilizable primer of the present invention with a target nucleic acid, such as DNA or RNA, followed by enzymatically extending the primer in the presence of a first of four different dideoxy nucleotides (chain terminators) to generate a mixture of primer extension products. The primer extension products each contain a primer and an extension segment. The extension products are denatured, immobilized, and washed to remove non- immobilized species present in the reaction. As in the embodiments described above, immobilization can occur before or after enzymatic extension, and is typically carried out prior to cleavage at the cleavable site. Subsequent to immobilization and removal of non-immobilized species, the primer extension products are cleaved to release the extension segments. The extension segments are sized by mass spectrometry, and the above steps are repeated with each of the three remaining different dideoxy nucleotides. The sequence of the target is then determined by comparing the sizes of the extension segments obtained from each of the four extension reactions. In a variation of the above, a single primer extension reaction is carried out using a mixture composed of more than one chain-terminating nucleotide, up to all four (e.g., dTTP, ddTTP, dATP, ddATP, dCTP, ddCTP, dGTP, and ddGTP). The resulting reaction mixture, containing up to all four-base specifically terminated products, is then analyzed using the mass data and known mass values for the four bases. Optionally, mass modified nucleotides may be utilized to enhance the resolution of the product mixture. Sequencing can also be carried out using the modified primers of the invention coupled with alternate sequencing methodologies which do not employ dideoxynucleosides. In one such embodiment, the sequence of a target nucleic acid, such as DNA or RNA, is determined by first hybridizing a primer with a target DNA, where the primer (i) is complementary to the target DNA; (ii) has a first region containing the 5' end of the primer and an immobilization attachment site, and (iii) has a second region containing the 3' end of the primer and a cleavable site. The 3' end of the primer is also capable of serving as a priming site for enzymatic extension. The primer is then extended with an enzyme in the presence of a first of four different deoxynucleotide α-thiotriphosphate analogs (dNTPαS) to generate a mixture of primer extension products containing phosphorothioate linkages. The phosphorothioate-containing extension products are then treated with a reagent that cleaves specifically at the phosphorothioate positions. Suitable reagents for promoting phosphorothioate-specific cleavage include 3' to 5' exonuclease, 2-iodoethanol, and 2,3-epoxy-l-propanol. Treating of the extension products is typically carried out under conditions that produce limited cleavage of the phosphorothioate linkages, resulting in the production of a group of primer extension degradation products. Alternatively, the primer extension reaction can be carried out using a limited amount of the α- thio-deoxynucleoside triphosphate analog, along with the corresponding conventional deoxynucleoside triphosphate (dNTP). The resulting extension products are then treated with a phosphorothioate-selective reagent as described above, under conditions effective to cleavage all of the phosphorothioate groups incorporated into the extension product (complete cleavage). The primer extension degradation products are immobilized at the immobilization attachment sites to produce immobilized primer extension degradation products (i.e., a nested set of fragments specific to the 5' end containing the primer), each containing a primer and an extension segment. In alternative embodiments of this aspect of the invention, immobilization may be carried out either i) prior to enzymatic extension, ii) after enzymatic extension, iii) prior to treating the phosphorothioate-containing primer extension products with a phosphorothioate- specific cleaving reagent, or (iv) or after such treating. Subsequent to immobilization, the primer extension degradation products are washed to remove non-immobilized species. Cleavage at the cleavable site results in the release of extension segments, which are then sized by mass spectrometry. Using the sequencing method of this aspect of the invention, the read length of any given extension segment is increased relative to the read length of its corresponding primer extension degradation product. The steps of hybridization, enzymatic extension, treatment with a phosphorothioate- cleaving reagent, immobilization, washing, cleaving, and sizing are then repeated with a second, third, and fourth of the four different dNTPαS analogs to determine the sequence of the target DNA by comparison of the sizes of the extension segments obtained from each of the four extension reactions. Optionally, the steps of hybridization, enzymatic extension, treatment with a phosphorothioate-cleaving reagent, immobilization, washing, and cleaving can be carried out in the presence of from 2-4 different dNTPαS analogs, followed by sizing of the resulting extension segments by mass spectrometry. According to yet another aspect, a method of fingerprinting is provided which utilizes the modified primers of the invention for obtaining a fingerprint of a target oligonucleotide. These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples. BRIEF DESCRIPTION OF THE FIGURES Figs. IA -1W show a native phosphodiester internucleotide linkage and exemplary internucleoside cleavable sites for use in the oligonucleotide composition of the present invention; Figs. 2A-2M include a number of exemplary immobilization attachment linkages for use in immobilizing the first region of a modified oligonucleotide primer; Figs. 3A and 3B illustrate time-of-flight mass spectra for samples of a modified oligonucleotide primer containing a cleavable ribose desorbed from a solid matrix of 3- hydroxypicolinic acid both before and after selective cleavage; Fig. 4 is a time-of-fiight mass spectrum of the cleavage product of an immobilized 18- mer containing a cleavable ribose at the 10-position; Fig. 5A-5E illustrate four alternate embodiments of an immobilized cleavable primer in accordance with the invention; Figs. 6A and 6B illustrate an exemplary method of determining the sequence of a target DNA molecule using the immobilizable, cleavable primers of the present invention; Figs. 7A and 7B illustrate the respective single gene mutation sites identified for two distinct genetic disorders suitable for detection using the modified primers of the present invention; Fig. 8 illustrates the immobilization of a modified primer via competitive hybridization of the first primer region to both a target molecule and a solid phase bound intermediary oligonucleotide, SPBIO; Fig. 9 illustrates the immobilization of a modified primer via hybridization to a SPBIO, where the modified primer contains a first primer region composed of a first portion complementary to the SPBIO and a downstream second portion complementary to a target molecule but not complementary to the SPBIO; Fig. 10 illustrates immobilization of an enzymatic extension product (composed of a modified primer and an extension segment) via base-pairing interaction to a target molecule bound to a solid support; Fig. 11 illustrates sequence-specific cleavage of an enzymatic extension product, where the modified primer contains a restriction recognition site in the first primer region and a cleavable site in the second primer region; Fig. 12 illustrates an exemplary sizing method of the present invention using first and second modified primers, where the first primer contains an enzyme-cleavable site and the second primer contains an immobilization attachment site; Fig. 13 is a MALDI time-of-flight mass spectrum of primer extension products obtained from an extension reaction using a primer containing a 5-thiol thymidine cleavable site and a 10- fold excess of primer. Primer extension products were immobilized via hybridization to a complementary biotinylated intermediary oligonucleotide bound to streptavidin coated beads and released by chemical cleavage for subsequent size analysis; Figs. 14A and 14B are MALDI time-of-flight mass spectra illustrating the utility of the present method in detecting single base substitutions (point mutations) between oligonucleotides, using base-specific digestion; and Figs. 15A and 15B are MALDI time-of-flight mass spectra of primer extension products obtained using (i) a cleavable primer according to the present invention (Fig. 15B) versus (ii) a full length primer (Fig. 15A), illustrating the difference in both resolution and read length of the resulting spectra. Fig. 16 illustrates sequence-specific cleavage of an enzymatic extension product, where the modified primer contains a class IIs restriction enzyme recognition site in the first primer region, composed of a 5' hairpin-type (self-complementary double stranded) domain, and a cleavable site in the second primer region. DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS The following terms, as used herein, have the meanings as indicated: In referring to a position within a single stranded oligonucleotide, a position that is "upstream" from a specified position is located 5' to this position, while a position that is "downstream" is located 3' to the point of reference. An "immobilization attachment site" or IAS is a site which may be present within an oligonucleotide primer for binding to a solid support material either directly, through an intervening spacer arm, or by specific hybridization to an intermediary oligonucleotide which is bound to a solid support. The immobilization attachment site may be located either upstream (i.e., 5' to) or downstream (i.e., 3' to) of the cleavable site and may require chemical modification prior to binding to the solid support. Alternatively, the immobilization attachment site may be contained within the extension segment resulting from an enzymatic extension reaction, or, may be contained within a target nucleic acid. The immobilization attachment site can be a select functional group for covalent bonding to a solid support, such as those representative functional groups shown in Figs. 2A-2K, and Fig. 2M. The immobilization attachment site can also be a ligand such as biotin, for attachment via a high-affinity non-covalent interaction with a solid support. Further, the immobilization attachment site can also be composed of a series of bases complementary to an intermediary oligonucleotide. Immobilization of the modified primer is effected, for example, by specific hybridization of the immobilization attachment site to an intermediary oligonucleotide, which is bound to a solid support. The intermediary oligonucleotide may also act as the template. The immobilization attachment site may be attached to the solid support by either chemical or enzymatic means. Upon attachment of the immobilization attachment site to a solid support, the resulting immobilization linkage is one which remains stable under the conditions employed for cleaving the cleavable site and does not inhibit base pair hybridization nor block the ability to extend the primer from its 3' end. Two nucleic acid fragments are considered to be "selectively hybridizable" if they are capable of specifically hybridizing to one another but not to other polynucleotides, under typical hybridization and wash conditions, as described, for example, in Maniatis, et al., pages 320-328, and 382-389. Two nucleic acid fragments are considered to be "complementary" if they are capable of specifically hybridizing to one another (i) under typical hybridization and wash conditions, as described, for example, in Maniatis, et al, pages 320-328, and 382-389, or (ii) using reduced stringency wash conditions that allow at most about 25-30% basepair mismatches, for example: 2 x SSC, 0.1% SDS, room temperature twice, 30 minutes each; then 2 x SSC, 0.1% SDS, 37°C. once, 30 minutes; then 2 x SSC room temperature twice, 10 minutes each. "Cleavable site" as used herein is a reactive moiety typically (i) located at or within about five nucleotides from the 3' end of a primer, and (ii) selectively cleavable by appropriate non- enzymatic or enzymatic means including chemical, thermal, or photolytic, to enable release of primer extension products that typically contain none or a relatively small number of base pairs of the modified primer. Cleavable site refers both to the selectively cleavable functional group as described above and also to protected forms thereof. The cleavable site may, for example, be (i) located along the polymer backbone (i.e., a modified 3'-5' internucleotide linkage in place of one of the phosphodiester groups), (ii) as a substituent on or replacement of one of the bases or sugars of the oligonucleotide primer, or (iii) as the 3' terminal residue (e.g., a ribonucleotide at the 3' end of the oligodeoxyribonucleotide primer). The cleavable site is stable under standard solid phase DNA synthesis conditions, during primer immobilization, hybridization, primer extension, and washing conditions. As used herein, a cleavable site may also be a nucleotide cleavable by an enzyme such as a nuclease. For example, the cleavable site may be a restriction endonuclease cleavable site, where the recognition sequence is located in the first primer region (i.e., upstream of the cleavage site). Exemplary restriction endonucleases for use in cleaving at the cleavable site include Bpml, Bsgl, BseR , BsmFl, and Fokl. A cleavable site may also be a nucleotide or series of nucleotides capable of blocking or terminating 5' to 3' enzyme-promoted digestion by an enzyme having 5' to 3' exonuclease activity, such as T7 gene 6 exonuclease (Amersham Life Sciences, Arlington Heights, IL). Representative blocking nucleotides are those containing a phosphorothioate, borano-phosphate, or peptide group. The blocking nucleotide/cleavable site should be one which does not inhibit enzymatic extension of the primer. As described herein, "fingerprinting" refers to a method of determining the positions of no more than two different bases in a target oligonucleotide strand, as opposed to "sequencing", which refers to a determination of the complete nucleotide sequence of (and also the corresponding amino acid sequence encoded by) a target oligonucleotide, including the identity and position of each nucleotide present in die target strand or its complement. "Non-covalent linkage" refers to any type of non-covalent bonding interaction, and is used herein primarily to describe different types of immobilization attachment sites. A non- covalent linkage includes base-specific hydrogen bonding interactions, such as those occurring between complementary nucleotide base pairs, or may refer to a high affinity ligand-protein interaction, such as the biotin/avidin or biotin/streptavidin interaction K ^ = 10 15 M" 1 ). "Extension segment" refers to the product resulting from in vitro enzymatic extension at the 3' end of a primer, excluding the portion of nucleotides originally present in the primer prior to extension. As used herein, "read length" refers to the number of nucleotides of a given target sequence for which new analytical data (e.g. , sizing, quantification, sequencing) can be obtained. New data refers to fragment information for primer extension products which excludes data derived from portions of the target DNA complementary to the primer(s) employed (e.g. , regions for which sequence information is already known). Read length is typically method dependent (i.e., a function of the detection method being employed). In some analytical methods size resolution may have an essentially finite upper limit, e.g., up to 100 nucleotides. One advantage of d e present invention is the ability to improve the amount of new or useful information about a target DNA sequence that can be obtained from a primer extension product, when the products are analyzed using such a method. For example, using the modified primers of the present invention, the read length of an exemplary extension segment would be determined as follows. A modified primer composed of 18 nucleotides complementary to a DNA target and having a cleavable linkage between nucleotides 17 and 18 (e.g., the cleavable site is within one nucleotide from the 3' end of the primer) is first annealed to the target strand, extended enzymatically (e.g., with a polymerase or ligase), and the resulting primer extension product (subsequent to immobilization) is cleaved at the cleavable site to produce an extension segment containing only one nucleotide derived from the primer. In carrying out sizing of the extension products, the read length is equal to the total number of nucleotides detected (X) minus the one nucleotide derived from the second region of the primer, or X-l. In contrast, prior to cleavage at the cleavable site, the product composed of the primer and the same set of extension segments would have a read length of X-l 8, where 18 equals the number of bases in the primer. Thus the amount of new or useful sequencing or size information for primer extension products obtained using the modified primers of the present invention is improved. π. OLIGONUCLEOTIDE COMPOSITION; SYNTHESIS OF MODIFIED PRIMERS A. FEATURES OF THE MODIFIED PRIMER The oligonucleotide primers of the present invention (i) are designed for optional attachment to a solid support in a manner that does not block the ability to extend the primer from its 3' end, and (ii) incorporate a cleavable moiety so that a 3' portion of the primer linked to an extension segment can be released from an upstream portion of the primer, 5' to the cleavable site. The upstream portion of the primer, referred to herein as the first primer region, typically contains a significant number of the total number of nucleotides present in the primer, so that upon cleavage at the cleavable site, the amount of new fragment information for primer extension products is maximized. The modified primer of the invention preferably contains an immobilization attachment site for attaching the primer to a solid support. For modified primers containing an internal immobilization attachment site (i. e. , a site contained within the primer itself), the immobilization attachment site or IAS is generally separated from the cleavable site by at least three nucleotides. Upon selective cleavage of the cleavable site, a large portion of the primer fragment remains affixed to the solid support. This enables the release of primer extension products that contain about five or fewer base pairs of the primer sequence, to extend the useful size analysis range (e.g., increased read lengths), as illustrated in Figs. 15A and 15B. Figs. 15A and 15B are mass spectra of products from primer extension reactions, illustrating the difference in fragment information obtained for cleaved primer extension segments according to the invention (Fig. 15B) versus non-cleaved full primer-extension segments (Fig. 15A). The details of the primer extension reactions and primer cleavage are described in Example 8. For sequencing applications, the modified primers can also provide more useful sequence information per fragment than extension products containing the entire primer. Exemplary oligonucleotide sequences for use as primers or probes in the oligonucleotide composition of the present invention typically have lengths ranging from about eight to thirty nucleotides, preferably between about ten and twenty five nucleotides. Typically, the oligonucleotide sequences are complementary to a site upstream, relative to the 5' end, of the target sequence of interest, based on known sequence information for the target molecule. The oligonucleotide sequence may additionally contain a label in the releasable primer fragment (e.g. , the second region), such as a radioactive or fluorescent tag, depending upon the method of sequence analysis employed. The modified primers of the present invention, having a 5' end and a 3' end, are generally composed of two separate nucleotide regions. In one embodiment of the invention as illustrated in Fig. 5A, die two regions are connected by a cleavable site, as indicated by "X". The heterocyclic bases, adenine, thymine, guanine, and cytosine are commonly indicated in the figures by the symbol "B". The first region, containing the 5' end of the primer, contains an immobilization attachment site, "I", for attachment to a solid support. In the embodiment shown in Fig. 5A, the modified primer is in immobilized form. The immobilization site may optionally be separated from the 5' end of the primer by a spacer arm, as indicated. Spacer arms for use in the present invention are generally six or more atoms in length. The number of nucleotides in me first region will vary, but typically will be at least about three nucleotides. In a preferred embodiment, die first primer region contains a significant portion of the nucleotides (e.g., typically from about 3-20 nucleotides) composing the modified primer. As shown, the cleavable linkage, "X", is a 3'-5'-internucleotide cleavable site which connects d e first region to the second region. The second region, which contains me 3' end of the primer, is composed of as few nucleotides as is feasible, al ough the number will vary depending on the primer employed. Preferably, d e second region contains from zero to five nucleotides and the total number of nucleotides in die modified primer will be between about eight and ύ irty, and preferably between ten and twenty five. The 3' end of the modified primer serves as a priming site for enzymatic extension. Fig. 5B illustrates an alternate embodiment of d e present invention in which a biotin molecule is connected to die 5' end of a modified primer. The biotin is attached to the 5' end of die primer through an extended spacer arm which serves to reduce steric hindrance. Biotin, a relatively small vitamin molecule that binds widi high affinity to both avidin and streptavidin, is one exemplary immobilization attachment site for use in the present invention. Biotinylated primers are available from commercial sources or may be synthesized by methods commonly employed in die art, typically utilizing a fiinctionalized biotin reagent containing a reactive group suitable for coupling. As in Fig. 5A above, an internucleotide cleavable linkage separates the two regions of the modified primer. The second region contains a 3' end suitable for enzymatic extension, which may take place either prior to or after immobilization to a solid support. Fig. 5C illustrates capture of the modified primer of Fig.5B prior to enzymatic extension on an avidin-functionalized solid support. In this embodiment of me invention, the modified primer is immobilized via a high affinity interaction between avidin and biotin, as indicated by Fig.5D illustrates an alternate embodiment of me invention in which the modified primer is attached to a solid support through an immobilization attachment site present as a substituent on one of the heterocyclic bases. As shown in Fig. 5D, the site for immobilization is an amino residue substituted at die 5 position of a uracil (Dattagupta, 1989), and more specifically, is a 5-allylamino substituent. The amino group may be in protected form (e.g., trifluoroacetamido) prior to attachment to the solid support. As indicated, immobilization to the solid support is through an amide linkage, aldiough any of a number of immobilization attachment linkages may be used, as will be described in more detail below. Coupling of the amino residue to a solid support is generally carried out by using an activated support material, such as an N- hydroxysuccinimide (NHS) ester fiinctionalized support. In die embodiment shown in Fig. 5D, die immobilized primer is in a branched or "T"- configuration. As in the above embodiments, die modified primer contains two regions separated by a cleavable linkage indicated by "X". The first region contains the 5'end of die primer and an immobilization attachment site as described above. Referring to die design of a "T"- configured primer as shown in Fig. 5D, generally, a large portion of the nucleotides composing the modified primer and required for sequence specific target binding are located 5' of d e "central" deoxyribose, indicated by an arrow. The second region contains die 3' end of die primer which serves as a priming site for enzymatic extension. In the exemplary modified primer shown, following hybridization to a target DNA, and enzymatic extension followed by denaturing and washing, selective cleavage of die cleavable site "X" releases die second region of die modified primer along wi the extension product (e.g. , the extension segment), while the first region containing a large portion of the nucleotides required for sequence specific target binding remains immobilized. Fig. 5E illustrates an exemplary modified primer containing a terminal cleavage site, as indicated by an (X). In this embodiment of the invention, me cleavable linkage is represented by a ribose moiety, although any of a number of terminal cleavable sites may be employed. As shown, the modified primer is in immobilized form and contains an immobilization attachment site 5' of the cleavable site. The first region contains the immobilization attachment site and the portion of the primer up to, but not including die ribose. The ribose, or, alternatively, the cleavable site, represents the second primer region and also serves as a priming site for enzymatic extension. Additional cleavable sites according to the invention include nucleotides cleavable by an enzyme such as a nuclease or glycosylase. Fig. 11 illustrates an exemplary cleavable primer containing a restriction recognition site in the first primer region and a cleavable site in the second primer region. As can be seen in Fig. 11, me primer 75 contains two regions, a first 5' region containing a restriction endonuclease recognition sequence 73 and a second region 76 containing a cleavable site 77, downstream or 3' to the recognition sequence. Preferably, the restriction endonuclease cleavable site is located at or within about five nucleotides from me 3' end of die primer. Representative restriction endonucleases for use in cleaving the modified primers of me invention include Bpml, Bsgl, Bse l, BsmFl, and Fokl, all of which make staggered cuts. A modified primer including a Bpml or Bsgl recognition site contains (i) a first region which includes the recognition sequence, 5'-CTGGAG-3' or 5'-GTGCAG-3, respectively, and (ii) located 16 bases downstream from the last nucleotide of the recognition sequence, in the second primer region, is the cleavable site. A modified primer containing a BseRl or BsmFl recognition site, 5'-GAGGAG-3' or 5'-GGGAC-3', respectively, contains a cleavable site located 10 bases downstream from the last nucleotide of die recognition sequence, while a primer containing a Fokl recognition sequence (5'-GGATG-3') possesses a cleavable site 9 bases downstream from e last nucleotide of the recognition sequence. Returning now to Fig. 11, the first region of the primer contains two separate domains. The first domain, 73, is composed of a series of bases recognizable by a restriction endonuclease as described above. The second domain of the first primer region, 76, is 3' to the restriction endonuclease recognition sequence and contains nucleotides complementary to a target DNA molecule 81 which acts as a template for enzymatic extension of die primer. The first domain of me first primer region 73 may optionally hybridize to die target molecule. After carrying out a primer extension reaction to form a primer extension product 79, as will be described in more detail below, die double stranded product is denatured 85, and an oligonucleotide 83 complementary to bom die first 73 and second 76 domains wi in the first primer region is added to die reaction mixture, preferably in an excess amount. Typically, the complementary oligonucleotide 83 contains about 15-25 nucleotides, sufficient to allow restriction enzyme recognition and cleavage at the cleavable site. Preferably, the restriction endonuclease cleavable site is at or near to the 3' end of die primer. The reaction mixture is then allowed to cool and reanneal 86. Due to die excess of complementary oligonucleotide 83 present, hybridization of the primer-extension product to die oligonucleotide complement is favored, as indicated at 87. Restriction endonuclease is men added to die mixture, as shown at 89, to promote cleavage at the cleavable site to release an extension segment 91, a small fragment of die complementary oligonucleotide 3' to the cleavable site 93, and a larger primer/complementary oligonucleotide fragment 5' of die cleavable site 95. A primer of the type described above may also contain an immobilization attachment site (IAS) downstream from die cleavable site, to enable immobilization of the extension segment. Introduction of an IAS should not adversely affect (i) sequence-specific binding of die template to me modified primer, (ii) sequence specific binding of die primer to the complementary oligonucleotide 83, (iii) enzymatic extension of die primer, or (iv) die cutting ability of the restriction enzyme. Generally, the extension product is immobilized and washed to remove reaction products (salts, enzymes, nucleotide fragments, reagents) prior to release and subsequent size and or sequence analysis. Other approaches include (i) me use of a primer or extension segment containing an immobilization attachment site, where, after enzymatically extending die primer and denaturing die double-stranded product, me single stranded primer-extension product is captured via binding at die immobilization attachment site, followed by removal of e template and addition of complementary oligonucleotide 83, as described above, or (ii) die use of a template modified to contain an immobilization attachment site, for capturing the template either prior to or after enzymatic extension, prior to addition of oligonucleotide 83. A variation of a cleavable primer of die type illustrated in Fig. 11 is shown in Fig. 16, where the first primer region contains a universal restriction recognition site witiiin a hairpin (Szybalski). Referring now to Fig. 16, the cleavable site 127 is a class IIs restriction endonuclease cleavable site, where die double stranded enzyme recognition sequence 129 is located in die first primer region (i.e., upstream of die cleavage site), and die first primer region contains a 5' hairpin-type (self-complementary double stranded) domain. The 5' hairpin domain 131 includes die double stranded recognition site 129 for die restriction enzyme. The second (single stranded) primer region contains (i) die cleavable site (i.e., restriction endonuclease cut site), and (ii) is composed of nucleotides complementary to a single stranded target 133, dius serving as a priming site for enzymatic extension. Following enzymatic extension of the primer (shown at 135), the product 137 is cleaved 139 by treatment with a suitable class ES restriction endonuclease to release fragments 141 and 143, followed by denaturation to release die single stranded extension segment for subsequent analysis, i.e., by mass spectrometry. As indicated in die figure at 145, die template may optionally be attached to a solid phase support at any stage during the process. In some instances, die cleavable site is a nucleotide capable of blocking or terminating 5' to 3' enzyme-promoted digestion by an enzyme having 5' to 3' exonuclease activity, such as T7 gene 6 exonuclease, ExoWlll, Reel, and spleen phosphodiesterase II. Such "blocking" nucleotides include nucleotides containing a phosphorothioate, borano-phosphate, or peptide group as will be described below. In a primer extension reaction utilizing a modified primer containing a blocking nucleotide as die cleavable site, following a primer extension reaction, the resulting product, composed of (i) a modified primer containing a blocking nucleotide, and (ii) an extension segment, is treated widi a nuclease such as an exonuclease having a 5' to 3' exonuclease activity. Nuclease treatment typically results in digestion of the first region of e primer to generate an extension segment composed of nucleotides downstream (i.e., 3') of the cleavable site. In all of the exemplary embodiments described above, cleavage of die cleavable site results in the release of newly synthesized primer extension products containing little or none of the nucleotide bases originally present in die modified primer. B. INTRODUCTION OF THE CLEAVABLE SITE The cleavable site (composed of a modified nucleotide) is typically introduced into an oligonucleotide probe by using one of following syndietic approaches. Depending upon the choice of cleavable site to be introduced, either a fiinctionalized nucleotide or a modified nucleotide dimer is first prepared, and dien selectively introduced into a growing oligonucleotide fragment during die course of primer syn esis. The primer containing the cleavable site may be prepared using solution syndiesis, or preferably, employing automated solid phase syndiesis conditions using a DNA syndiesizer. In forming a modified dimer, two suitably protected nucleotides are coupled to each odier to form a modified 3'-5'-internucleotide linkage. The dimer containing the cleavable site (or a protected form tiiereof) is then incorporated into the oligonucleotide primer during syndiesis to form a modified oligonucleotide containing a cleavable site. The cleavable site is chemically cleavable under select conditions but is stable under standard solid phase DNA synthesis, solid support attachment, primer extension and hybridization conditions. Alternatively, functional ization is carried out on a single nucleotide to introduce a reactive group suitable for forming a cleavable site upon reaction widi a second nucleotide molecule or during primer syndiesis. Aldiough functional ization may take place at sites widiin the base or the sugar of the nucleotide, typically, modification will be carried out to result in an oligonucleotide primer containing a specific cleavable site in place of one of the phosphodiester linkages of the resulting polymer. Preferred non-internucleotide locations for modification or introduction of a cleavable site include C(5) of tiiymine and N(4) of cytosine, as diese two base sites are readily chemically manipulated wi out preventing base pairing. A number of exemplary internucleoside cleavable sites for use in the oligonucleotide composition of e present invention are illustrated in Figs. 1B-1W. Fig. IA is an illustration of an unmodified, native 3,-5,-phosphodiester linkage. A cleavable site or linkage for use in the invention is one which may be introduced at a specific position widiin the oligonucleotide sequence, preferably at or within about five nucleotides from the 3' end of the primer, and is selectively cleaved under conditions which do not permit cleavage of the immobilization attachment site. In one preferred embodiment, e cleavable site is located at e 3' end of the primer. The cleavable linkage should also be one that is chemically accessible. Chemically cleavable internucleotide linkages for use in the present invention include but are not limited to die following, as illustrated in Figs. 1B-1W, respectively: dialkoxysilane, Fig. IB; j8-cyano ether, Fig. 1C; 5'-deoxy-5'-aminocarbamate, Fig. ID; 3'deoxy-3'-aminocarbamate, Fig. IE; urea, Fig. IF; 2'cyano-3',5'-phosphodiester, Fig. 1G; 3'-(S)-phosphorodιioate, Fig. 1H; 5'-(S)-phosphorothioate, Fig. II; 3'-(N)-phosphoramidate, Fig. U; 5'-(N)-phosphoramidate, Fig. IK; α-amino amide, Fig. 1L; vicinal diol, Fig. 1M; ribonucleoside insertion, Fig. IN; 2'- amino-3',5'-phosphodiester, Fig. 10; allylic sulfoxide, Fig. IP; ester, Fig. 1Q; silyl e ier, Fig. 1R; didiioacetal, Fig. IS; 5'-thio-formal, Fig. IT; α-hydroxy-methyl-phosphonic bisamide, Fig. IU; acetal, Fig. IV; and 3'-thio-formal, Fig. 1W. Other chemically cleavable linkages include me ylphosphonate and phosphotriester. Cleavable linkages suitable for non-chemical cleavage mediods such as photolysis or thermolysis include nitrobenzyl edier (NBE), cis-syn thymidine dimer (Nadji, et al, 1992), and cyclohexene. Nucleoside dimers containing the cleavable linkages illustrated in Figs. 1B-1W are synthesized using standard nucleic acid chemistry known to one of skill in the art (Hobbs, 1990; Townsend, et al, 1986). Alternatively, one may directly synthesize a modified nucleoside containing either a 5'- or 3'-reactive group (or protected form diereof) for use in standard solid phase synthesis to introduce the desired cleavable linkage. 2'-Functionalized nucleosides are typically prepared from die corresponding ribonucleoside starting materials. An internucleotide /3-cyano ether linkage, as shown in Fig. 1C, may be formed by reaction of a suitably protected nucleoside witii a 5'-(2-cyanoallyl) functional ized 3'-phosphoramidite. Selective cleavage is effected by a jS-elimination reaction upon treatment with base, promoted by me presence of the jδ-cyano substituent. A nucleoside dimer containing a 3'-(O)-carbamate internucleoside bond is prepared by any of a number of syndietic approaches including reaction between the corresponding 3'-acyI chloride and a 5'-amino-modified nucleoside. Alternatively, a 3'-modified isocyanate nucleoside is prepared and subsequently reacted witii die 5'-hydroxyl of a suitably protected nucleoside. A nucleoside dimer containing a 5'-(O)-carbamate cleavable linkage is prepared from the imidazole carbamate precursor. Oligonucleosides containing me iyl phosphonate linkages are prepared using solid support based synthesis with phosphonamidite reagents used in place of the standard phosphoramidites (Agrawal and Goodchild, 1987). Phosphotriesters are somewhat labile under basic deblocking conditions, however, this cleavable group may be introduced into an oligonucleotide backbone by using mild reaction conditions or more labile amine protecting groups (Miller, et al, 1971). Memanol or ethanol in die presence of tosyl chloride is used to esterify the internucleoside phosphate group (Moody, etal., 1989); methyl methanesulfonatemay also be used as a mediylating agent (Koole, et al, 1987). Preferred cleavable sites for use in me modified oligonucleotide composition include dialkoxysilane, ribose, 3'- and 5'-phosphoramidate, and 3'-and 5'-phosphorothioate. In one embodiment of die present invention, the cleavable site contained in the modified oligonucleotide primer is dialkoxysilane (Ogilvie, et al, 1986; Seliger, et al., 1987; Cormier, et al, 1988). Synthesis of a primer containing a dialkoxysilane internucleotide linkage is described in Example 1 A. Al ough die preparation of a diisopropylsilyl-linked dinucleoside is described in Example IA, alkyl groups for use as substituents on silicon are not limited to isopropyl and may be either straight chain or branched alkyl groups. Further, the two alkyl substituents on silicon may be identical, as in die case of diisopropylsilyl, or may be different. A variety of dialkylsilylating reagents are available from Petrarch Systems, Bertram, PA. In the synthetic approach outlined in Example 1 A, a reactive 3'-O-silyl ether intermediate is first prepared, followed by formation of a nucleoside dimer containing a 3'-5'-diisopropylsilyl internucleoside bridging group. Formation of a 3'-silyl triflate intermediate is carried out by reacting a 5'-(O)-dimethoxytrityl(DMT)-protected nucleoside, such as 5'-(O)-DMT-thymidine or theN-protected nucleosides N6-benzoyl-2'-deoxy-5'-(O)-DMT-adenosine, N4-benzoyl-2'-deoxy- 5'-(O)-DMT-cytidine, or N2-isobutryl-2'-deoxy-5'-(O)-DMT-guanosine, with an O-protected silane reagent. In Example IA, the protected nucleoside is treated with the reactive silane, bis(frifluoromethane-sulfonyl)diisopropylsilane, in the presence of die sterically hindered base, 2,6-di-tert-butyl-4-methylpyridine, to promote formation of the desired 3'-(O)-diisopropylsilyl triflate intermediate. Use of a bulky base such as the tri-substituted pyridine reagent helps to prevent formation of the undesired symmetrical nucleoside dimer formed by condensation of unreacted nucleoside widi die triflate intermediate (Saha, et al, 1993). Following introduction of the desired 3'-O-silyl ether group, die 3'-O-diisopropylsilyl triflate intermediate is reacted widi unprotected nucleoside to form the desired nucleoside dimer containing a 3'(O),5'(O)-dialkoxysilane cleavable site. The protected dimer may then be further fiinctionalized, for instance, by conversion of die 3'-hydroxyl to die corresponding 2-cyanoethyl- N,N-diisopropylphosphoramidite for use in automated solid phase syndiesis utilizing standard phosphoramidite chemistry to provide die desired primer sequence. Selective cleavage of the dialkoxysilane site is effected by treatment with fluoride ion (Corey and Snider, 1972) Anodier preferred selectively cleavable functionality for use in the invention is phosphorothioate. The preparation of primers containing a 3'(S)-phosphorothioate or a 5'(S)- phosphorothioate internucleotide linkage is described in Examples IB and 1C, respectively. In accordance widi die modified oligonucleotide composition of die invention, the phosphorodiioate internucleotide linkage is selectively cleaved under mild oxidative conditions (Cosstick, et al., 1989). In one syndietic approach for preparing primers containing a 3'(S)-phosphorothioate cleavable site as described in Example IB, a protected 3'-thio-substituted nucleoside starting material, such as 5-O-MMT-3'-S-benzoyI-3'-dιymidine (Cosstick, et al., 1988), is first deprotected by treatment with base to form die debenzoylated tiiiol, 5'-(O)-MMT-3'- diiodiymidine, followed by conversion to die corresponding reactive diiophosphoramidite by reaction with 2-cyanoethyl-N,N-diisopropylaminophosphormonochloridite. The reactive diiophosphoramidite is coupled to a second nucleoside molecule to form the corresponding tiiiophosphite dimer, followed by oxidation of die phosphorus center to form the fully protected 3'-(S)-phosphorothioate-linked dimer. In order to promote coupling of me diiophosphoramidite to a second nucleoside molecule such as 3'-O-acetyldιymidine and prevent undesired self-condensation side reactions, an acidic activating agent, 5-(p<2r..-nitrophenyl)tetrazole, is used. The thiophosphite dimer is oxidized widi a suitable oxidant such as tetrabutylammonium oxone or tetrabutylammonium periodate to form die fully protected (P-(O)-2-cyanoedιyl-3'-acetyl) dimer containing a protected form of the desired internucleoside linkage. Deprotection is readily carried out under standard conditions as described in Example IB. As discussed above, die nucleoside dimer, containing a 3'- (S)phosphorothioate cleavable linkage may be readily incorporated into an oligonucleotide primer using standard solid phase phosphoramidite chemistry. Alternatively, one may use the reactive diiophosphoramidite directiy to introduce die desired 3'-(S)-phosphorodιioate linkage into an oligonucleotide primer during solid phase synthesis. For introduction of a fiinctionalized nucleoside containing a 3'-(S)- thiophosphoramidite during solid phase syndiesis on controlled pore glass, during the coupling cycle for introducing the tiiio-modified nucleoside, the thio-modified nucleoside, dissolved in acetonitrile saturated with 5-(pαrα-nitrophenyl)tetrazole, is injected into the column containing the solid support, and the coupling efficiency is monitored by release of trityl cations. After preparing the desired immobilized, cleavable primer in accordance widi die present invention, and carrying out the desired hybridization and primer extension reactions, the phosphorothioate internucleotide site is cleaved by treatment with a mild oxidizing agent such as aqueous silver nitrate. Preparation of die corresponding 5'-(S)phosphorothioate modified oligonucleotide is carried out in a somewhat different fashion than diat described above for me 3'-(S)- phosphorothioate and is described in detail in Example 1(C). The approach makes use of a key 5-thio-modified nucleoside intermediate for incorporation of die desired 5'-(S)-phosphorodιioate cleavable linkage during solid phase oligonucleotide synthesis (Mag, et al., 1991 ; Sproat, et al. , 1987). Synthesis of the nucleoside building block containing a protected 5'-tiιio group is carried out by first preparing die 5'-tosylate of thymidine by treatment with tosyl chloride, followed by conversion of the 5'-tosylate to 5'-(S-trityl)-mercapto-5'-deoxythymidine. 5'-Tosyl-thymidine is converted to 5'-(S-trityl)-mercapto-5'-deoxydιymidine by treatment with a five-fold excess of sodium trityldiiolate, which is prepared in-situ by deprotonation of tritylmercaptan with sodium hydroxide. In the above synthetic step, a sulfur atom is introduced into the 5'-position of a nucleoside, forming the S-trityl precursor of the desired key intermediate. Subsequent phosphitylation at the 3'-position with 2-cyanoedιoxy-bis-(N,N-diisopropylamino)phosphine in e presence of tetrazole results in the desired functional ized nucleoside, 5'-(S-trityl)-mercapto-5'- deoxythymidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphite. The 5'-(S)-protected nucleoside intermediate is introduced into an oligonucleotide primer using standard solid-phase phosphoramidite chemistry by first coupling it to a deprotected polymer-bound oligonucleotide. The phosphite linkage is dien oxidized wid aqueous I 2 , and the S-trityl group is cleaved with silver nitrate and reduced widi didiiothreitol to form a reactive thiol. The thiol is then coupled to a 2'-deoxynucleoside-3'-phosphoramidite, followed by oxidation of the thiophosphite linkage to yield the desired 5'-phosphorotiιioate cleavable site. Selective cleavage of the phosphorodiioate site may also be effected by treatment with an aqueous solution of either silver nitrate (AgNO 3 ) or mercuric chloride (HgClj). Anodier functional group for use as a cleavable site in the modified oligonucleotide composition of the invention is phosphoramidate. Oligonucleotides bearing phosphoramidate internucleotide linkages can be prepared chemically by standard, solid phase DNA syndiesis (Bannwarth, 1988). Preparation of primers containing a 5'(N)-phosphoramidate internucleotide linkage is described in Example ID. In the syntiietic approach described in Example ID, a 5'- amino-modified nucleoside is eidier purchased commercially or synthesized by conversion of die 5'-hydroxyl group to die corresponding azide, followed by reduction over a palladium/carbon catalyst (Yamamoto, et al, 1980). The 5'-amino group is then protected by treatment with 4-memoxytritylchloride, followed by reaction with b i s (d i isop ropyl ammonium)tetrazolide and (2- cyanoedιoxy)bis(diisopropylamino)phosphine to form the corresponding 3'-(2-cyanoethyl)-N,N- diisopropylphosphoramidite-functionalized nucleoside. This reactive nucleoside, containing a 5'- protected amino function, is then selectively introduced into an oligonucleotide fragment during standard solid phase DNA syndiesis utilizing phosphoramidite chemistry, to form the desired 5'- phosphoramidate bond. Selective cleavage of the phosphoramidate bond is carried out under mild acidic conditions, such as by treatment widi 80% acetic acid. Phosphoramidate linkages are more labile in me ribo series than in me deoxyribo series (Tomasz, et al, 1981). Anomer functional group for use as a cleavable site in die present oligonucleotide composition is ribose. Modified primers containing a cleavable ribose are described in Examples 3-5. Ribose, containing suitable 0-protecting groups, is selectively introduced into a growing oligomer fragment during automated solid phase syndiesis using standard phosphoramidite chemistry. Selective cleavage is carried out by treatment widi dilute ammonium hydroxide, as described in Examples 3 and 5. C. ATTACHMENT TO SOLID SUPPORT In accordance witii one aspect of the invention, the oligonucleotide primers (i) may be designed for attachment to a solid support in a manner mat does not block die ability to extend the primer from its 3' end, and (ii) incorporate a cleavable moiety so mat a 3' portion of die primer (linked to an extension product) can be released from an optionally immobilized 5' por¬ tion. The oligonucleotide primers of die invention are preferably designed for binding to a solid support material eitiier directly, tiirough an intervening spacer arm, or by specific hybridization to an intermediary oligonucleotide which is bound to a solid support (SPBIO). Immobilization may occur at a location upstream (i.e., 5' to) or downstream (i.e., 3' to) of the cleavable site. Attachment to a solid phase may also take place via an attachment site (i) contained widiin a nucleic acid extension segment resulting from an enzymatic extension reaction, or, (ii) contained widiin a target nucleic acid. The immobilization attachment site can be a select functional group for covalent bonding to a solid support, such as tiiose representative functional groups shown in Figs. 2A-2K, and Fig. 2M. The immobilization attachment site can also be a ligand such as biotin, for attachment via a high-affinity non-covalent interaction with a solid support. Further, die immobilization attachment site can also be composed of a series of bases complementary to an intermediary oligonucleotide bound to a solid support, as illustrated in Figs. 8 and 9. Referring now to Fig. 8, a primer 15 having a cleavable site 17 as described above is (i) hybridized to a single stranded, target DNA sequence 21 utilizing conditions under which die target will anneal stably to the primer, and (ii) enzymatically extended to form an extension segment 19. The extension product is then exposed to an intermediary oligonucleotide which is bound to a solid support 23. The intermediary oligonucleotide is complementary to all or at least me first region of the primer. In the embodiment illustrated in Fig. 8, die sequence of die intermediary oligonucleotide is homologous witii at least a portion of die target molecule 21, so mat both the intermediary oligonucleotide and the target are competing to hybridize to an overlapping region of the primer. In instances in which die sequence of the extension product is known, the sequence of die intermediary oligonucleotide may be designed to be complementary to a portion of the extension segment rather tiian to the primer, or, to a region containing portions of both the primer and me extension segment. In employing is approach for immobilization, the concentration of target molecule relative to intermediary oligonucleotide is preferably reduced in order to favor hybridization of me primer extension product to die intermediary oligonucleotide. Hybridization of the extension product to template is tiiermodynamically favored, since the template is capable of hybridizing to the full length of the extension product. The concentration of target nucleic acid can be reduced by a number of mediods, including specific chemical or enzymatic digestion which leaves die primer extension product intact. Selective digestion of the template may be effected by any of a number of methods, including die following: (i) use of a deoxyuridine-containing template; (ii) use of an RNA template to provide DNA extension products; (iii) use of a template containing modified internucleoside linkages; or (iv) exonuclease-promoted digestion of template. Each is described in greater detail below. In employing die first approach, a nucleic acid fragment which has been site selectively modified (Longo, et al, 1990) to contain deoxyuridine in place of deoxythymidine is used as a template for the primer extension reaction. Following enzymatic extension, the template- containing reaction mixture is treated widi uracil DNA glycosylase (Amersham Life Sciences, Arlington Heights, IL) to fragment the template molecule at positions modified to contain deoxyuridine. Uracil DNA glycosylase excises deoxyuracil from dU-containing DNA by cleaving the N-glycosidic bond between the uracil base and the sugar phosphate backbone. In the second approach, an RNA template is used to provide DNA extension products. The template is then selectively removed by digestion using RNase, such as RNase A. Alternatively, as indicated by (iii) above, a template molecule containing modified internucleoside linkages such as a phosphoramidate or phosphorotiiioate is used. Following extension of the primer, the template is digested by chemically-promoted cleavage at me modified linkage positions. The choice of template-modified internucleoside linkage and template-digestion reagent will depend upon die type of cleavable site present in the primer. Digestion of template is typically carried out under conditions which leave die primer cleavable site intact. Cleavage of a phosphorothioate linkage (5'-(O)-P(S)O 2 ) can be effected by treatment with glycidol or iodoetiianol (Olsen), while selective cleavage of a phosphoramidate bond is typically carried out under mild acidic conditions, such as by treatment with 80% acetic acid. In utilizing exonuclease-promoted digestion of template (as indicated by (iv) above), me primer extension product is modified to contain a suitable exonuclease-resistant blocking group as described previously. Upon exonuclease treatment with either a 3'-5' specific or a 5'-3' specific exonuclease, the "protected" primer extension product then remains intact, due to the presence of die blocking group. In utilizing a 3'-5' exonuclease (e.g., snake venom phosphodiesterase or exonuclease III), a suitable blocking group is placed at the 3' terminus of the extension product to prevent enzyme-promoted degradation. The relative concentration of template to solid-bound intermediary oligonucleotide, can also be minimized, for example, (i) by performing cycle synthesis procedures witii limited amounts of template (e.g., cycle sequencing or strand displacement amplification), or (ii) adding a large excess (e.g., 10 to 100-fold) of intermediary oligonucleotide to die reaction mixture. Returning now to Fig. 8, under conditions which favor hybridization of the primer extension product to the solid phase bound intermediary oligonucleotide, the primer extension product is immobilized 25 by hybridization to e solid phase bound intermediary oligonucleotide 23 to form captured product 27 and free (i.e., single stranded template). The 5' end of die solid phase bound intermediary oligonucleotide may terminate before or after e cleavable site of die modified primer. After immobilization as described above, excess reaction products are removed by washing 29 to provide a purified immobilized product 31. The immobilized product is cleaved 33 to release die extension segment 35 for subsequent analysis. In accordance with the present invention, immobilization of a cleavable extended primer by hybridization to an intermediary solid phase bound oligonucleotide is described in Example 6. Briefly, a modified M13 reverse primer containing a 5'-(S)-thymidine cleavable group (SEQ ID NO: 12) was (i) hybridized to a single-stranded target, and (ii) enzymatically extended in the presence of dideoxythymidine to produce a set of dideoxythymidine- terminated extension fragments at a 8: 1 ratio of primer to template. The single stranded extension products were then annealed to an intermediary oligonucleotide (SEQ ID NO: 13) complementary to the M13 reverse primer and biotinylated at die 3' end. The extension product-intermediary oligonucleotide hybrids were then immobilized by addition of streptavidin-coated magnetic beads, washed, and the extension product released by silver-nitrate promoted cleavage of e 5'-(S)-thymidine cleavable group. The extension segments were analyzed by MALDI time-of-flight mass spectrometry, as shown in Fig. 13. As can be seen, extension segments with read lengtiis up to at least about 33 base pairs can be detected with good resolution. In a variation of the above approach, as shown in Fig. 9, the primer 39 is designed to contain an immobilization attachment site 38 contained in the first primer region composed of a series of bases complementary to an intermediary oligonucleotide bound to a solid support. However, in this embodiment, the intermediary oligonucleotide does not share homology with the template molecule 45, so that the intermediary oligonucleotide and template do not compete witii one anotiier for hybridization to the primer. As illustrated in Fig. 9, following enzymatic extension to form extension segment 43, the primer is immobilized by specific hybridization to die solid phase bound intermediary oligonucleotide 37. This design of primer, template, and intermediary oligonucleotide allows for simultaneous, non-competitive hybridization of die primer to botii the template and intermediary oligonucleotide. The solid phase bound extension product is washed and the template is eliminated 47 to provide a purified immobilized product 49, which is tiien cleaved 51 at cleavable site 41 to release extension segment 53 for subsequent size and/or sequence analysis as described for Fig. 8 above. Alternatively, a primer of the present invention may be bound to a solid phase via hybridization to a target nucleic acid which is immobilized, as shown in Fig. 10. In utilizing this approach, the target molecule 61 acts as both a template for enzymatic extension of the primer 55 and as an intermediary for solid phase binding of the primer. The template can be attached to die solid phase either before or after carrying out enzymatic extension of the primer to form the extended primer segment 59. As has been described, immobilization of the primer extension product allows for ready removal of excess enzyme, salts, etc., 63, to provide a purified immobilized primer extension product 65. Prior to analysis, die extended primer is denatured from die template 67 and released into solution. Cleavage at die cleavable site 57 promotes release of an extension segment 71 and a fragment composed of the first primer region 69. Depending upon tiie design of the primer and die mode of product analysis, the presence of primer fragment 69 may adversely impact the quality of die subsequent product analysis. In these instances, the same methods used to eliminate template, as described above, may be used to eliminate fragment 69. In cases in which e extension segment is sized by mass spectrometry, the mass of fragment 69 can, in some instances, be selected to avoid interference with product peaks in the resulting mass spectra, and may also be used to provide an internal mass standard. Upon attachment of the immobilization attachment site to a solid support, the resulting immobilization linkage is generally one which remains stable under the conditions employed for cleaving die cleavable site and does not inhibit base pair hybridization nor block die ability to extend die primer from its 3' end. In the modified primer of die invention, die immobilization attachment site is typically separated from the cleavable site by at least three nucleotides. In a preferred embodiment, upon selective cleavage of die cleavable site, a large portion of die primer fragment remains affixed to die solid support. This enables die release of primer extension products tiiat typically contain about five or fewer base pairs of die primer sequence, to provide more useful sequence information per fragment than extension products containing die entire primer. The modified primers of tiie present invention may, for example, be used for detecting a genetic disorder for which the nucleotide sequence of both die wild type and mutant alleles are known. A modified primer for this purpose will have a 5' end and a 3' end and contain from about 8-30 base pairs complementary to die gene sequence upstream from the known mutation site. Preferably, the 3' end of the primer is complementary to a site upstream from the known mutation region by at least about ten base pairs, to provide verifying sequence information on either side of die mutation region. In accordance witii one aspect of the invention, die modified primer also contains (i) an immobilization site for attachment to a solid support and (ii) a cleavable site. One primer design according to die present invention is one in which die immobilization site is located 5' of me cleavable site which is preferably located at or widiin about five base pairs from die 3' end of the primer. This modified primer is men used as a probe to distinguish me presence of DNA containing the mutant sequence of interest. The primer is (i) hybridized to an unknown, single- stranded, target DNA sequence utilizing conditions under which both the mutant and die normal sequences will anneal stably to the primer, and (ii) enzymatically extended. Primer immobilization may optionally take place either before or after chain extension. Following chain extension and release of die immobilized primer extension products by selective cleavage of die cleavable linkage, the primer extension products are analyzed to determine die sequence across die known mutation region and identification of the genetic disorder, if present. An exemplary modified primer containing 20 deoxynucleotide residues and specific for its ability to detect a known genetic disorder is shown in Figs. 6A and 6B. As indicated, die modified primer contains a first region containing an immobilization attachment site, "I", mat is 5' of the cleavable site, "X", and consists of a total of 16 nucleotide residues. The second region contains tiie 3' end of the primer and contains 4 nucleotides (C-T-G-C). The cleavable linkage, X, connects the first and second regions. In illustrating this aspect of the invention, the modified primer as shown in Fig. 6A (top) and having the sequence presented as SEQ ID NO:2, is first hybridized to a single stranded DNA target having the sequence presented as SEQ ID NO:3, as shown in Fig. 6A. Typically, die hybridization medium contains (i) denatured, unknown (target) DNA from a human or other biological source, (ii) the modified probe, and (iii) annealing buffer, such as 5X "SEQUENASE" Buffer (200 mM Tris-HCl, pH 7.5, 100 mM MgCl 2 , 250 mM NaCl) (United States Biochemical Corporation, Cleveland, OH). The annealing reaction is carried out by warming the above mixture to 65 °C for two minutes, and tiien allowing the mixture to cool slowly to room temperature over a period of about thirty minutes (Maniatis, etal, 1982; Ausubel, etal, 1989). Following hybridization, d e modified primer is extended on die single stranded template widi deoxynucleotides and randomly terminated witii dideoxynucleosides using DNA polymerase (e.g., "SEQUENASE" DNA Polymerase, Version 1.0 or 2.0) to perform DNA synthesis (Primings, et al, 1980; Sanger, 1975). As indicated in Fig. 6A, extension occurs from the 3' end of die modified primer. The primer extension products are denatured from the target, typically using heat or a chemical denaturant such as formamide, to provide a mixture of both primer extension products and target DNA. (See for example "PROTOCOLS FOR DNA SEQUENCING WITH SEQUENASE T7 DNA POLYMERASE", Version 1.0 or 2.0, 4th ed., United States Biochemical, or "CIRCUMVENT Thermal Cycle Dideoxy DNA Sequencing Kit Instruction Manual", New England Biolabs, Inc., Beverly MA). As shown in Fig. 6B, the primer extension products are tiien bound to the solid support at die immobilization attachment site, although immobilization may optionally be carried out prior to enzymatic extension and/or denaturation. By immobilizing the extended primers, the target DNA strands which remain free in solution are readily removed, along with excess reagents, ions, enzymes and die like, in a series of wash steps. Generally, me solid substrate is washed widi large volumes of a wash solution (e.g., 10 mM TrisHCl, 1 mM EDTA; or pure water) at room temperature. The solid particles, containing immobilized primer extension products and free of impurities, are then submitted to conditions effective to selectively cleave die cleavable site while maintaining die first primer region having the sequence presented as SEQ ID NO:4 affixed to the solid support as shown in Fig. 6B. As indicated in the particular embodiment illustrated in Fig. 6B, selective cleavage results in release of primer extension products containing only four nucleotides from the original modified primer. The supernatant containing the released extension segments is suitable for subsequent analysis. Immobilization simplifies purification of the primer extension products for subsequent analysis. As discussed above, undesirable enzymes, salts, reagents, and sequencing targets are washed away prior to selective cleavage of the extension product. In immobilizing the modified primers of the present invention, the first region of the primer may be attached to die solid support material eitiier prior to or after introduction of die cleavable site. Any of a number of methods commonly employed in the art may be utilized to immobilize an oligonucleotide on a solid support (Saiki, et al, 1989; Zhang, et al., 1991; Kremsky, et al, 1987; Van Ness, et al, 1991; Ghosh, et al, 1987; Gingeras, et al., 1987; Khrapko, et al, 1991). Solid support materials for use in the invention include cellulose, nitrocellulose, nylon membranes, controlled-pore glass beads, acrylamide gels, polystyrene matrices, activated dextran, avidin/streptavidin-coated polystyrene beads, agarose, polyetiiylene, fiinctionalized plastics, glass, silicon, aluminum, steel, iron, copper, nickel, silver and gold. Some substrates may require functional ization prior to attachment of an oligonucleotide. Solid substrates tiiat may require such surface modification include aluminum, steel, iron, copper, nickel, gold, and silicon. In one approach, die solid substrate material is fiinctionalized by reaction with a coupling agent, such as a zircoaluminate. Zircoaluminates generally contain both oxo and hydroxy bridges and are characterized by high thermal and hydrolytic stability. Such compounds, due to tiieir highly metallic nature, are particularly reactive with metal surfaces such as the metallic solid supports described above. Bi-functional zircoaluminates containing a variety of organofunctional groups are commercially available (e.g., "MANCHEM" Zircoaluminates, Rhδne-Poulenc Latex & Specialty Polymers, Cranbury, NJ). Upon attachment to a solid support, the oligonucleotide, typically DNA, should couple efficiently to the solid support material. Further, the immobilized DNA should be botii stable upon immobilization and accessible for base hybridization and other potential derivatization reactions. The immobilization attachment site should remain stable under die conditions employed for selectively cleaving the cleavable site in the modified oligonucleotide composition of the invention. Coupling of an oligonucleotide to a solid support may be carried out tiirough a variety of immobilization attachment functional groups. Immobilization attachment sites for use in the present invention include those illustrated in Figs. 2A-2M. Attachment of the support material to die oligonucleotide may occur by reaction between die reactive site on die support and a reactive site contained widiin die oligonucleotide or via an intervening linker or spacer molecule. Altiiough any suitable functional group fulfilling the desired criteria above may be used to attach the oligonucleotide to the support, preferred linkages include disulfide (Fig. 2G), carbamate (Fig. 2B), hydrazone (Fig. 2J), ester (Figs. 2C, 21 and 2K, where Y equals oxygen), (N)-functionalized thiourea (Fig. 2D), fiinctionalized maleimide (Fig. 2A, where Y equals sulfur, oxygen, or nitrogen), streptavidin or avidin/biotin (Fig. 2L), mercuric-sulfide (Fig. 2E), gold- sulfide (Fig. 2M), amide (Figs. 2C, 21 and 2K, where Y equals nitrogen), iolester (Figs. 2C, 21 and 2K, where Y equals sulfur). Other suitable functionalities for attaching to a solid support material include azo, ether, and amino. The immobilization attachment site may be located (i) as a substituent along die modified primer backbone (e.g., derivatization occurring at a terminal 5'-hydroxyl position), (ii) as a substituent on one of the bases or sugars of the modified primer, (iii) in the first region of die primer, composed of a series of bases complementary to a solid phase bound intermediary oligonucleotide, (iv) within a nucleic acid extension segment resulting from an enzymatic extension reaction, or (v) contained within a target nucleic acid. Immobilization via a base pairing interaction between the primer and a solid phase bound intermediary oligonucleotide (SPBIO) is shown in Figs. 8 and 9. Indirect immobilization of the primer extension product via a base pairing interaction to solid phase bound template is shown in Fig. 10. Solid support materials for use in coupling to an oligonucleotide include fiinctionalized supports such as the 1 , 1 '-carbonyldiimidazole activated supports available from Pierce (Rockford, IL) or fiinctionalized supports such as those commercially available from Chiron Corp. (Emeryville, CA). Solid supports for use in the present invention include matrix materials such as 6% cross-linked agarose, Trisacryl GF-2000 (a hydrophilic matrix material) and TSK HW- 65F, all activated with l,l'-carbonyldiimidazole (Pierce). Immobilization is typically carried out by reacting a free amino group of an amino-modified oligonucleotide widi die reactive imidazole carbamate of the solid support. Displacement of the imidazole group results in formation of a stable N-alkyl carbamate linkage between the oligonucleotide and the support as shown in Fig. 2B. Coupling is usually carried out at pHs ranging from 9-11 although a pH range from 9.5-10 is preferable. Coupling to pH sensitive materials may be carried out in buffer at pHs around 8.5. Amino-modified oligonucleotides for use in attaching to a solid support may be syndiesized using standard solid phase DNA syndiesis memodologies employing, for example, the modified nucleoside phosphoramidite Amino-Modifier-dT (Glen Research, Sterling VA), which contains a base labile trifluoroacetyl group protecting a primary amine attached to thymine via a 10-atom spacer arm, phosphoramidite 5'-Amino-Modifier C6 (Glen Research, Sterling VA), which contains a primary amino group protected widi an acid labile monometiioxytrityl group,orN-trifluoroacetyl-6-aminohexyl-2-cyanoethylN',N'-isopropylphosphoramidite(Applied Biosystems, Foster City, CA). Although amino-containing oligonucleotides are most commonly prepared using phosphoramidite chemistry, any other metiiod which leads to oligonucleotides containing primary amine groups may also be used. Amino-modified oligonucleotides are readily transformed to the corresponding thiol or carboxyl-terminated derivatives for use in immobilization or spacer arm attachment reactions requiring 5'-functionalities other than amino. Amino-modified oligonucleotides may be converted to die corresponding carboxyl derivatives by reaction widi succinic anhydride (Bischoff, et al, 1987). If desired, the carboxyl-derivatized primer may be coupled to a bifunctional linker such as 1 ,6-diaminohexane prior to attachment to the solid support by carrying out die coupling reaction in die presence of an activating agent such as a water soluble carbodiimide. Thiol-modified oligonucleotides may be prepared by treating die deprotected 5'-amino group of a fiinctionalized oligonucleotide witii dithiobis(succinimidylpriopionate), followed by sulfhydryl deprotection witii ditiiioerythritol (Bischoff, et al, 1987). Oligonucleotides containing free amino, thiol, and hydroxyl functions may also be coupled to supports by utilizing epoxide ring-opening reactions (Maskos, et al, 1992). One such exemplary epoxy-activated solid support is available from Pierce (Rockford, IL) and contains 1,4-butanediol diglycidyl ether-activated agarose. Coupling reactions are typically carried out at pHs from 7.5-13, depending upon the stability of the molecule to be immobilized. In immobilization reactions carried out witii the above Pierce support, the resulting immobilized oligonucleotide is separated from the solid support by a 13-atom hydrophilic spacer arm. In another immobilization approach, aldehyde groups of a modified oligonucleotide are coupled to hydrazide groups on a solid matrix as shown in Fig. 2J. A primary hydroxyl group on an oligonucleotide is first oxidized to the corresponding aldehyde, typically with a mild oxidant such as sodium periodate. The oligonucleotide is tiien coupled to a hydrazide-containing matrix such as Pierce's CarboLink™ Hydrazide. The coupling reaction is performed at neutral pH. Alternatively, the immobilization reaction is carried out using a thiol-derivatized oligonucleotide which is coupled to a fiinctionalized matrix such as Pierce's Immobilized p- Chloromercuribenzoate (Fig. 2E). The support is a cross-linked agarose containing an ethylene diamine spacer and coupling takes place via affinity binding between the mercury and sulfur atoms. Similarly, as shown in Fig. 2M, immobilization may be carried out by anchoring a 5'- tiiiolated primer to a gold surface (Hegner, et al, 1993a). Using this approach, the modified primer is chemisorbed onto a gold surface (e.g., the solid support) via thiolate bonding. The preparation of polycrystalline gold surfaces has been previously described (Hegner, et al., 1993b). Functional ization may also be carried out using a homo- or hetero- bifunctional cross- linker, such as Pierce's Sulfo-SMCC. Cross-linkers for use in die present invention will typically contain spacer arms between about 3-20 angstroms in length. Cross-linkers for use in coupling an oligonucleotide to a solid support will typically contain functional groups for targeting reactive primary amines, sulfhydryls, carbonyls, and carboxyls. Cross-linking agents for reaction with primary amino groups will typically contain terminal amidoester groups or N- hydroxysuccinimidyl esters. An exemplary linker such as Pierce's Sulfo-SMCC contains a reactive carboxyl group at one end for coupling to amine-derivatized solid supports such as hexylamine-derivatized polystyrene beads. The other end of die linker molecule contains a reactive maleimide molecule which readily reacts witii oligonucleotides containing nucleophilic groups such as hydroxy, tiiio, or amino. Cross-linkers for reaction with sulfhydryl groups typically contain terminal maleimide groups, alkyl or aryl halides, α-haloacyls or pyridyl disulfides. A variety of chemical cross-linking agents are available from Pierce (Rockford, IL). Alternatively, coated plates, such as those available from Pierce, may be used to immobilize the modified oligonucleotide. Examples of plates for use in immobilizing the oligonucleotides of the invention include activated plates such as Pierce's Reacti-Bind™ Maleic Anhydride Activated Polystyrene Plates and Reacti-Bind™ Streptavidin Coated Polystyrene Plates. A primary amino-containing oligonucleotide is immobilized on die former plate surface by covalent attachment through a stable amide bond formed by reaction between the free amino group of the oligonucleotide and the reactive anhydride (Fig. 2H). The latter plates are effective for affinity binding of biotinylated oligonucleotides. Gold-coated plates may also be utilized for binding to tiiiol-derivatized primers. Biotinylated oligonucleotides for use in immobilization to streptavidin or avidin-coated solid supports are prepared as described in Example 2A and shown in Fig. 2L. A variety of biotinylation reagents are commercially available (e.g., Pierce) which are fiinctionalized to react widi molecules such as modified oligonucleotides containing primary amino, sulfhydryl, or carbohydrate groups. Returning to Example 2A, an amino-modified primer is treated witii biotin or with a modified form of biotin containing an intervening spacer arm, such as NHS-SS-Biotin (Pierce), or NHS-LC-Biotin (Pierce), a biotin derivative containing an eleven carbon spacer arm between biotin and a terminal N-hydroxylsuccinimide activated carboxyl group. The biotinylated primer is then immobilized by attachment to a streptavidin-coated support. Due to die strong non- covalent biotin/ streptavidin interaction, the immobilized primer is considered to be essentially irreversibly bound to the solid support. This is one preferred immobilization attachment for use in the present invention, as the resulting immobilized complex is unaffected by most extremes of pH, organic solvents, and otiier denaturing agents (Green, 1975). An alternative to avidin(streptavidin)-biotin immobilization is incorporation of a digoxigenin molecule (Sigma, St. Louis, MO) in die modified primer widi subsequent capture using anti-digoxigenin antibodies. Enzymatic methods may also be utilized for coupling an oligonucleotide to a solid support (Goldkorn, et al, 1986). In one exemplary embodiment, a poly(dA) tail is added to die 3' ends of a double stranded DNA using 3'terminal transferase. The (dA)-tailed DNA is then hybridized to oligo(dT)-cellulose. To covalently link die DNA to die solid support, die hybridized sample is first reacted witii a Klenow fragment of DNA polymerase I, followed by treatment with T4 DNA ligase. The unligated strand of DNA is separated from die immobilized strand by heating followed by extensive washing. The method results in ssDNA covalently linked by its 5' end to a solid support. The modified primers of die present invention may also be affixed onto a gold surface. In utilizing this immobilization approach, oligonucleotides modified at die 5'-end witii a linker arm terminating in a thiol group are chemisorbed witii high affinity onto gold surfaces (Hegner, etal, 1993a). Thiolated primers, available tiirough post solid-phase syndiesis modification using commercially available reagents (Pierce, Rockford IL), are immobilized on a thin layer of gold eitiier prior to or following enzymatic extension. The gold layer is deposited onto the sample stage for direct analysis by mass spectrometry following internal cleavage and evaporation. Alternatively, the resulting extension segments may be transferred onto an alternate surface prior to analysis. m. REACTIONS EMPLOYING THE IMMOBILIZED CLEAVABLE OLIGONUCLEOTIDE COMPOSITION A. HYBRIDIZATION AND EXTENSION The method employed for determining the sequence of a target oligonucleotide strand will often involve Sanger-type sequencing using the modified cleavable primers of the present invention. Immobilization of the modified primer on the solid support may take place either before or after the enzymatic extension reactions. Utilizing the Sanger DNA sequencing procedure, dideoxynucleosides of each of the four bases are obtained for inclusion into the reaction mixture. The dideoxy nucleotides are incorporated into DNA by, for example, E. coli DNA polymerase since they have a normal 5' triphosphate group. Once incorporated into the growing DNA strand, the dideoxynucleoside triphosphate (ddNTP) cannot form a phosphodiester bond witii the next incoming dNTP and growth of the DNA chain is terminated. A typical DNA sequencing reaction using the Sanger method proceeds as follows. The reaction consists of a target DNA strand to be sequenced, a modified primer containing a cleavable site in accordance witii the invention, and that is complementary to the end of me target strand, a carefully controlled ratio of one particular dideoxynucleoside witii its normal deoxynucleotide counterpart, and die tiiree other deoxynucleoside triphosphates. The modified primer may or may not be immobilized to the solid support at tins point. (Immobilization may occur either before of after die enzymatic extension reactions, depending on a number of experimental factors). DNA polymerase is added and normal polymerization begins from the primer. Upon incorporation of a ddNTP, die growth of the chain is stopped. A series of different strands results, the lengths of which are dependent on the location of a particular base relative to the end of die DNA. The target strand is usually distributed into four DNA polymerase reactions, each containing one of the four ddNTPs and a modified primer of the present invention. The extension reaction is then carried out as described above. Sanger-type DNA sequencing is generally carried out using a DNA sequencing kit such as "SEQUENASE" Version 1.0 or Version 2.0 T7 DNA Polymerase (United States Biochemical, Cleveland OH). The "SEQUENASE" Version 1.0 kit uses a chemically-modified DNA polymerase derived from bacteriophage T7 DNA polymerase in which the high 3'-5' exonuclease activity of the native T7 DNA polymerase is inactivated. In using the USB "SEQUENASE" kit, double stranded templates are first denatured (if one is using double stranded template), and the primer is then hybridized or annealed to me target by heating for 2 min at 65 °C, followed by slow cooling to less man 35 °C over about 15-30 minutes. Supercoiled plasmid DNAs are denatured by treatment with sodium hydroxide, neutralized, and etiianol-precipitated in order to anneal die primer for sequencing. Termination mixtures containing the different ddNTPs are prepared for use in die termination reactions. The annealed DNA mixture is optionally labeled, and die labeled reaction mixture is added to each of die four termination tubes. In the present invention, extension reactions are carried out to produce a distribution of products ranging from near zero to several hundreds of base pairs in lengdi. Optionally, a stop solution (containing formamide, EDTA, bromophenol blue, and xylene cyanol FF) is added to stop the reactions prior to analysis of the resultant samples. For reactions in which the modified primers were not immobilized to a solid support prior to enzymatic extension, immobilization is carried out as described in Section IIB above. The 'immobilized extended primers are then washed to remove excess enzymes, ions, salts, and other impurities. In one embodiment, die extended primers are immobilized onto the surface of microtitre wells. Immobilization to the solid support facilitates product purification and subsequent isolation by cleavage of die primer at the cleavage site followed by removal in e supernatant. Alternatively, DNA sequencing may be carried out using deoxynucleotide a- tiiiotriphosphates, dNTPαSs (available from United States Biochemical, Cleveland OH), followed by limited exonuclease-promoted base-specific digestion, such as widi Exonuclease III (New England BioLabs, Beverly MA) or snake venom phosphodiesterase (Boehringer Mannheim, Mannheim, Germany) (Olsen, D., et al, 1993). Cleavage of DNA fragments specifically at positions of incorporated phosphorothioate groups may also be carried out using chemical reagents such as 2-iodoethanol or 2,3-epoxy-l-propanol (Nakamaye, et al., 1988). Briefly, the sequencing of a target DNA sequence using d e modified primers of die present invention via the incorporation of phosphorothioate nucleosides is carried out as follows. A target DNA sequence is hybridized witii a modified primer as described above. The primer is men extended enzymatically in die presence of one deoxynucleotide α-tiiiotriphosphate (dNTPαS) to generate a mixture of primer extension products containing phosphorothioate linkages. The primer extension products are then treated with a reagent tiiat (i) cleaves specifically at the phosphorothioate linkages, such as 2-iodoetiιanol or 2,3-epoxy-l-propanol, or (ii) digests the DNA downstream from the phosphorotiiioate linkage, such as a 3'-5' exonuclease, under conditions resulting in die production of a nested set of base-specific primer extension degradation products. Optionally, die primer extension degradation products are immobilized at die immobilization attachment sites to produce immobilized primer extension degradation products, each containing a primer and an extension segment. Alternatively, immobilization may be carried out eitiier i) prior to enzymatic extension, ii) after enzymatic extension, or iii) prior to treating the phosphorotiiioate-containing primer extension products witii a phosphorothioate- specific cleaving reagent. Subsequent to immobilization, the primer extension degradation products are washed to remove non-immobilized species. Cleavage at the cleavable site results in the release of extension segments, which are then sized by mass spectrometry. Using the sequencing method of this aspect of die invention, die read length of any given extension segment is increased relative to die read length of its corresponding primer extension degradation product. The steps of hybridization, enzymatic extension, treatment with a phosphorothioate- cleaving reagent, immobilization, washing, cleaving, and sizing are then repeated with a second, third, and fourth of the four different dNTPαS analogs to determine the sequence of the target DNA by comparison of the sizes of die extension segments obtained from each of the four extension reactions. The methods and modified primers described herein may also be used for obtaining a fingerprint of a target oligonucleotide. As described herein, fingerprinting refers to a method of determining the positions of no more than two different bases in a target oligonucleotide strand, as opposed to sequencing, which refers to a determination of the complete nucleotide sequence of (and also, in die case of a gene exon, die corresponding amino acid sequence encoded by) a target nucleic acid, including the identity and position of each nucleotide present in the target strand or its complement. DNA or RNA fingerprinting, which requires less reagents, can provide a rapid and cost effective alternative to sequencing, and may be used in a number of different applications, e.g., identification of one or more infectious agents in a genomic sample from a subject, screening cDNA libraries, screening genes from human or non- human genomes to detect mutations, polymorphisms, and for forensic applications. One preferred method for determining a single or 2-base fingerprint of an oligonucleotide extension segment, generated using die modified primers of die present invention, is mass spectrometry. In determining a fingerprint of a target oligonucleotide, a base-specific nested fragment set, containing base-specific terminated fragments derived from the target molecule, is produced for subsequent analysis. As referred to herein, a nested set is defined as a mixture of biopolymers (e.g., DNA, RNA, peptides, or carbohydrates) for which all of the components of the mixture have a common terminus and are produced from a single polymeric sequence. The base-specific nested fragment set may be produced, for example, by base-specific chain termination using the Sanger method or by selective chemical cleavage, to be described below. In instances where amplification of a target molecule is desired, amplification is carried out using any of a number of conventional amplification methods including PCR, SPA (single primer amplification, NASBA (nucleic acid sequence-based amplification), TMA (transcription-mediated amplification), and SDA (strand-displacement amplification). In fingerprinting methods employing a size fractionating device for product analysis, such as a mass spectrometer, die resulting single-base fingerprint can often provide indirect information regarding d e otiier bases present in the target sequence. For example, the difference in mass (Δm) corresponding to die positions of two peaks in a mass spectrum may also reveal the composition of the intervening bases, to be described as follows. To determine a single base fingerprint of a target oligonucleotide, such as a tiiymidine fingeφrint, a thymidine-specific nested fragment set is produced as described above. The resulting fragment family, containing a number of thymidine-terminated nucleotide fragments, is typically purified and analyzed by a size fractionation method, such as mass spectrometry. To increase mass spectrometric performance, mass-modified nucleotides can be utilized. The resulting mass spectrum contains a number of peaks, each corresponding to die mass of a particular thymidine-terminated fragment present in the product mixture. The differences in mass between the various thymidine-terminated fragments is then correlated witii die calculated mass of various combinations of nucleotides, preferably witii the assistance of a computer program containing the molecular weights of the modified primer, the portion of die primer 3' of the cleavable site, and each of the various nucleotides, to identify the combination of nucleotides intervening between the known thymidine positions. As an illustration, in considering the difference in mass between two given fragment peaks, an exemplary mass difference of 1,276 mass units corresponds uniquely to die following base composition: (two G) + A + T. A single base fingeφrint can thus be used to (i) identify die locations of a particular base widiin a target sequence, and (ii) determine die base composition widiin localized regions of a target sequence. For applications requiring a greater level of detail, a second base-specific nested set is produced and analyzed to produce a second fingeφrint as described above. Different base- specific nested sets can be produced in a single reaction vessel, or in separate reactions, depending upon die metiiod utilized and die corresponding reagents required. The different base- specific nested sets (e.g. , thymidine-terminated fragments and cytidine-terminated fragments) may be analyzed separately, or as a mixture. Use of a single base fingeφrint for detection of a point mutation is illustrated in Example 7. Briefly, the cytosine fingeφrints of two distinct single stranded DNA targets (SEQ ID NO: 14 and SEQ ID NO: 15) having sequences differing only at positions 16 and 19, relative to die 3' end (counting upstream after die priming region), were determined using dideoxycytosine triphosphate to produce a family of cytosine-terminated nucleotide fragments, followed by analysis of the resulting reaction product mixtures by mass spectrometry (Fig. 14A, 14B). The exact mass values corresponding to die differences between select peaks in each of die spectra were calculated, confirming the presence of two single nucleotide substitutions (point mutations) at positions 16 and 19. In an alternative approach, a base-specific nested fragment set is produced by selectively cleaving a DNA or RNA molecule modified to contain selectively cleavable groups (e.g., dUTP or amino fiinctionalized nucleoside triphosphates) at positions corresponding to a particular base. The resulting uridine-modified oligonucleotide is then treated widi uracil DNA glycosylase to form a set of fragments, preferably a nested set captured onto a solid phase. Similarly, a 5'- amino-modified target molecule is cleaved by treatment with acid. Preferably, the above fingeφrinting method employs die cleavable primers of me invention to remove die majority of the primer from the primer extension fragments. By reducing the mass of the analyte fragments, the distribution of products is shifted into the region where mass spectrometry has higher mass accuracy and resolution. Additionally, it may be useful to mass modify die different nucleotides by introducing any of a number of mass modifying functionalities (e.g., by replacing thymidine with 5'-bromouridine, or utilizing an immobilization attachment site, spacer arm, or alternative internucleotide linkage, as described above) to enhance die mass difference between die different nucleotides. In a second fingeφrinting approach, a restriction endonuclease is used to generate a non- random fragmentation pattern, useful, for example, for detecting mutations. In an alternate embodiment of the invention, target DNA for sequencing or fingeφrinting is amplified using the polymerase chain reaction or PCR (Mullis, 1987; Mullis, et al, 1987; Kusukawa, et al, 1990; Gyllensten, 1989). Briefly, PCR amplification is typically carried out by tiiermal cycling a cocktail containing die target DNA of interest, a mixture of deoxynucleotide triphosphates (dNTPs), a reaction buffer, each of two primers, and an extension enzyme such as Taq DNA polymerase (United States Biochemical, Cleveland OH) (Erlich, 1989; Innis, 1990). A PCR run profile typically consists of a 5-minute denaturation step at 94°C, followed by 30 cycles of 15 seconds at 94°C, 15 seconds at the annealing temperature, and 1 minute at 72°C. Following thermal cycling, the samples can be maintained at 4 C C until removal from the thermal cycler. Annealing temperatures range from about 55 C C to 65°C, although most target sequences amplify well at 60°C. Amplification is followed by hybridization with the modified primers of the present invention, enzymatic extension and sequencing of the products as described above. Fig. 12 illustrates PCR amplification of a template molecule using the modified primers of the present invention. As an alternative to using dideoxy chain terminators, PCR can be combined with dUTP incoφoration to produce a nested set terminated at the sites of dUTP incoφoration by treatment with uracil DNA-glycosylase. PCR can also be combined with phosphorothioate methods, as described above. In accordance with the present invention, amplification can be carried out using first and second primers, where one of the primers, i.e., the first primer 95, contains a cleavable site 99, and anodier primer, i.e., the second primer 105, contains an immobilization attachment site for binding to a solid support. The second primer is composed of a 5' end and a 3' end, is homologous to the target nucleic acid, and includes a first segment containing die 3' end of me second primer, and a second segment containing the 5' end of the primer and an immobilization attachment site. These first and second primers are combined witii a target nucleic acid 103 to generate primer/nucleic acid complexes and converted to double-stranded fragments 109 in the presence of a polymerase and deoxynucleoside triphosphates, as indicated at 107. The sizing method can be carried out using a large excess of target nucleic acid to generate substantial amounts of primer extension product, or alternatively, may be coupled widi various rounds of amplification. Upon achieving a desired amount of product 109, extension products containing die second primer are immobilized 117 by attachment at the immobilization attachment site, either before or after cleavage at the cleavable site. The extension product is tiien cleaved 111 at die cleavable site to generate a mixture which includes a double-stranded product 115. Non-immobilized cleaved fragments are removed 119, preferably by washing, and die purified double-stranded product 121 is denatured 123 to release die extension segment 125, which is sized by mass spectrometry, where the read length of the extension segment is increased relative to die read lengtii of the primer/nucleic double stranded fragments. As will be appreciated, the cleavable site of die first primer and the immobilization attachment site of the second primer include those of the types described above. In die exemplary embodiment illustrated in Fig. 12, the first primer 95 contains a restriction enzyme recognition site 97 in the first primer region 101, and a cleavable site 99 in the second primer region, and die second primer contains an immobilization attachment site for attachment to a solid support. Cleavage at the cleavable site is carried out by addition of a restriction endonuclease selective for the recognition site contained in die first primer region to provide (i) released fragments containing die first region of the first primer and (ii) a double- stranded product, which is immobilized prior to denaturing for release of the desired extension segment. As illustrated in Fig. 12, restriction endonucl ease-promoted cleavage results in the release of a double stranded product, 115, and a short double stranded fragment, 113, containing the first region of the primer. B. CLEAVAGE Cleavage of the selectively cleavable site is carried out as described in Section II A and in Examples 1 A-D and Example 3. Returning to this aspect of the invention, internucleoside silyl groups such as trialkylsilyl ether and dialkoxysilane are cleaved by treatment with fluoride ion. Base-cleavable sites for use in the present invention include S-cyano ether, 5'-deoxy-5'- aminocarbamate, 3 '-deoxy-3 '-aminocarbamate, urea, 2'-cyano-3',5'-phosphodiester, 2'-amino- 3',5'-phosphodiester, ester and ribose. Thio-containing internucleotide bonds such as 3'-(S)- phosphorothioate and 5'-(S)-phosphorothioate are cleaved by treatment with silver nitrate or mercuric chloride. Acid cleavable sites for use in the present invention include 3'-(N)- phosphoramidate, 5'-(N)-phosphoramidate, ditiiioacetal, acetal and phosphonic bisamide. An a- aminoamide internucleoside bond is cleavable by treatment witii isothiocyanate, and titanium is used to cleavea2'-amino-3',5'-phosphodiester-O-ortho-benzyl internucleosidebond. Vicinal diol linkages are cleavable by treatment with periodate. Thermally cleavable groups include allylic sulfoxide and cyclohexene while photo-labile linkages include nitrobenzylether and thymidine dimer. Cleavage conditions are utilized which leave the immobilization attachment site intact, so that a major portion of the primer remains affixed to the solid support. Preferably, cleavage of die cleavable site results in primer extension products containing five or fewer base pairs from die primer sequence. This maximizes die amount of sequence information provided upon subsequent analysis. C. ANALYSIS Any of a number of size fractionating devices may be used to determine die sequence of a target oligonucleotide fragment. Size fractionation methods for use in die present invention include gel electrophoresis, such as polyacrylamide or agarose gel electrophoresis, capillary electrophoresis, mass spectrometry, and HPLC. In methods employing gel electrophoresis sizing and analysis, die DNA fragments are typically labeled with either radioisotopes or with attached fluorophores, and visualized using autoradiography or fluorescence detection, respectively. The modified primers of die present invention are particularly advantageous when tiiey are used to generate oligonucleotide fragments whose sizes are to be resolved using technologies mat currently have difficulty resolving fragments of over about 100 base pairs differing by one nucleotide in length, such as mass spectrometry. One preferred metiiod for oligonucleotide analysis using die modified oligonucleotide composition of e invention is mass spectrometry, and particularly matrix-assisted laser desoφtion ionization (MALDI) mass spectrometry, preferably carried out on a time-of-flight (TOF) mass spectrometer (Wu, et al, 1993). MALDI mass spectrometry provides a rapid and efficient method for oligonucleotide sequencing. MALDI-TOF mass spectrometry may be used to provide unfragmented mass spectra of mixed-base oligonucleotides containing more than 100 base pairs. Moreover, mass spectral resolution of sequences currently to at least about 40 base pairs in length may be attained. In this method, pulsed ultraviolet laser light is used to desorb an oligonucleotide out of an absorbing solid matrix, which causes creation of free, unfragmented, charged oligomers. Mass analysis is done in a time-of-flight mass spectrometer. Singly charged molecular ions are typically the most abundant species and fragment ions are minimized. In preparing the sample for analysis, the analyte is mixed into a matrix of molecules which resonantly absorb at the laser wavelengtii. Solid matrix materials for this use include 3- hydroxypicolinic acid (Wu, et al, 1993), α-cyano-4-hydroxycinnamic acid Ofoungquist, et al., 1994), nicotinic acid (Hillenkamp, 1988), and ice (Nelson, et al, 1989), although a preferred material is 3-hydroxypicolinic acid. Examples 4 and 5 include detailed descriptions of MALDI-TOF mass spectral analyses of modified oligonucleotide compositions according to die present invention. Example 8, in conjunction witii Figs. 15A and 15B, illustrates die difference in fragment information obtained for cleaved primer extension segments according to the invention (Fig. 15B) versus non-cleaved full primer-extension segments (Fig. 15A). As described in Example 3, a synthetic 17-mer DNA probe containing a cleavable ribose in the 7-position was selectively cleaved by ammonium hydroxide treatment. Mass spectra of the intact mixed base primer prior to (Fig. 3A) and after (Fig. 3B) ammonium hydroxide treatment reveal die selective cleavage of the ribose linkage. As shown in Fig. 3A, two sizable peaks were observed for die intact 17-mer corresponding to die di-protonated molecular ion [M+2H] 2+ and the protonated molecular ion [M+H] + . Following ammonium hydroxide treatment, peaks corresponding to the expected cleavage products, the 7-mer, the 10-mer, and intact 17-mer, were readily observable and identifiable, as illustrated in Fig. 3B. Similarly, mass spectral analysis was carried out on a biotinylated 18-mer containing a ribose in the 10 position and captured on streptavidin-coated beads, as described in Example 5. The immobilized primer was washed after surface binding, followed by treatment with ammonium hydroxide to effect selective cleavage of the immobilized primer at the ribose site. Figure 4 illustrates the mass spectrum of the 8-mer resulting from selective cleavage of the ribose site within the immobilized primer. IV. UTILITY A. GENOMIC SEQUENCING The method of the present invention may be used for both "shotgun-type" sequence analysis and "directed walk". In the shotgun approach, a random sequence of DNA is selected and used to prime on an unknown target. This approach uses large numbers of primers to increase the possibility of successfully hybridizing with an unknown target sequence. In one embodiment of this approach, a multi-well assay format is used where each well has a different primer and the same substrate (i.e., the target DNA molecule) is added to each well under hybridization conditions. The primers in the wells are the modified primers of the present invention where immobilization to die well surface is through the primer immobilization site. Primer extension reactions are carried out. Extension products are only formed in wells where complementary sequences exist between the primer and die substrate. Each well is examined for the presence of extension products. Extension products are tiien sequenced and sequences assembled for any given target DNA molecule based on the known sequences of die primers that yielded extension products and base sequence overlap from die extension product sequences (i.e. , alignment of the extension product sequences). In using the modified primers of the present invention, die amount of sequence information for the extension segments is maximized over that obtained widi similar techniques employing conventional primers, due to cleavage and removal of a large portion of die primer prior to fragment analysis (e.g., increased read lengths). Further, die metiiod, when coupled with analysis by mass spectrometry, is fast and can provide large amounts of data in a relatively short period of time. In a related approach, die present method may be used to sequence or fingeφrint short reads of cDNA inserts for puφoses of gene mapping and identification. These short reads identify each insert uniquely and are called Expressed Sequence Tags (ESTs) or Expressed Fingeφrint Tags (EFTs). In preparing a cDNA library, cDNA copies of mRNAs are first inserted into a standard cloning vector such aspBlueScript. A modified primer according to die present invention is designed to hybridize to the pBlueScript vector sequence with its 3' end immediately adjacent to die cDNA insert. Primer extension and sequencing or fingeφrinting reactions are then carried out which read into die insert and identify its sequence or fingeφrint. In order to identify a unique length of sequence, a minimum read lengtii for die extension segment is typically 30 bases, although a preferred read length is at least about 40 bases. In an alternative embodiment, an array of die immobilized, cleavable primers can be formulated (Fodor, et al, 1991; Southern, et al, 1992). In this aspect of the invention, the array consists of the modified primers of die present invention where die cleavable linkage is, for example, a photocleavable linkage (e.g., backbone nitrobenzyl group) and the primer is attached to the support matrix tiirough die immobilization site of the modified primer. In this embodiment, die target DNA molecule is hybridized to die primers, primer extension reactions carried out and die different sequence primers are sequentially cleaved and the presence or absence of an extension product is determined. When extension products are detected ώeir sequences can be determined as described above. In the directed walk approach, a known DNA sequence is used as the primer sequence, thus providing an initiation point for sequencing in botii directions away from the known region. Each newly identified sequence is then used to direct the synthesis of new primers to enable progression of me sequence walk. B. DIAGNOSTICS A number of synthetic oligonucleotides are available or may be readily syntiiesized which are complementary to target nucleic acid sequences (e.g., RNA or DNA) and may be used as probes to detect die presence of certain bacteria, viruses, fungi, parasites and the like. The oligonucleotide composition of the present invention may be used, for example, to detect the presence of specific DNA or RNA sequences or fingeφrints corresponding to me following targets (i) heφes viruses such as cytomegalovirus (CMT), Epstein Barr, and Simplex Heφesvirus; (ii) hepatitis viruses such as hepatitis A, B, G, and D; (iii) papillomaviruses such as human papilloma virus 6, 11, 16, 18, and 33; (iv) retroviruses such as human immunodeficiency virus 1 (HIV I), HIV II, human T-cell lymphoblastic virus I (HTLV I), HTLV II; (v) animal viruses such as pig parvovirus and pig mycoplasma hypneumoniae, parvoviruses such as parvovirus B 19; (vi) picornaviruses such as rhinovirus (enterovirus) and rhinovirus HRV 2-14; (vii) bacteria such as mycobacterium avium, mycobacterium tuberculosis, chlamydia trachomatis, escherichia coli, streptococci and staphylococci; and (viii) parasites such as trypanosoma, toxoplasma, and plasmodia. Modified primers of the present invention having the primer sequence, sequences specifically hybridizable to nucleic acids from the microorganism of interest, are hybridized to nucleic acids in a sample. Primer extension reactions and isolation of extension products is carried out as described above. Presence of die extension product indicates die presence of nucleic acid from the microorganism in the sample. The modified primers and sizing method of die present invention provide a method for rapid, high throughput screening for the presence of specific, target sequences or fingeφrints using mass spectrometry. In a related embodiment, the modified primers of die invention can be used to identify pathogens by direct sequencing or fingeφrinting. In one such approach, a particular region of genomic DNA is identified which has segments that are common among a large population of patiiogens (e.g., conserved regions) located next to regions tiiat contain unique sequences for each pathogen (e.g., variable regions). One such exemplary sequence is from DNA mat is transcribed into bacterial 16 S ribosomal RNA, or 16 S rRNA (Olsen, G.J., et al, 1992). All 16 S-like rRNAs contain the same core structure. Nucleotides which are conserved in 90% of the available bacterial 16 S rRNA sequences have been identified (Schmidt, et al., 1994). Pathogen identification using rRNAs as described above is carried out as follows. In accordance widi the present invention, a primer is constructed to hybridize to a select region of me 16 S rRNA consensus sequence, for example, sequence 1047-1065 in 16 S rRNA, where i) the primer has the sequence 5'-ACGACANCCATGCANCACC-3'(SEQ ID NO:9), and ii) reads into the hypervariable region, e.g. , sequence 995-1046. Upon analysis of the primer extension segments by mass spectrometry, a single pathogen, if present, can be uniquely identified by determining die sequence or fingeφrint along the hypervariable region, with a desirable read length of at least 20 bases, and preferably, at least 40. Alternatively, instead of selecting a conserved region adjacent a hypervariable region, a series of unique primers can be created tiiat will hybridize to a hypervariable or unique region of a selected patiiogen. Enzymatic extension of these primers provides sequence or fingeφrint information about an adjacent segment of the hypervariable region. This methodology enables specific identification of each pathogen present in a mixed population. Utilizing this approach, one may target different hypervariable regions for each target patiiogen. This approach may be preferred for identifying viruses for which there is often very little conservation among other viruses or bacteria. In addition to determining the presence of a nucleic acid in a sample from such microorganisms, die present invention facilitates die determination of die specific sequences or fingeφrints present in a sample. For example, specific variants of HIV or trypanosomes can be identified by fingeφrinting or sequencing as well as d e presence or absence of genes responsible for antibiotic resistance. The modified primers can likewise be used in diagnostic mediods where mutant sequences are distinguished from wild-type sequences by sequence variations, such as, deletions, insertions, point mutations. Numerous potential target sites can be evaluated by mis method including target sites selected from DNA sequences that vary in length (e.g., BCR/ABL) as well as those that vary in sequence (e.g., sickle cell anemia). The sizing methodology of die present invention is particularly suited for the former application (e.g., target sites of varying length). Hybridizations of d e modified primers to target nucleic acids are carried out according to standard procedures, with suitable adjustment of the hybridization conditions to allow modified primer hybridization to the target region. Exemplary genetic disorders for detection using the modified primers of the present invention include sickle cell anemia and α,-antitrypsin deficiency (Watson, et al, 1992). As shown in Fig. 7A, sickle cell anemia results from a mutation that changes a glutamic acid residue (coded by the triplet GAG) for a valine residue (coded by GTG) at position 6 in the /S-globin chain of hemoglobin. This base change (A to T) destroys die recognition sequence for a number of restriction enzymes, including Mstlf. A modified primer for detecting this disorder would typically contain a cleavable site as indicated in Fig. 7A, located about 2 nucleotides from the end of the primer and preferably about 10-20 nucleotides upstream from the known mutation site. Also detectable using die modified primers of the present invention is αl-antitrypsin deficiency, a disorder characterized by uninhibited production of elastase, a protease which destroys the elastic fibers of the lung causing patients to suffer from pulmonary emphysema. The αl-antitrypsin gene has been cloned and as shown in Fig. 7B, die mutant gene, a fragment of which is presented by SEQ ID NO:7, corresponds to a single base change tiiat leads to an amino acid substitution (glutamine to lysine) at residue 342 as indicated by SEQ ID NO: 8. A portion of the wild-type αl-antitrypsin gene, as shown in Fig. 7B(top), is presented by SEQ ID NO:5. A fragment of die protein produced in individuals having die wild type αl-antitrypsin gene is presented by SEQ ID NO:6. Other diseases for which die corresponding gene mutations have been identified and which may be detected using die modified primers of die present invention include Duchenne muscular dystrophy, factor X deficiency, hemophilia and phenylketonuria. Modified primers used for detecting such disorders typically contain a cleavable site located near die end of me primer, where the end of die primer is upstream from die known mutation site (e.g., within about 20 base pairs from a mutation site detected in a 40- mer). Detection of a point mutation using the methods described herein is provided in Example 7 and further illustrated in Figs. 14A and 14B. Another diagnostic example is the detection of BCR-ABL transcripts, which are found in the majority of chronic myelogenous leukemia (CML) patients and in Ph + acute lymphocytic leukemia patients, and are believed to be necessary for die maintenance of leukemic phenotype (Szczylik, et al, 1991; Gale, et al; Collins, et al, 1984; Daley, et al.). The BCR-ABL transcripts are the result of a translocation of the proto-oncogene ABL (chromosome 9) to the breakpoint cluster region (BCR) (chromosome 22), resulting in the formation of BCR-ABL hybrid genes. In tins embodiment, the modified primers of the present invention would have their 3' end before the breakpoint region. Primer extension reactions would tiien proceed across die breakpoint region if present, or continue through the normal transcript region if no breakpoint was present. The sequence of such primer extension products are diagnostic of whedier a breakpoint fusion exists in any given sample of nucleic acids. The modified primers can also be employed in DNA amplification reactions (e.g., Mullis; Mullis, et al) for detecting die presence of specific sequences in samples by sizing or sequencing or for preparing quantities of DNA for sequencing reactions. In this embodiment of the invention, modified primers containing immobilization sites that can be attached to die solid support following amplification are particularly useful (e.g., biotin and digoxigenin). Amplified products can be captured, the modified primers cleaved, and die resulting amplification products isolated. In particular, die present method may be utilized to identify patiiogens by the sizing of PCR products. Briefly, primers are first selected to hybridize widi a sequence unique to the target pathogen(s) of interest. The primers are chosen for use in a multiplex situation (e.g., one in which several different patiiogens may be present) to produce PCR products of varying sizes, with each size correlating to a unique PCR product for a specific patiiogen. Such an experiment for determining d e presence of tiiree different patiiogens (e.g., Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus) is carried out by adding to a sample containing, in addition to a DNA analyte, modified primers for each of die above patiiogens designed to produce PCR amplification products having sizes of, for example, 65, 70, and 75 base pairs, respectively. The PCR products are reduced in size by as many as 20-25 nucleotides by cleavage at die cleavable site (in die modified primer). This results in shifting the corresponding peaks into a more readily resolvable range of d e mass spectrometer and permits multiplexing of greater numbers of PCR products. The cleaved-amplification products are detected and sized using mass spectrometry, according to die method of the present invention. The following examples illustrate, but in no way are intended to limit tiie scope of the present invention. Materials and Mediods Protected nucleotide H-phosphonates such as Bz-DMT-deoxyadenosineH-phosphonate, iBu-DMT-deoxyguanosine H-phosphonate, fully protected deoxynucleoside phosphor amidites, protected deoxynucleoside diesters and triesters, nucleotide dimers, and solid phase supports may be purchased from Sigma Chemical Co. (St. Louis, MO). Bis(trifluoromethanesulfonyl)diisopropyIsilane may be purchased from Petrarch Systems Inc. (Bertram, PA). Phosphoramidites may be purchased from Applied Biosystems (Foster City, CA). Standard chemical reagents and solvents may be purchased from Aldrich Chemical Company (St. Louis, MO). Example 1 Preparation of a Modified Oligonucleotide Containing a 3'-5'-Cleavable Linkage Nucleoside dimers containing the following 3'-5'-internucleoside cleavable linkages are prepared as follows. A. 3'.5'-Dialkoxysilane Internucleoside Linkage The 3'-O-functionalized nucleoside intermediate, 3'-O-(diisopropylsilyl)-2'- deoxynucleoside triflate (1) is prepared by first adding bis(trifluoromethanesulfonyl)diisopropylsilane (1 mmol) to an equimolar amount of the sterically hindered base, 2,6-di-tert-butyl-4-medιylpyridine, dissolved in dry acetonitrile, under an inert atmosphere. The resulting solution is cooled to -40°C in a cooling batii, to which is added a solution of a 5'-(O)-protected nucleoside, 5'-(dimethoxytrityI)-2'-deoxynucleoside (0.9 mmol) and 2,6-di-tert-butyl-4-methylpyridine (0.25 mmol) in dimediylformamide over a 10 minute period. The resulting reaction mixture is stirred at -40°C for 1 hour and tiien allowed to warm to room temperature. The 3'-O-diisopropylsilyl triflate product is isolated by precipitation from water, with yields typically ranging from 90-100%. Isolation is not required, and preferably, die reactive intermediate is coupled directly widi unprotected nucleoside to form the desired dimer. The above procedure is used to form the 3'-silyl derivatives of the protected nucleosides 5'-(O)-dimethoxytrityl-thymidine, N6-benzoyl-2'-deoxy-5'-(O)-DMT-adenosine,N4-benzoyl-2'- deoxy-5'-(O)-DMT-cytidine, and N2-isobutyl-2'-deoxy-5'-(O)-dimethoxytrityl-guanosine with minimal formation of die undesired 3',3' symmetrical dimers. Intermediate (1) is reacted widi nucleoside, such as thymidine, by stirring a mixture of (1) and nucleoside for approximately 1 hour at room temperature. The coupled dimer is isolated by adding the reaction mixture dropwise to a vigorously-stirred ice/water mixture. The mixture is filtered to give a white solid, which is then dried and purified by column chromatography on silica gel (eluent: ethyl acetate/hexane gradient). The protected dimer, 5'-(O)-DMT-3'-(O)-(5'- (O)-nucleosidyldiisopropylsilyl)thymidine (2), is typically isolated in yields ranging from 50- 75%. The prepared dimers are then fiinctionalized for use in automated solid phase syndiesis to form primers containing a dialkoxysilane cleavable site. The dimer, such as (2) above, is converted to the corresponding 3'-(2-cyanoetiιyl-N,N- diisopropylphosphoramidite) by dissolving die 5'-DMT dimer in tetrahydrofuran and adding the resulting solution dropwise to a stirred solution containing 4-DMAP (4-dimethylaminopyridine), diisopropylediylamine and 2-cyanoethyl-N,N-diisopropylphosphoramidochloridite in THF under nitrogen and maintained at room temperature. The reaction mixture is stirred for 2 h, added to etiiyl acetate, washed widi brine, and dried over magnesium sulfate. The crude product is tiien purified by column chromatography on silica using 1:1 ethyl acetate/hexane as die eluent. The phosphoramidite-functionalized dimer is tiien employed for use in automated solid phase syndiesis using a programmable DNA synthesizer to form an oligonucleotide primer containing a 3'-5'-diisopropylsilyl ether cleavable site. Cleavage: After carrying out the desired hybridization and extension reactions widi an immobilized primer containing a dialkoxysilane internucleotide linkage as described above, me silyl-ether (Si-O) linkage is selectively cleaved by treatment with fluoride ion (Green, 1975) to release die extension product, typically containing no more than about five nucleotides derived from die modified primer molecule. B. 3'(S ' )-Phosphorothioate Internucleoside Linkage The fiinctionalized nucleoside, 5'-(O)-monomethoxytrityl-3'-(S)-thiophosphoramidite, is prepared as follows. The 3'-S-functionalized starting material, 5'-(O)-monomethyoxytrityl-3'- (S)-benzoyl-thymidine (3), is prepared according to die metiiod of Cosstick, et al. (Cosstick, et al, 1988). Debenzoylation is carried out by treating a solution of 5'-(O)-monomethoxytrityl-3'- (S)-benzoylthymidine dissolved in argon-saturated ethanol and maintained at 5°C with ION sodium hydroxide. The resulting solution is stirred for approximately 1 h. The product, 5'-O- MMT-3'-thiothymidine (4), is purified by column chromatography on silica gel. The 5'-(O)- MMT-3'-(S)-thymidine (4) is en converted to the corresponding diiophosphoramidite by reaction with 2-cyanoethyl-N,N-diisopropylaminophosphomonochloridite under standard conditions (McBride, et al, 1983). The 3'-(S)-phosphoramidite (5) is suitable for coupling to a second nucleoside to form a dimer containing a cleavable phosphorothioate site or for use in automated solid phase synthesis to prepare an oligonucleotide containing a phosphorothioate internucleoside linkage. Chemical synthesis of the phosphorothioate dimer is carried out as follows. A solution of 3'-(O)-acetylthymidine in acetonitrile is added dropwise over a 20 minute period to a stirred solution of die 3'-(S)-phosphoramidite (5) in acetonitrile saturated widi 5-(4-nitrophenyl)tetrazole. Use of the tetrazole activating agent reduces die probability of the self-condensation reaction occurring between two diiophosphoramidite molecules. The resulting thiophosphite dimer (6) is oxidized in situ by quenching the reaction mixture with 2,6-lutidine, followed by addition of an oxidant such as TBA periodate in dichloromethane. The fully protected phosphorotiiioate dimer (7) is deprotected by treatment with t-butylamine, 80% aqueous acetic acid, followed by concentrated aqueous ammonia to yield die 3-(S)-phosphorothioate-linked thymidine dimer (8). Formation of an oligonucleotide probe containing a 3'-(S)-phosphorothioate cleavable linkage is performed by solid phase syndiesis on controlled pore glass using a DNA syntiiesizer. The protocol and reaction conditions for conducting die solid phase reaction cycle are adjusted according to tiie primer products desired using standard solid phase phosphoramidite procedures. The fiinctionalized 3'-(S)-phosphoramidite nucleoside (5), prepared as described above, is utilized to introduce the 3'-(S)-phosphorothioate moiety into the oligonucleotide primer in the presence of the fiinctionalized tetrazole reagent, 5-(para-nitrophenyl)tetrazole. Cleavage: After carrying out hybridization, extension, and washing of an immobilized modified primer containing a 3'-(S)-phosphorothioate internucleotide bond, selective cleavage of the phosphorus-sulfur bond is carried out by treatment witii aqueous silver nitrate. C. 5'(SVPhosphorothioate Internucleoside Linkage Syndiesis of an oligonucleotide containing a 5'-phosphorotiιioate internucleoside linkage is carried out by first syntiiesizing a derivatized phosphoramidite, 5'-(S)-trityl-deoxytiιymidine-3'- (O)-(2-cyanoethyl-N,N-diisopropylamino)phosphite as described below. 5'-(O)-/?-toluenesulfonyl tiiymidine (9) is prepared by reacting thymidine witii one equivalent of -toluenesulfonyl chloride in pyridine. The reaction mixture is stirred for 3 h at room temperature, cooled in ice and quenched by addition of water. Following dissolution in ethyl acetate, and sequential washing witii sodium bicarbonate and brine, the solution is dried over sodium sulfate and the solvent is removed in vacuo. The desired 5'-tosylate product (9) is readily recrystallized from ethyl acetate/methanol, thus avoiding the need for a protecting group at die 3'-OH position. The 5'-tosylate is then converted to the corresponding 5'-(S)-trityl-deoxytiιymidine (10). A solution of die 5'-(O)-tosylthymidine (9) in ethanol is added to a reaction flask containing a five-fold molar excess of 7.0 M sodium hydroxide and triphenylmethyl mercaptan in ethanol (which generates the corresponding reactive thiolate in situ). The reaction mixture is refluxed for 8 hours under an inert atmosphere, filtered to remove residual solids, dissolved in etiiyl acetate, and die solution is washed, dried, and evaporated in vacuo. The crude product is purified by chromatography on silica gel using a methanol/methylene chloride gradient. Thedesiredreactivenucleosidephosphoramidate,5'-(S)-trityl-deoxythymidine-3'-(O)-(2- cyanoethyl-N,N-diisopropylamino)phosphite (11), is prepared by treating a solution of the protected nucleoside, 5'-(S)-trityl)-deoxythymidine (10), in dry 1:1 acetonitrile/methylene chloride with an equimolar amount of tetrazole, followed by addition of a 1.5 molar excess of 2-cyanoetiιyoxy-bis-(N,N-diisopropylamino)phosphine. The reaction mixture is stirred for about 1 hour at room temperature and subsequently quenched by addition of butanol. The solution is diluted with ethyl acetate, washed, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product is purified by flash chromatography. Incoφoration of the desired 5'(S)-phosphorothioate cleavable site into an oligonucleotide probe is carried out by utilizing standard solid phase phosphoramidite chemistry. D. 5'(N)-Phosphoramidate Internucleoside Linkage Oligonucleotide fragments containing a 5'-(N) phosphoramidate internucleotide bond are prepared as follows. Thymidine is transformed to die corresponding 5'-azide derivative (12) by treatment witii sodium azide and triphenylphosphine in carbon tetrabromide according to tiie procedure of Hasa, et al (Hasa, et al, 1976). Reduction of 5'-azido-deoxytiιymidine (12) is then carried out by hydrogenation over Pd/C catalyst to form die corresponding 5'-amino derivative (13). Formation of the corresponding 5'-N-protected nucleoside is carried out by dissolving (13) (25 mmol) in anhydrous pyridine (150 ml), to which is added 4-DMAP (17 mmol), trietiiylamine (17 mmol), and 4-methoxytrityl chloride (60 mmol), and die resulting reaction mixture is stirred for 2 h at room temperature. Methanol is added to die reaction flask and tiie resulting mixture is added to a solution of saturated sodium bicarbonate, extracted widi chloroform, and the organic extracts dried over anhydrous sodium sulfate. The organic layer is evaporated to dryness and the resulting crude residue is purified by column chromatography over silica gel to yield 5'-amino-5'-deoxy-5'-(N)-(4-methoxytrityl)thymidine (13). (Cleavage of the N-MeOTr protecting group is carried out by treatment witii 3% dichloroacetic acid in 1,2- dichloroetiiane). The desired functionalized nucleoside, 5'-amino-5'-deoxy-5'-(N)-(4- methoxytrityl)thymidine-3'-(2-cyanoethyl)-N,N-diisopropylphosphoramidite (14), containing a reactive phosphoramidite moiety suitable for incoφoration into an oligonucleotide fragment is synthesized as follows. The 5'-amino-protected tiiymidine (13) (4 mmol) is dissolved in anhydrous metiiylene chloride (60 ml), to which is added dry bis(diisopropylammonium)tetrazolide (3 mmol) and (2- cyanoethoxy)bis(diisopropylamino)phosphine (8 mmol). The mixture is stirred for 1 hour at room temperature, poured into a saturated sodium bicarbonate solution, and extracted several times with chloroform. The combined organic extracts are rinsed witii brine, dried, and evaporated to dryness. The crude residue is dissolved in a minimal amount of methylene chloride and precipitated by addition of pentane to yield a mixture of the diastereomeric product, 14. The functionalized nucleoside 14 is tiien selectively introduced into an oligonucleotide fragment to form an oligonucleotide containing a 5'-(N)- phosphoramidate cleavable site. The 5'-amino nitrogen of thymidine derivative 14 is die reactive center at which phosphoramidate bond formation takes place. The modified nucleoside 14 is introduced in die course of a standard cycle into a growing DNA fragment synthesized on a solid support as follows. Insertion of die phosphoramidate group is performed at a specific site to form the desired nucleotide fragment containing a selectively cleavable site. Formation of the following modified exemplary sequence: d(T-T-C-A-T-G-C-A-A- (phosphoramidate)-T-C-C-G-A-T-G) (SEQ ID NO:l) is performed as follows. The DNA fragment is syntiiesized in a stepwise fashion beginning from the hexamer sequence d(C-C-G-A- T-G) (Gromova, 1987). The hexamer is synthesized on controlled pore glass as solid support using standard procedures (Bannwarth, et al, 1986; Bannwarth, 1987). The introduction of key intermediate 14 is performed during a standard cycle utilizing slightly longer times for the coupling of 14 and for deblocking of die 4-methoxytrityl protecting group. Following cleavage of the 5'-MeOTr group of 14, the syndiesis is continued using standard phosphoramidites as the building units to form the desired 16-mer sequence. The support material is treated witii concentrated ammonia at 56°C overnight to cleave the 16-mer product from die solid support. Following removal of die support material, the ammonia is removed by vacuum evaporation, and the remaining residue is dissolved in water/dioxane and subsequently precipitated by addition of THF. The resulting 16-mer is then purified by either gel electrophoresis or HPLC. Selective chemical cleavage of the phosphoramidate internucleotide bond is carried out under mild acidic conditions to form the corresponding phosphate and amino-functionalized fragments. Treatment with 80% acetic acid at room temperature for between 2-6 hours results in selective cleavage of die internucleotide phosphoramidate bond while leaving the unmodified portion of the DNA fragment intact. Example 2 Attachment to the Solid Support A. Streptavidin Affinity Immobilization A modified primer from Example 1 above containing a cleavable site is immobilized by attachment to a fiinctionalized solid support material. In some cases tiie cleavable site-containing primer is modified as described below. For attaching an oligonucleotide primer to a streptavidin-coated support a biotinylated primer is typically used. Biotinylation is carried out as follows. A primer containing a cleavable site is prepared as in Example 1, with a minor modification: d e primer is synthesized to contain a reactive amino site for biotinylation. An amino group is introduced during solid phase syndiesis using a standard DNA synthesizer, such as Applied Biosystems 393 DNA/RNA Synthesizer. To selectively introduce die internal amino function, the modified nucleoside phosphoramidite Amino-Modifier dT, containing a base labile trifluoroacetyl group protecting a primary amine attached to thymine witii a 10 atom spacer arm, is added at an appropriate phase of the DNA syndiesis cycle. Upon completion of die oligonucleotide synthesis, the primer is cleaved from the support by standard methods. The remaining base-protecting groups as well as the trifluoroacetyl amino protecting group are removed by treatment with fresh, concentrated ammonium hydroxide at 40° for 15-17h. The solution is dried by rotary evaporation, and die residue is redissolved in 200 μl of water. The amino-modified primer (approximately 0.25 μmol) is reacted with NHS-LC-Biotin (Pierce, Rockford IL) which has an 11 carbon spacer between the biotin group and die N- hydroxylsuccinimide activated carboxyl group. Aliquots of a 50mM NHS-LC-biotin solution in DMF are added to die primer solution containing 0.1 M sodium bicarbonate/sodium carbonate buffer at pH 9 over a 1.5h period. The solution is maintained at room temperature overnight and the biotinylated primer is tiien purified by reverse phase HPLC. The biotinylated primer is then immobilized by attachment to streptavidin-coupled magnetic beads (Dynabeads M-280, Dynal, Inc., Great Neck, NY) as described in Dynabeads M-280 Technical Handbook: Magnetic DNA Technology 6, Dynal Inc. A neodymium-iron- boron magnet is used to immobilize the beads during supernatant removal and washing steps. B. Immobilization via a Thiourea Linkage 5'-Amino-modified oligonucleotide primers containing a cleavable linkage are prepared as described in Examples 1 and 2A above. Glass slides are activated in a two-stage process for coupling to amino-fimctionalized oligonucleotides. The glass surface is first functionalized by reaction widi aminopropyltrimethoxysilane to form an amino-derivatized surface. To carry out the amino- fiinctional ization, clean microscope slides are immersed for 2 minutes in a 1% solution of 3- aminopropyltrimethoxysilane solution in 95% acetone/water. The slides are then washed several times with acetone (5 minutes per wash), and dried for 45 minutes at 110°C. Following amino-derivatization, the glass slides are treated widi excess p- phenylenediisothiocyanate to convert the amino groups to amino-reactive phenylisothiocyanate groups suitable for coupling to amino-functionalized oligonucleotides. The amino-derivatized glass plates are treated for 2 hours with a solution of 0.2% 1,4-phenylene diisotiiiocyanate solution in 10% pyridine/DMF, followed by washing with methanol and acetone. A 2 mM solution of the amino-modified primer in sodium carbonate/bicarbonate buffer (2 μL) is applied directly to the activated glass plate surface and die resulting slides are then incubated at 37°C in a covered Petri dish containing a minimal amount of water for about 2 h. The plates containing thiourea-I inked primer are en washed sequentially witii 1 % ammonium hydroxide, and water, followed by air drying at room temperature. C. Immobilization via Hg-S Affinity Binding An amino-modified oligonucleotide primer is prepared as described above. Conversion of die 5'-amino group to a thiol is carried out by reacting 5.0 A^ units of the amine-containing primer dissolved in 1.0 ml of 0.2 molar 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (pH 7.7) with 1.6 ml of 10 mM dithiobis(succinimidylpropionate) in dry acetonitrile for 1 hour at 20°C. An additional 1.0 ml of 10 mM dithiobis(succinimidylpriopionate) in acetonitrile is then added to the reaction vessel and the resulting mixture is stirred for an additional hour. Addition of ditiiioerythritol (3.5 ml of a 20 mM solution in 0.2M Tris buffer) is followed by stirring for 1 hour at 37°C. The tiiiol-derivatized primer solution is concentrated under vacuum to form a concentrate which is further purified using reverse phase HPLC followed by lyophilization. In an alternate approach for synthesizing thiol-modified oligonucleotide primers, tiie 5'- phosphate of an oligonucleotide primer is esterified witii 6-mercapto-hexanol using a β- cyanoethyl-phosphoramidite C6-Thiol-Modifier (Clontech Laboratories, Inc., Palo Alto, CA). The tiiiol-derivatized primer is then immobilized on/7-chloromercuribenzoate derivatized agarose by mixing a solution of the thiol-derivatized primer (0.25 A^ units) dissolved in 140 μl of 1.0 M sodium chloride, 1.0 mM etiiylenediaminetetraacetic acid disodium salt (EDTA), and 50 mM Tris HCl (pH 8) with 50 μl of p-chloromercuribenzoate derivatized agarose for 3 minutes at 20°C. Example 3 Selective Cleavage of a Synthetic DNA Probe Containing a Cleavable Ribose A synthetic DNA probe containing a cleavable ribose site was selectively cleaved by ammonium hydroxide treatment. The 17-mer, having the sequence: 5'-AAA TAC ATC riboGCT TGA AC-3' (SEQ ID NO: 10) was prepared to contain a cleavable ribose in the 7- position. The modified probe was treated with aqueous 3% ammonium hydroxide for 15 minutes at room temperature (pH 10) to effect selective cleavage of the ribose moiety. Example 4 Mass Spectral Analysis of the Selective Cleavage Products of a Ribose- Containing DNA Probe The cleavage products from Example 3 were analyzed using matrix assisted laser desoφtion witii concomitant ionization (MALDI) in conjunction with time-of-flight (TOF) mass spectrometry. The experimental apparatus used for analyzing die sample fragments was composed of an excitation source, a sample manipulator and a TOF mass spectrometer. The excitation sources used for desoφtion were a Nd:YAG laser (model DCR-1, Spectra-Physics, Mountain View, CA) with spatially filtered 5 ns pulses frequently tripled to 355 nm or quadrupled to 266 nm and a 35-ps pulse Nd:YAG laser (model PY610C-10, Continuum, Santa Clara, CA) operating at 355 nm. Both of the lasers were operated at a 10 Hz repetition rate, witii a 5 nm pulse widtii. The desoφtion laser beam maintained at an incident angle of 45° was focused onto die sample with a 250 mm focal lengtii quartz lens to an elliptical spot size of approximately 100 by 150 μm. A Glan-laser polarizer (Newport Coφoration, Fountain Valley, CA) was placed in a rotation stage in die beam path for continuously variable attenuation, allowing adjustment of the polarized Nd: YAG laser energy density from below 1 mJ/cm 2 to 100 mJ/cm 2 . The optimum energy density for despoφtion was found to be in the range of 2 to 20 mJ/cm 2 . Sample preparation was carried out as follows. The oligonucleotide fragments were dissolved in deionized water at room temperature to concentrations of about 50 μmol/liter. A separate saturated solution of 3-hydroxypicolinic acid (3-HPA) in 50% water/acetonitrile was freshly prepared, and the two solutions were mixed to provide a sample solution containing 3HPA and analyte in a molar ratio of about 10,000: 1. A 2 μL aliquot of the sample solution was pipetted onto die sample stage and spread to an area of 2mm diameter. The sample was dried under a gentle flow of nitrogen prior to insertion into die vacuum system. The sample stage, consisting of either a smooth silver foil or a polished silicon wafer, was mounted on a manipulator which allows three translational and one rotational degrees of freedom. The experiments were carried out at room temperature. The sample region was evacuated by a 300 liter per second turbomolecular pump. The drift and detection regions were evacuated using a cryopump with nominal 1500 liter per second pumping speed. The base pressure of the chamber was 3 x 10 "9 Torr, and the normal working pressure, within about five minutes of sample introduction, was 5 x 10" 8 Torr. The ions produced during desoφtion were extracted peφendicular to the sample surface into die time-of-flight mass spectrometer by biasing the sample with a voltage of 28 kV, with the drift and extraction potentials at ground. The sample-extractor distance was 5 mm, and an einzel lens about 5 cm from the sample was used to focus die ions. Botii linear and reflecting TOF mass spectrometric geometries were examined. For reflecting TOF-MS, a two stage electrostatic reflector was used and the effective drift patii was 2.0 m. A dualmicrochannel plate detector was used. The detector was placed beside die electrostatic deflector due to space constraints of the vacuum chamber. Deflecting voltage was applied to horizontal deflecting plates and die beam patii was bent in order to direct tiie ions to the detector for die linear geometry. The total flight distance was 1 m for the linear geometry. The four degree bend was sufficient to block the line-of-sight between the ion creation region and die detector to prevent any energetic neutral flux created in the ionization region from reaching the detector. For reflecting TOF measurements, die beam path was bent in die opposite direction. To avoid detector saturation caused by the high abundance of ionized matrix molecules in experiments performed at higher laser powers, the low mass matrix ions were deflected away from the detector by a pulsed electric field of 200 V/cm. The signal output of the microchannel plates was amplified and tiien digitized witii a time resolution of 10 to 50 ns/channel and typically summed over 100 laser pulses. Mass calibration was performed by analyzing a variety of known masses, such as alkalis at low mass, isotopically resolved fullerenes, mixtures of gramicidin S, bovine insulin, horse heart cytochrome C, and horse heart myoglobin. The resulting time-of-flight mass spectra are illustrated in Figs. 3A and 3B. Figure 3A is a mass spectrum of the 17-mer syndietic mixed base primer containing a cleavable ribose linkage prior to ammonium hydroxide treatment. Two sizable peaks were observed for the intact 17-mer corresponding to die di-protonated molecular ion [M+2H] 2+ and the protonated molecular ion, [M+H] + . The resulting oligomer fragments obtained following ammonium hydroxide treatment were tiien analyzed, as shown in Fig. 3B. As indicated in the mass spectrum, peaks corresponding to the expected cleavage products, the 7-mer, the 10-mer, and intact 17-mer, were readily observable (and identifiable). Example 5 Capture and Selective Cleavage of a Biotinylated Primer Having a Cleavable Ribose in die 10 Position A biotinylated 18-mer containing a ribose in the 10 position, 5'-biotin-ATCTTCCTG- ribo-GCAAACTCA-3', SEQ ID NO.l l, (Keystone Laboratories, Inc., Menlo Park, CA), was captured on streptavidin-coated beads (DynaBeads M-280, Dynal, Inc., Great Neck, NY). The immobilized primer was men washed after surface binding, followed by treatment with ammonium hydroxide as described in Example 3 above to effect selective cleavage of the immobilized primer at the ribose site. The modified primer, containing d e biotin immobilization attachment site and die cleavable ribose site, was analyzed both prior to capture and subsequent to selective cleavage. The samples were analyzed using MALDI in conjunction with TOF mass spectrometry, as described in Example 4 above. Figure 4 illustrates the mass spectrum of the 8-mer resulting from selective cleavage of the ribose site widiin die immobilized primer. Example 6 Immobilization of a Cleavable Extended Primer by Hybridization to an Intermediary Solid-Phase Bound Oligonucleotide A modified M13 reverse primer containing a 5'-(S)-thymidine located 5 nucleotides from the 3' end, having die sequence presented herein as SEQ ID NO: 12, was hybridized to a single stranded target molecule (SEQ ID NO: 14) in hybridization medium containing annealing buffer, 10X "THERMOSEQUENASE" Buffer (260 mM Tris-HCl, pH 9.5, 65 mM MgCl . ) (Amersham Life Sciences, Arlington Heights, IL). The annealing reaction was carried out by warming die above mixture to 65 °C for two minutes, and then allowing the mixture to cool slowly to room temperature over a period of about thirty minutes (Maniatis, etal, 1982; Ausubel, etal, 1989). Following hybridization, the modified primer was extended using DNA polymerase ("THERMOSEQUENASE" DNA Polymerase) in the presence of a mixture of deoxynucleotides and dideoxynucleosides to produce a set of oligonucleotide fragments corresponding to die locations of adenine widiin die target (i.e., thymidine within die reaction product). The reaction was performed using standard cycle sequencing protocols and an 8:1 ratio of primer to template. Due to die primer-to-template ratio employed, d e resulting set of primer extension products were primarily (89%) in single stranded form. Following primer extension, an intermediary oligonucleotide complementary to the M13 reverse primer and biotinylated at the 3' end, having SEQ ID NO: 13, was added to die mixture and annealed to the primer using a standard heat/cool annealing process: 95° C for 2 minutes 30 seconds; 25 cycles at 95° C for 15 seconds, 45° C for 20 seconds, 55° C for 10 seconds, 70° C for 20 seconds), 5 cycles at 95° C for 30 seconds, and 70° C for 20 seconds, followed by cooling from 95° C to 70° C for 1 minute at die rate of 0.1° C per second, and subsequent maintenance of die sample at 4° C. Streptavidin coated magnetic beads (MPG-Steptavidin, CPG, Inc., Lincoln Park, NJ) were then added to the mixture to capture the biotinylated intermediary oligonucleotide/extended primer hybrid. The immobilized product was washed to remove enzymes, triphosphates and salts in a multistep wash process. The sample was then treated witii silver nitrate (5 μL, 0.02 mM, Aldrich, Milwaukee, WI) and DTT to release die extension segments into solution. This solution was (i) separated from the solid phase bound intermediary oligonucleotide-first primer region complex, (ii) mixed witii 3-hydroxypicolinic acid, (iii) dried onto a silicon plate and (iv) analyzed by MALDI TOF mass spectrometry as described in Example 4 above. The released extension segments are shown in Fig. 13. As seen in Fig. 13, the method allows detection of oligonucleotide extension segments with read lengths up to at least about 33 base pairs with good resolution. Example 7 Detection of Point Mutationfs') Using a Single Base Fingeφrint Two DNA templates, one a syntiietic 73-mer (presented herein as SEQ ID NO: 14) with a sequence identical to wild-type M13 plasmid, corresponding to template "16-C/19-G", and the other a mutant plasmid, (having a partial sequence included herein as SEQ ID NO: 15), referred to as template "16-A/19-T", were used in primer extension reactions. The sequences of die templates differed only at base positions 16 and 19, relative to the 3' end (counting upstream from the end of the priming region), with the first template possessing a cytosine at position 16 and a guanine at position 19, as presented in SEQ ID NO: 14, while the second template, corresponding to SEQ ID NO: 15, contained an adenine and a tiiymine substituted at positions 16 and 19, respectively. Each of die templates was subjected to enzymatic extension in the presence of ddC using a primer with the sequence presented herein as SEQ ID NO: 16. The resulting product mixtures, containing ddC-terminated oligonucleotide fragments derived from the parent templates, were then analyzed by MALDI TOF mass spectrometry as described above and illustrated in Fig. 14A (corresponding to the reaction product(s) derived from template 16-C/19-G, SEQ ID NO: 17) and Fig. 14B (corresponding to die reaction produces) derived from template 16-A/19-T, SEQ ID NO: 18). As can be seen from the resulting spectra of the reaction products, the exact mass values corresponding to the differences between select peaks in each of the spectra were calculated, confirming the presence of two single nucleotide substitutions at positions 16 and 19 from the 5' end. As demonstrated in Figs. 14A-14B, the measured mass values for the peak-to-peak differences are shown in large type, while die actual/theoretical mass values are shown in small type. The G-to-T base substitution is indicated by the difference in the Δm values for template 16-C/19-G (mass peak b - mass peak a= 618.9) versus template 16-A/19-T (mass peak f - mass peak e= 593). The observed mass difference of 25 (618.9 minus 593.4) corresponds to the difference in mass between guanine (MW= 151) and tiiymine (MW= 126). Confirmation of a single base substitution occurring at position 19 as a result of a C to A mutation was similarly determined (e.g., Δm d . b versus Δm g . f ). The single base substitution at position 19 was further confirmed by the absence of a peak at position 19 in the spectrum corresponding to template 16- A/19-T (Fig. 14B). Example 8 Comparison of Primer Extension Reactions Using Cleavable Versus Full Primers A modified M13 reverse primer containing a 5'-biotin group and a thiol-tiiymidine located 5 nucleotides from the 3' end of the primer (SEQ ID NO: 16) was hybridized to a single stranded target molecule (SEQ ID NO: 14), followed by enzymatic extension in the presence of a mixture of deoxynucleotides and dideoxy-T to produce a set of oligonucleotide fragments corresponding to the locations of adenine within die target, following the procedure described in Example 6. Following die ddT extension reaction, the biotinylated primer/extension product was captured (immobilized) on streptavidin-coated magnetic beads. The bead-immobilized primer/extension products were then subjected to a series of wash steps to remove template, enzyme, triphosphates and additional salts. The streptavidin-coated magnetic beads, now containing immobilized primer/extension products, were tiien divided into two tubes. The contents of the first tube was treated witii silver nitrate and DTT, to cleave the primer at the thiol-thymidine located 5 nucleotides from the 5'- end of the primer and to release the extension segments into solution. The contents of second tube were boiled to effect disruption of me biotin/streptavidin bond and release die full primer/extension products into solution. The two samples were then separately mixed witii 3-hydroxypicolinic acid, dried, and analyzed by MALDI TOF mass spectrometry as described above. The resulting mass spectra are shown in Figs. 15A (full length primer-extension segments) and 15B (cleaved primer-extension segments having increased read lengtii). As can be seen, the resolution quality and read length of spectra of cleaved primer-extension segment samples (Fig. 15B) according to die present method are superior to tiiose of the full primer- boil/release sample (Fig 15A). The broad peak centered around base no. 15 in the uncleaved primer sample (Fig. 15A) is due to primer dimerization, and is an artifact that occasionally occurs when tiie sample includes a large amount of primer. Cleavage of die primer removes mis artifact, as can be seen in Fig. 15B. While die invention has been described widi reference to specific methods and embodiments, it will be appreciated tiiat various modifications and changes may be made witiiout departing from the invention. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: SRI INTERNATIONAL (ii) TITLE OF INVENTION: Oligonucleotide Sizing Using Cleavable Primers (iii) NUMBER OF SEQUENCES: 18 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Dehlinger S Associates (B) STREET: P.O. Box 60850 (C) CITY: Palo Alto (D) STATE: CA (E) COUNTRY: U.S.A. (F) ZIP: 94306 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: unknown (B) FILING DATE: 30-APRIL-1996 (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/445,751 (B) FILING DATE: 22-MAY-1995 (C) APPLICATION NUMBER: unassigned (Docket No. 8255-0015.30) (D) FILING DATE: 26-APRIL-1996 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Evans, Susan T. (B) REGISTRATION NUMBER: 38,443 (C) REFERENCE/DOCKET NUMBER: 8255-0015.30 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415-324-0880 (B) TELEFAX: 415-324-0960 (2) INFORMATION FOR SEQ ID Nθ:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Exemplary 16-mer with phosphoramidate linkage (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 9..10 (D) OTHER INFORMATION: /note= "sequence contains phosphoramidate bond between nucleotides 9 and 10" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: TTCATGCAAT CCGATG 16 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Immobilized cleavable 20-mer primer ( ix) FEATURE : (A) NAME/KEY: misc_feature (B) LOCATION: 16..17 (D) OTHER INFORMATION: /note= "primer containing a first region with an immobilization attachment site, a cleavable site "x" between nucleotides 16 and 17" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ACTCCTGTGG AGAACTCTGC 20 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Single stranded target, complement to seg. id no. 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GCAGAGTTCT CCACAGGAGT 20 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Immobilized primer subsequent to selective cleavage (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ACTCCTGTGG AGAACT 16 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: portion of alphal antitrypsin gene, posns 333-352(wild type) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: ACCATCGACG AGAAAGGGA 19 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: frag of product expressed by alphal-antitrypsin gene (wild) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Thr lie Asp Glu Lys Gly Thr 1 5 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: port, of alpha-1 antitrypsin gene w/ point mutation (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ACCATCGACA AGAAAGGGA 19 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: port, of product expressed by mutant alphal-antitrypsin gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Thr lie Asp Lys Lys Gly Thr 1 5 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: primer for conserved region 1047-1065 in 16S rRNA (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 7..8 (D) OTHER INFORMATION: /note= "the primer contains a 1:1 mixture of dT and dG incorp. at positions 7 and 15" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: ACGACATCCA TGCATCACC 19 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: 17-mer w/ cleavable ribose site (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 9..10 (D) OTHER INFORMATION: /note= "probe contains a cleavable ribose at position 10 from 5' end (position 7 from 3' end)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: AAATACATCG CTTGAAC 17 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: biotinylated 18-mer contg ribose at position 10 ( ix ) FEATURE : (A) NAME/KEY: misc_feature (B) LOCATION: 9..10 (D) OTHER INFORMATION: /note= "18-mer is biotinylated at 5' end and contains a ribose at position 10 from 5' end" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: ATCTTCCTGG CAAACTCA 18 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Modified M13 reverse primer w/ 5'-(S)thymidine (ix) FEATURE: (A) NAME/KEY: misc_structure (B) LOCATION: 17..18 (D) OTHER INFORMATION: /note= "reverse primer contains a 5'-(S)thymidine at position 18" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CACACAGGAA ACAGCTATGA CC 22 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: 3'-biotin intermed oligo complementary to M13 rev primer (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 16..17 (D) OTHER INFORMATION: /note= "intermediary oligonucleotide is biotinylated at 3' end" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TAGCTGTTTC CTGTGTG 17 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 73 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: 73-mer w/ sequence identical to wild type M13 plasmid ( i ) FEATURE : (A) NAME/KEY: misc_feature (B) LOCATION: 51..68 (D) OTHER INFORMATION: /note= "priming region corresponds to nucleotides 51-68; C and G of interest at positions 35 and 32, respectively" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CAGCTTTTGT TCCCTTTAGT GAGGGTTAAT TGCGCGCTTG GCGTAATCAT GGTCATAGCT 60 GTTTCCTGTG TGA 73 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 73 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: mutant plasmid, template "16-A/19-T" (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 51..68 (D) OTHER INFORMATION: /note= "priming region corresponds to nucleotides 51-68; mutations at positions 35 (A) and 32 (T)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: CAGCTTTTGT TCCCTTTAGT GAGGGTTAAT TTCGAGCTTG GCGTAATCAT GGTCATAGCT 60 GTTTCCTGTG TGA 73 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: 5' biotinylated M13 reverse primer, 5' (S)T at posn 14 (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 13..14 (D) OTHER INFORMATION: /note= "primer is biotinylated at 5' end and contains a 5'(S)-T at positio..." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CAGGAAACAG CTATGACC 18 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Extension product from template "16-C/19-G" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: ATGATTACGC CAAGCGCGCA ATTAACCCTC ACTAAAGGGA ACAAA 45 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: extension product from template 16-A/19-T (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ATGATTACGC CAAGCTCGAA ATTAACCCTC ACTAAAGGGA ACAAA 45

Claims: IT IS CLAIMED: 1. A method for determining die size of a primer extension product, comprising: (a) hybridizing a primer witii a target nucleic acid, where said primer (i) is complementary to said target nucleic acid; (ii) has a first region containing die 5' end of the primer, and (iii) has a second region containing the 3' end of the primer, where die 3' end is capable of serving as a priming site for enzymatic extension and where said second region contains a cleavable site, (b) extending the primer enzymatically to generate a mixture containing a product composed of the primer and an extension segment; (c) cleaving at the cleavable site to release the extension segment; and (d) sizing die extension segment by mass spectrometry, where the read length of the extension segment is increased relative to the read length of the product of (b). 2. The method of claim 1, where said target nucleic acid is immobilized. 3. The method of claim 2, where the target nucleic acid is immobilized prior to said extending. 4. The method of claim 2, where the target nucleic acid is immobilized prior to said cleaving. 5. The method of claim 1, where the cleavable site is a nucleotide capable of blocking 5' to 3 'enzyme-promoted digestion, and where said cleaving is carried out by digesting the first region of the primer with an enzyme having a 5' to 3' exonuclease activity. 6. The method of claim 1 , where the cleavable site is located at or within about five nucleotides from the 3' end of said primer. 7. The method of claim 6, where the second region of said primer is a single nucleotide tiiat also contains the cleavable site. 8. The method of claim 7, where said second region is a ribonucleotide. 9. The method of claim 1, where the cleavable site is selected from the group consisting of dialkoxysilane, 3'-(S)-phosphorothioate, 5'-(S)-phosphorothioate, 3'-(N)- phosphoramidate, 5'-(N)phosphoramidate, uracil, and ribose. 10. The method of claim 1, where the enzyme for extending die primer in step (b) is a DNA polymerase. 11. The method of claim 1, where the enzyme for extending die primer in step (b) is a ligase. 12. The method of claim 4, which further comprises separating the product of (b) from said immobilized target nucleic acid prior to said cleaving step. 13. The method of claim 1 , where said sizing is by time-of-flight mass spectrometry. 14. The method of claim 1, where said extending is carried out in the presence of a nucleotide containing (i) an immobilization attachment site and (ii) a releasable site, where the extension segment includes said immobilization attachment site and said releasable site. 15. The method of claim 14, further comprising, prior to said sizing, immobilizing said extension segment at die immobilization attachment site, and releasing said extension segment at said releasable site. 16. A method for determining die size of a primer extension product, comprising: (a) hybridizing a primer widi a target nucleic acid, where said primer (i) is complementary to said target nucleic acid; (ii) has a first region containing the 5' end of die primer, and an immobilization attachment site, and (iii) has a second region containing die 3' end of die primer, where die 3' end is capable of serving as a priming site for enzymatic extension and where said second region contains a cleavable site, (b) extending die primer enzymatically to generate a mixture containing a product composed of die primer and an extension segment; (c) cleaving at the cleavable site to release the extension segment, where prior to said cleaving die primer is immobilized at said immobilization attachment site; and (d) sizing the extension segment by mass spectrometry, where the read length of the extension segment is increased relative to die read length of die product of (b). 17. The method of claim 16, where the cleavable site is located at or widiin about five nucleotides from die 3' end of said primer. 18. The metiiod of claim 17, where the second region of said primer is a single nucleotide tiiat also contains the cleavable site. 19. The method of claim 18, where said second region is a ribonucleotide. 20. The method of claim 16, where the cleavable site is selected from the group consisting of dialkoxysilane, 3'-(S)-phosphorothioate, 5'-(S)-phosphorothioate, 3'-(N)- phosphoramidate, 5'-(N)phosphoramidate, uracil, and ribose. 21. The method of claim 16, where the enzyme for extending die primer in step (b) is a DNA polymerase. 22. The method of claim 16, where the enzyme for extending die primer in step (b) is a ligase. 23. The method of claim 16, which further comprises washing the immobilized product prior to said cleaving step. 24. The metiiod of claim 16, wherein said sizing is accomplished by matrix-assisted laser desoφtion ionization mass spectrometry. 25. The method of claim 24, where said sizing is by time-of-flight mass spectrometry. 26. The method of claim 24, wherein said extension segment is embedded in a chemical matrix prior to said sizing. 27. The metiiod of claim 16, where said primer is immobilized on a solid support by attachment at the immobilization attachment site to an intervening spacer arm bound to die solid support. 28. The method of claim 27, wherein said intervening spacer arm is six or more atoms in length. 29. The method of claim 16, wherein die immobilization attachment site occurs as a substituent on one of the bases or sugars of the primer. 30. The method of claim 16, where said immobilization attachment site is biotin or digoxigenin. 31. The method of claim 16, where said primer is immobilized on a solid support selected from the group consisting of glass, silicon, polystyrene, aluminum, steel, iron, copper, nickel, silver and gold. 32. The method of claim 16, where the immobilization attachment site of the primer is composed of a series of bases complementary to an intermediary oligonucleotide, and where said primer is immobilized by specific hybridization of die immobilization attachment site to the intermediary oligonucleotide bound to a solid support. 33. A method for determining die size of a primer extension product, comprising: (a) combining first and second primers with a target nucleic acid, under conditions that promote hybridization of die primers to the nucleic acid, generating primer/nucleic acid complexes, where said first primer (i) has a 5' end and a 3' end, (ii) is complementary to said target nucleic acid, (iii) has a first region containing the 5' end of die first primer and (iv) has a second region containing die 3' end of the first primer, where said 3' end is capable of serving as a priming site for enzymatic extension and where said second region contains a cleavable site, and where said second primer (i) has a 5' end and a 3' end, (ii) is homologous to said target nucleic acid, (iii) has a first segment containing die 3' end of die second primer, and (iv) has a second segment containing die 5' end of the second primer and an immobilization attachment site, (b) converting the primer /nucleic acid complexes to double-stranded fragments in the presence of a DNA polymerase and deoxynucleoside triphosphates, (c) amplifying the number of primer-containing fragments by successively repeating the steps of (i) denaturing said double-stranded fragments to produce single-strand fragments, (ii) hybridizing said single stranded fragments widi said first and second primers to form strand/primer complexes, (iii) generating amplification products from die strand/primer complexes in the presence of DNA polymerase and deoxynucleoside triphosphates, and (iv) repeating steps (i) to (iii) until a desired degree of amplification has been achieved, (d) immobilizing amplification products containing die second primer via said immobilization attachment site, (e) removing non-immobilized amplified fragments, (f) cleaving said amplification products at die cleavable site, to generate a mixture including a double-stranded product, (g) denaturing the double-stranded product to release die extension segment, and (h) sizing the extension segment by mass spectrometry, where the read length of die extension segment is increased relative to die read length of the amplified strand-primer complexes of (c). 34. A metiiod for determining die size of a primer extension product, comprising: (a) combining first and second primers widi a target nucleic acid, under conditions tiiat promote hybridization of the primers to the nucleic acid, generating primer/nucleic acid complexes, where said first primer (i) is complementary to said target nucleic acid; (ii) has a first region containing the 5' end of the primer and an immobilization attachment site, and (iii) has a second region containing the 3' end of the primer, where the 3' end is capable of serving as a priming site for enzymatic extension and where said second region contains a cleavable site, and where said second primer is homologous to said target nucleic acid, (b) converting die primer/nucleic acid complexes to double-stranded fragments in the presence of a DNA polymerase and deoxynucleoside triphosphates, (c) amplifying die number of primer-containing fragments by successively repeating the steps of (i) denaturing said double-strand fragments to produce single-stranded fragments, (ii) hybridizing said single stranded fragments with said first and second primers to form strand/primer complexes, (iii) generating double-stranded amplification fragments from the strand/primer complexes in the presence of DNA polymerase and deoxynucleoside triphosphates, and (iv) repeating steps (i) to (iii) until a desired degree of amplification has been achieved, (d) denaturing die amplification fragments to generate a mixture including a product composed of die first primer and an extension segment; (e) immobilizing amplified fragments containing the first primer, utilizing said immobilization attachment site, and removing non-immobilized amplified fragments, (f) cleaving said first primer at die cleavable site to release the extension segment; and (g) sizing the extension segment by mass spectrometry, where the read lengtii of the extension segment is increased relative to the read lengtii of the product of (d). 35. A method for determining die DNA sequence of a target DNA sequence, comprising: (a) hybridizing a primer with a target DNA, where said primer (i) is complementary to said target DNA; (ii) has a first region containing die 5' end of die primer and an immobilization attachment site, and (iii) has a second region containing the 3' end of the primer, where the 3' end is capable of serving as a priming site for enzymatic extension and where said second region contains a cleavable site, (b) extending die primer widi an enzyme in the presence of a first of four different dideoxy nucleotides to generate a mixture of primer extension products each product containing a primer and an extension segment; (c) cleaving at die cleavable site to release die extension segments, where prior to said cleaving the primers are immobilized at said immobilization attachment sites; (d) sizing the extension segments by mass spectrometry, where the read lengtii of the extension segment is increased relative to the read lengtii of the product of (b), (e) repeating steps (a) through (d) with a second, third, and fourth of the four different dideoxy nucleotides, and (f) determining die DNA sequence of said target DNA by comparison of the sizes of the extension segments obtained from each of die four extension reactions. 36. A metiiod for determining die DNA sequence of a target DNA sequence, comprising (a) hybridizing a primer witii a target DNA, where said primer (i) is complementary to said target DNA; (ii) has a first region containing the 5' end of the primer and an immobilization attachment site, and (iii) has a second region containing the 3' end of die primer, where the 3' end is capable of serving as a priming site for enzymatic extension and where said second region contains a cleavable site, (b) extending die primer with an enzyme in the presence of a first of four different deoxynucleotide α-thiotriphosphate analogs (dNTPαS) to generate a mixture of primer extension products containing phosphorothioate linkages, (c) treating the primer extension products with a reagent that cleaves specifically at the phosphorothioate linkages, where said treating is carried out under conditions producing limited cleavage, resulting in die production of a set of primer extension degradation products, (d) washing the primer extension degradation products, where prior to said washing, tiie primer extension degradation products are immobilized at said immobilization attachment sites, each immobilized primer extension degradation product containing a primer and an extension segment, where said washing is effective to remove non-immobilized species, (e) cleaving at the cleavable site to release die extension segments, (f) sizing die extension segments by mass spectrometry, where die read length of any given extension segment is increased relative to the read lengtii of its corresponding primer extension degradation product, (g) repeating steps (a) through (f) with a second, third, and fourth of the four different dNTPαSs, and (h) determining the DNA sequence of said target DNA by comparison of the sizes of the extension segments obtained from each of the four extension reactions. 37. The method of claim 36, wherein the reagent of step (c) is selected from the group consisting of exonuclease, 2-iodoethanol, and 2,3-epoxy-l-propanol. 38. A metiiod for determining a single base fingeφrint of a target DNA sequence, comprising: (a) hybridizing a primer with a target DNA, where said primer (i) is complementary to said target DNA; (ii) has a first region containing the 5' end of die primer and an immobilization attachment site, and (iii) has a second region containing die 3' end of the primer, where the 3' end is capable of serving as a priming site for enzymatic extension and where said second region contains a cleavable site, (b) extending the primer with an enzyme in the presence of a dideoxynucleoside triphosphate corresponding to said single base, to generate a mixture of primer extension products, each product containing a primer and an extension segment; (c) cleaving at die cleavable site to release die extension segments, where prior to said cleaving d e primers are immobilized at said immobilization attachment sites; (d) sizing die extension segments by mass spectrometry, where the read lengtii of any given extension segment is increased relative to the read length of its corresponding primer extension product of (b), and (e) determining die positions of said single base in said target DNA by comparison of the sizes of the extension segments. 39. A method for determining an adenine fingeφrint of a target DNA sequence, comprising: (a) hybridizing a primer with a DNA target, where said primer (i) is complementary to said target DNA; (ii) has a first region containing the 5' end of the primer and an immobilization attachment site, and (iii) has a second region containing die 3' end of die primer, where the 3' end is capable of serving as a priming site for enzymatic extension and where said second region contains a cleavable site, (b) extending die primer witii an enzyme in the presence of deoxyadenosine triphosphate (dATP), deoxytiiymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxyuridine triphosphate (dUTP), to generate a mixture of primer extension products containing dUTP at positions corresponding to dATP in said target, each product containing a primer and an extension segment; (c) treating the primer extension products with uracil DNA-glycosylase to fragment specifically at dUTP positions, resulting in the production of a set of primer extension degradation products, (d) washing die primer extension degradation products, where prior to said washing, die primer extension degradation products are immobilized at said immobilization attachment sites, each immobilized primer extension degradation product containing a primer and an extension segment, where said washing is effective to remove non-immobilized species, (e) cleaving at die cleavable site to release die extension segments, (f) sizing the extension segments by mass spectrometry, where the read length of any given extension segment is increased relative to the read length of its corresponding primer extension degradation product, and (g) determining die positions of adenine in said target DNA by comparison of die sizes of d e released extension segments.

Inventors: BECKER CHRISTOPHER HANK, MONFORTE JOSEPH ALBERT, POLLART DANIEL JOSEPH, SHALER THOMAS ANDREW, BECKER, CHRISTOPHER, HANK, MONFORTE, JOSEPH, ALBERT, POLLART, DANIEL, JOSEPH, SHALER, THOMAS, ANDREW
Applicants: STANFORD RES INST INT, SRI INTERNATIONAL
Application year: 1996
Main classification: C12Q1
IPC codes: B82B1, C12N15, C12Q1