DNA DIAGNOSTICS BASED ON MASS SPECTROMETRY

Abstract: The invention provides fast and highly accurate mass spectrometer based processes for detecting a particular nucleic acid sequence in a biological sample. Depending on the sequence to be detected, the processes can be used, for example, to diagnose a genetic disease or chromosomal abnormality; a predisposition to a disease or condition, infection by a pathogenic organism, or for determining identity or heredity.

Description: DNA DIAGNOSTICS BASED ON MASS SPECTROMETRY Background of the Invention The genetic information of all living organisms (e.g. animals, plants and microorganisms) is encoded in deoxyribonucleic acid (DNA). In humans, the complete genome is comprised of about 100.000 genes located on 24 chromosomes (The Human Genome. T. Strachan. BIOS Scientific Publishers. 1992). Each gene codes for a specific protein which after its expression via transcription and translation, fulfills a specific biochemical function within a living cell. Changes in a DNA sequence are known as mutations and can result in proteins with altered or in some cases even lost biochemical activities; this in turn can cause genetic disease. Mutations include nucleotide deletions. insertions or alterations (i.e. point mutations). Point mutations can be either "missense". resulting in a change in the amino acid sequence of a protein or "nonsense" coding for a stop codon and thereby leading to a truncated protein. More than 3000 genetic diseases are currently known (Human Genome Mutations, D.N. Cooper and M. Krawczak. BIOS Publishers. 1993). including hemophilias, thalassemias, Duchenne Muscular Dystrophy (DMD). Huntington's Disease (HD). Alzheimer's Disease and Cystic Fibrosis (CF). In addition to mutated genes, which result in genetic disease, certain birth defects are the result of chromosomal abnormalities such as Trisomy 21 (Down's Syndrome). Trisomy 13 (Patau Syndrome), Trisomy 18 (Edward's Syndrome). Monosomy X (Turner's Syndrome) and other sex chromosome aneuploidies such as Klienfelter's Syndrome (XXY). Further, there is growing evidence that certain DNA sequences may predispose an individual to any of a number of diseases such as diabetes. arteriosclerosis, obesity, various autoimmune diseases and cancer (e.g. colorectal. breast. ovarian, lung). Viruses, bacteria, fungi and other infectious organisms contain distinct nucleic acid sequences, which are different from the sequences contained in the host cell. Therefore. infectious organisms can also be detected and identified based on their specific DNA sequences. Since the sequence of about 16 nucleotides is specific on statistical grounds even for the size of the human genome, relatively short nucleic acid sequences can be used to detect normal and defective genes in higher organisms and to detect infectious microorganisms (e.g. bacteria, fungi, protists and yeast) and viruses. DNA sequences can even serve as a fingerprint for detection of different individuals within the same species. (Thompson. J.S. and M.W. Thompson, eds.. Genetics in Medicine. W.B. Saunders Co.. Philadelphia. PA ( 1986). Several methods for detecting DNA are currently being used. For example, nucleic acid sequences can be identified by comparing the mobility of an amplified nucleic acid fragment with a known standard by gel electrophoresis. or by hybridization with a probe, which is complementary to the sequence to be identified. Identification, however, can only be accomplished if the nucleic acid fragment is labeled with a sensitive reporter function (e.g. radioactive (32p. 35s). fluorescent or chemiluminescent). However, radioactive labels can be hazardous and the signals they produce decay over time. Non-isotopic labels (e.g. fluorescent) suffer from a lack of sensitivity and fading of the signal when high intensity lasers are being used. Additionally, performing labeling, electrophoresis and subsequent detection are laborious, time-consuming and error-prone procedures. Electrophoresis is paπicularly error-prone, since the size or the molecular weight of the nucleic acid cannot be directly correlated to the mobility in the gel matrix. It is known that sequence specific effects, secondary structures and interactions with the gel matrix are causing artefacts. In general, mass spectrometry provides a means of "weighing" individual molecules by ionizing the molecules in vacuo and making them "fly" by volatilization. Under the influence of combinations of electric and magnetic fields, the ions follow trajectories depending on their individual mass (m) and charge (z). In the range of molecules with low molecular weight, mass spectrometry has long been part of the routine physical- organic repertoire for analysis and characterization of organic molecules by the determination of the mass of the parent molecular ion. In addition, by arranging collisions of this parent molecular ion with other particles (e.g., argon atoms), the molecular ion is fragmented forming secondary ions by the so-called collision induced dissociation (CID). The fragmentation pattern/pathway very often allows the derivation of detailed structural information. Many applications of mass spectrometric methods are known in the art. particularly in biosciences. and can be found summarized in Methods in Enzvmology. Vol. 193: "Mass Spectrometry" (J.A. McCloskey. editor). 1990. Academic Press. New York. Due to the apparent analytical advantages of mass spectrometry in providing high detection sensitivity, accuracy of mass measurements, detailed structural information by CID in conjunction with an MS/MS configuration and speed, as well as on-line data transfer to a computer, there has been considerable interest in the use of mass spectrometry for the structural analysis of nucleic acids. Recent reviews summarizing this field include K. H. Schram. "Mass Spectrometry of Nucleic Acid Components. Biomedical Applications of Mass **. * ** Spectrometry" 2ά, 203-287 (1990); and P.F. Crain. "Mass Spectrometric Techniques in Nucleic Acid Research." Mass Spectrometry Reviews 9. 505-554 (1990). However, nucleic acids are very polar biopolymers that are very difficult to volatilize. Consequently, mass spectrometric detection has been limited to low molecular weight synthetic oligonucleotides by determining the mass of the parent molecular ion and through this, confirming the already known oligonucleotide sequence, or alternatively, confirming the known sequence through the generation of secondary ions (fragment ions) via CID in an MS/MS configuration utilizing, in particular, for the ionization and volatilization. the method of fast atomic bombardment (FAB mass spectrometry) or plasma desorption (PD mass spectrometry). As an example, the application of FAB to the analysis of protected dimeric blocks for chemical synthesis of oligodeoxynucleotides has been described (Kόster et al. Biomedical Environmental Mass Spectrometry 14. 1 1 1-1 16 (1987)). Two more recent ionization/desorption techniques are electrospray/ionspray (ES) and matrix-assisted laser desorption/ionization (MALDI). ES mass spectrometry has been introduced by Fenn et al. ( J. Phvs. Chem. g£, 4451 -59 ( 1984); PCT Application No. WO 90/14148) and current applications are summarized in recent review articles (R.D. Smith et al., Anal. Chem. £2, 882-89 (1990) and B. Ardrey. Electrospray Mass Spectrometry, Spectroscopy Europe.4, 10- 18 ( 1992)). The molecular weights of a tetradecanucleotide (Covey et al. "The Determination of Protein. Oligonucleotide and Peptide Molecular Weights by Ionspray Mass Spectrometry," Rapid Communications in Mass Spectrometry. 2, 249-256 (1988)). and of a 21-mer (Methods in Enzvmology. 193. "Mass Spectrometry" (McCloskey. editor), p. 425. 1990, Academic Press. New York) have been published. As a mass analyzer. a quadrupole is most frequently used. The determination of molecular weights in femtomole amounts of sample is very accurate due to the presence of multiple ion peaks which all could be used for the mass calculation. MALDI mass spectrometry. in contrast, can be particularly attractive when a time-of-flight (TOF) configuration is used as a mass analyzer. The MALDI-TOF mass spectrometry has been introduced by Hillenkamp el al. ("Matrix Assisted UV-Laser Desorption/ionization: A New Approach to Mass Spectrometry of Large Biomolecules." Biological Mass Spectrometry (Burlingame and McCloskey. editors). Elsevier Science Publishers. Amsterdam, pp. 49-60. 1990.) Since, in most cases, no multiple molecular ion peaks are produced with this technique, the mass spectra, in principle, look simpler compared to ES mass spectrometry. Although DNA molecules up to a molecular weight of 410.000 daltons have been desorbed and volatilized (Williams et al. "Volatilization of High Molecular Weight DNA by Pulsed Laser Ablation of Frozen Aqueous Solutions." Sc ence, 2___, 1585-87 (1989)), this technique has so far only shown very low resolution (oligothymidylic acids up to 18 nucleotides. Huth-Fehre et al.. Rapid Communications in Mass Spectrometry. £, 209-13 (1992); DNA fragments up to 500 nucleotides in length K. Tang et al.. Rapid Communications in Mass Spectrometry. 8_, 727-730 ( 1994): and a double-stranded DNA of 28 base pairs (Williams et al.. "Time-of-Flight Mass Spectrometry of Nucleic Acids by Laser Ablation and Ionization from a Frozen Aqueous Matrix." Rapid Communications in Mass Spectrometry. 4. 348-351 ( 1990)). Japanese Patent No. 59-131909 describes an instrument, which detects nucleic acid fragments separated either by electrophoresis. liquid chromatography or high speed gel filtration. Mass spectrometric detection is achieved by incorporating into the nucleic acids, atoms which normally do not occur in DNA such as S. Br. I or Ag. Au. Pt. Os. Hg. Summary of the invention The instant invention provides mass spectrometric processes for detecting a particular nucleic acid sequence in a biological sample. Depending on the sequence to be detected, the processes can be used, for example, to diagnose (e.g. prenatally or postnatally) a genetic disease or chromosomal abnormality; a predisposition to a disease or condition (e.g. obesity, artherosclerosis, cancer), or infection by a pathogenic organism (e.g. virus, bacteria, parasite or fungus); or to provide information relating to identity, heredity, or compatibility (e.g. HLA phenotyping). In a first embodiment, a nucleic acid molecule containing the nucleic acid sequence to be detected (i.e. the target) is initially immobilized to a solid support. Immobilization can be accomplished, for example, based on hybridization between a portion of the target nucleic acid molecule, which is distinct from the target detection site and a capture nucleic acid molecule, which has been previously immobilized to a solid support. Alternatively, immobilization can be accomplished by direct bonding of the target nucleic acid molecule and the solid support. Preferably, there is a spacer (e.g. a nucleic acid molecule) between the target nucleic acid molecule and the support. A detector nucleic acid molecule (e.g. an oligonucleotide or oligonucleotide mimetic), which is complementary to the target detection site can then be contacted with the target detection site and formation of a duplex, indicating the presence of the target detection site can be detected by mass spectrometry. In preferred embodiments, the target detection site is amplified prior to detection and the nucleic acid molecules are conditioned. In a further preferred embodiment, the target detection sequences are arranged in a format that allows multiple simultaneous detections (multiplexing), as well as parallel processing using oligonucleotide arrays ("DNA chips"). In a second embodiment, immobilization of the target nucleic acid molecule is an optional rather than a required step. Instead, once a nucleic acid molecule has been obtain from a biological sample, the target detection sequence is amplified and directly detected by mass spectrometry. In preferred embodiments, the target detection site and/or the detector oligonucleotides are conditioned prior to mass spectrometric detection. In another preferred embodiment, the amplified target detection sites are arranged in a format that allows multiple simultaneous detections (multiplexing), as well as parallel processing using oligonucleotide arrays ("DNA chips"). In a third embodiment, nucleic acid molecules which have been replicated from a nucleic acid molecule obtained from a biological sample can be specifically digested using one or more nucieases (using deoxyribonucleases for DNA or ribonucleases for RNA) and the fragments captured on a solid support carrying the corresponding complementary sequences. Hybridization events and the actual molecular weights of the captured target sequences provide information on whether and where mutations in the gene are present. The array can be analyzed spot by spot using mass spectrometry. DNA can be similarly digested using a cocktail of nucieases including restriction endonucleases. In a preferred embodiment, the nucleic acid fragments are conditioned prior to mass spectrometric detection. In a fourth embodiment, at least one primer with 3' terminal base complementarity to an allele (mutant or normal) is hybridized with a target nucleic acid molecule, which contains the allele. An appropriate polymerase and a complete set of nucleoside triphosphates or only one of the nucleoside triphosphates are used in separate reactions to furnish a distinct extension of the primer. Only if the primer is appropriately annealed (i.e. no 3' mismatch) and if the correct (i.e. complementary) nucleotide is added, will the primer be extended. Products can be resolved by molecular weight shifts as determined by mass spectrometry. In a fifth embodiment, a nucleic acid molecule containing the nucleic acid sequence to be detected (i.e. the target) is initially immobilized to a solid support. Immobilization can be accomplished, for example, based on hybridization between a portion of the target nucleic acid molecule, which is distinct from the target detection site and a capture nucleic acid molecule, which has been previously immobilized to a solid support. Alternatively, immobilization can be accomplished by direct bonding of the target nucleic acid molecule and the solid support. Preferably, there is a spacer (e.g. a nucleic acid molecule) between the target nucleic acid molecule and the support. A nucleic acid molecule that is complementary to a portion of the target detection site that is immediately 5' of the site of a mutation is then hybridized with the target nucleic acid molecule. The addition of a complete set of dideoxynucleosides or 3'-deoxynucleoside triphosphates (e.g. pppAdd. pppTdd. pppCdd and pppGdd) and a DNA dependent DNA polymerase allows for the addition only of the one dideoxynucleoside or 3'-deoxynucleoside triphosphate that is complementary to X. The hybridization product can then be detected by mass spectrometry. In a sixth embodiment, a target nucleic acid is hybridized with a complementary oligonucleotides that hybridize to the target within a region that includes a mutation M. The heterodupiex is then contacted with an agent that can specifically cleave at an unhybridized portion (e.g. a single strand specific endonuclease), so that a mismatch, indicating the presence of a mutation, results in the cleavage of the target nucleic acid. The two cleavage products can then be detected by mass spectrometry. In a seventh embodiment, which is based on the ligase chain reaction (LCR), a target nucleic acid is hybridized with a set of ligation educts and a thermostable DNA ligase, so that the ligase educts become covalently linked to each other, forming a ligation product. The ligation product can then be detected by mass spectrometry and compared to a known value. If the reaction is performed in a cyclic manner, the ligation product obtained can be amplified to better facilitate detection of small volumes of the target nucleic acid. Selection between wildtype and mutated primers at the ligation point can result in the detection of a point mutation. The processes of the invention provide for increased accuracy and reliability of nucleic acid detection by mass spectrometry. In addition, the processes allow for rigorous controls to prevent false negative or positive results. The processes of the invention avoid electrophoretic steps: labeling and subsequent detection of a label. In fact it is estimated that the entire procedure, including nucleic acid isolation, amplification, and mass spec analysis requires only about 2-3 hours time. Therefore the instant disclosed processes of the invention are faster and less expensive to perform than existing DNA detection systems. In addition, because the instant disclosed processes allow the nucleic acid fragments to be identified and detected at the same time by their specific molecular weights (an unambiguous physical standard), the disclosed processes are also much more accurate and reliable than currently available procedures. Brief Description of the Figures FIGURE 1 A is a diagram showing a process for performing mass spectrometric analysis on one target detection site (TDS) contained within a target nucleic acid molecule (T). which has been obtained from a biological sample. A specific capture sequence (C) is attached to a solid support (SS) via a spacer (S). The capture sequence is chosen to specifically hybridize with a complementary sequence on the target nucleic acid molecule (T). known as the target capture site (TCS). The spacer (S) facilitates unhindered hybridization. A detector nucleic acid sequence (D). which is complementary to the TDS is then contacted with the TDS. Hybridization between D and the TDS can be detected by mass spectrometry. FIGURE IB is a diagram showing a process for performing mass spectrometric analysis on at least one target detection site (here TDS 1 and TDS 2) via direct linkage to a solid support. The target sequence (T) containing the target detection site (TDS 1 and TDS 2) is immobilized to a solid support via the formation of a reversible or irreversible bond formed between an appropriate functionality (L') on the target nucleic acid molecule (T) and an appropriate functionality (L) on the solid support. Detector nucleic acid sequences (here Dl and D2), which are complementary to a target detection site (TDS 1 or TDS 2) are then contacted with the TDS. Hybridization between TDS 1 and Dl and or TDS 2 and D2 can be detected and distinguished based on molecular weight differences. FIGURE 1 C is a diagram showing a process for detecting a wildtype (Dwt) and/ or a mutant (Dmut) sequence in a target (T) nucleic acid molecule. As in Figure 1 A, a specific capture sequence (C) is attached to a solid support (SS) via a spacer (S). In addition, the capture sequence is chosen to specifically interact with a complementary sequence on the target sequence (T), the target capure site (TCS) to be detected through hybridization. However, if the target detection site (TDS) includes a mutation. X. which changes the molecular weight, mutated target detection sites can be distinguished from wildtype by mass spectrometry. Preferably, the detector nucleic acid molecule (D) is designed so that the mutation is in the middle of the molecule and therefore would not lead to a stable hybrid if the wildtype detector oligonucleotide (Dwt) is contacted with the target detector sequence, e.g. as a control. The mutation can also be detected if the mutated detector oligonucleotide (Dmut) with the matching base at the mutated position is used for hybridization. If a nucleic acid molecule obtained from a biological sample is heterozygous for the particular sequence ( i.e. contain both Dwt and Dmut). both Dwt and Dmut j|i be bound to the appropriate strand and the mass difference allows both D v and Dmut to be detected simultaneously. FIGURE 2 is a diagram showing a process in which several mutations are simultaneously detected on one target sequence by employing corresponding detector oligonucleotides. The molecular weight differences between the detector oligonucleotides Dl . D2 and D3 must be large enough so that simultaneous detection (multiplexing) is possible. This can be achieved either by the sequence itself (composition or length) or by the introduction of mass-modifying functionalities M l - M3 into the detector oligonucleotide. FIGURE 3 is a diagram showing still another multiplex detection format. In this embodiment, differentiation is accomplished by employing different specific capture sequences which are position-specifically immobilized on a flat surface (e.g. a 'chip array'). If different target sequences Tl - Tn are present, their target capture sites TCS1 - TCSn will interact with complementary immobilized capture sequences C 1 -Cn. Detection is achieved by employing appropriately mass differentiated detector oligonucleotides Dl - Dn. which are mass differentiated either by their sequences or by mass modifying functionalities Ml - Mn. FIGURE 4 is a diagram showing a format wherein a predesigned target capture site (TCS) is incorporated into the target sequence using PCR amplification. Only one strand is captured, the other is removed (e.g., based on the interaction between biotin and streptavidin coated magnetic beads). If the biotin is attached to primer 1 the other strand can be appropriately marked by a TCS. Detection is as described above through the interaction of a specific detector oligonucleotide D with the corresponding target detection site TDS via mass spectrometry. FIGURE 5 is a diagram showing how amplification (here ligase chain reaction (LCR)) products can be prepared and detected by mass spectrometry. Mass differentiation can be achieved by the mass modifying functionalities (Ml and M2) attached to primers (PI and P4 respectively). Detection by mass spectrometry can be accomplished directly (i.e. without employing immobilization and target capturing sites (TCS)). Multiple LCR reactions can be performed in parallel by providing an ordered array of capturing sequences (C). This format allows separation of the ligation products and spot by spot identification via mass spectrometry or multiplexing if mass differentiation is sufficient. FIGURE 6A is a diagram showing mass spectrometric analysis of a nucleic acid molecule, which has been amplified by a transcription amplification procedure. An RNA sequence is captured via its TCS sequence, so that wildtype and mutated target detection sites can be detected as above by employing appropriate detector oligonucleotides (D). FIGURE 6B is a diagram showing multiplexing to detect two different (mutated) sites on the same RNA in a simultaneous fashion using mass-modified detector oligonucleotides Ml-Dl and M2-D2. FIGURE 6C is a diagram of a different multiplexing procedure for detection of specific mutations by employing mass modified dideoxynucleoside or 3'-deoxynucleoside triphosphates and an RNA dependent DNA polymerase. Alternatively. DNA dependent RNA polymerase and ribonucleotide triphosphates can be employed. This format allows for simultaneous detection of all four base possibilities at the site of a mutation (X). FIGURE 7A is a diagram showing a process for performing mass spectrometric analysis on one target detection site (TDS) contained within a target nucleic acid molecule (T), which has been obtained from a biological sample. A specific capture sequence (C) is attached to a solid support (SS) via a spacer (S). The capture sequence is chosen to specifically hybridize with a complementary sequence on T known as the target capture site (TCS). A nucleic acid molecule that is complementary to a portion of the TDS is hybridized to the TDS 5' of the site of a mutation (X) within the TDS. The addition of a complete set of dideoxynucleosides or 3'-deoxynucleoside triphosphates (e.g. pppAdd. pppTdd. pppCdd and pppGdd) and a DNA dependent DNA polymerase allows for the addition only of the one dideoxynucleoside or 3'-deoxynucleoside triphosphate that is complementary to X. FIGURE 7B is a diagram showing a process for performing mass spectrometric analysis to determine the presence of a mutation at a potential mutation site (M) within a nucleic acid molecule. This format allows for simultaneous analysis of both alleles (A) and (B) of a double stranded target nucleic acid molecule, so that a diagnosis of homozygous normal, homozygous mutant or heterozygous can be provided. Allele A and B are each hybridized with complementary oligonucleotides ((C) and (D) respectively), that hybridize to A and B within a region that includes M. Each heterodupiex is then contacted with a single strand specific endonuclease. so that a mismatch at M, indicating the presence of a mutation, results in the cleavage of (C) and/or (D), which can then be detected by mass spectrometry. FIGURE 8 is a diagram showing how both strands of a target DNA can be prepared for detection using transcription vectors having two different promoters at opposite locations (e.g. the SP6 and the T7 promoter). This format is particularly useful for detecting heterozygous target detection sites (TDS). Employing the SP6 or the T7 RNA polymerase both strands could be transcribed separately or simultaneously. Both RNAs can be specifically captured and simultaneously detected using appropriately mass-differentiated detector oligonucleotides. This can be accomplished either directly in solution or by parallel processing of many target sequences on an ordered array of specifically immobilized capturing sequences. FIGURE 9 is a diagram showing how RNA prepared as described in Figures 6. 7 and 8 can be specifically digested using one or more ribonucleases and the fragments captured on a solid support carrying the corresponding complementary sequences. Hybridization events and the actual molecular weights of the captured target sequences provide information on whether and where mutations in the gene are present. The array can be analyzed spot by spot using mass spectrometry. DNA can be similarly digested using a cocktail of nucieases including restriction endonucleases. Mutations can be detected by different molecular weights of specific, individual fragments compared to the molecular weights of the wildtype fragments. FIGURE 10A shows a spectra resulting from the experiment described in the following Example 1. Panel i) shows the absorbance of the 26-mer before hybridization. Panel ii) shows the filtrate of the centrifugation after hybridization. Panel iii) shows the results after the first wash with 50mM ammonium citrate. Panel iv) shows the results after the second wash with 50mM ammonium citrate. FIGURE 1 OB shows a spectra resulting from the experiment described in the following Example 1 after three washing/ centrifugation steps. FIGURE IOC shows a spectra resulting from the experiment described in the following Example 1 showing the successful desoφtion of the hybridized 26mer off of beads. FIGURE 1 1 shows a spectra resulting from the experiment described in the following Example 1 showing the successful desoφtion of the hybridized 40mer. The efficiency of detection suggests that fragments much longer than 40mers can also be desorbed. FIGURE 12 shows a spectra resulting from the experiment described in the following Example 2 showing the successful desoφtion and differentiation of an 18-mer and 19-mer by electrospray mass spectrometry. the mixture (top), peaks resulting from 18-mer emphasized (middle) and peaks resulting from 19-mer emphasized (bottom) FIGURE 13 is a graphic representation of the process for detecting the Cystic Fibrosis mutation ΔF508 as described in Example 3. FIGURE 14 is a mass spectrum of the DNA extension product of a ΔF508 homozygous normal. FIGURE 15 is a mass spectrum of the DNA extension product of a ΔF508 heterozygous mutant. FIGURE 16 is a mass spectrum of the DNA extension product of a ΔF508 homozygous normal. FIGURE 17 is a mass spectrum of the DNA extension product of a ΔF508 homozygous mutant. FIGURE 18 is a mass spectrum of the DNA extension product of a ΔF508 heterozygous mutant. FIGURE 19 is a graphic representation of various processes for performing apolipoprotein E genotyping. FIGURE 20 shows the nucleic acid sequence of normal apolipoprotein E (encoded by the E3 allele) and other isotypes encoded by the E2 and E4 alleles. FIGURE 21 A shows a composite restriction pattern for various genotypes of apolipoprotein E. FIGURE 21 B shows the restriction pattern obtained in a 3.5% MetPhor Agarose Gel for various genotypes of apolipoprotein E. FIGURE 21 C shows the restriction pattern obtained in a 12% polyacrylamide gel for various genotypes of apolipoprotein E. FIGURE 22A is a chart showing the molecular weights of the 91. 83. 72, 48 and 35 base pair fragments obtained by restriction enzyme cleavage of the E2. E3 and E4 alleles of apolipoprotein E. FIGURE 22B is the mass spectra of the restriction product of a homozygous E4 apolipoprotein E genotype. FIGURE 23 A is the mass spectra of the restriction product of a homozygous E3 apolipoprotein E genotype. FIGURE 23B is the mass spectra of the restriction product of a E3/E4 apolipoprotein E genotype. FIGURE 24 is an autoradiograph of a 7.5% polyacrylamide gel in which 10% (5μl) of each PCR was loaded. Sample M: pBR322 Alul digested: sample 1 : HBV positive in serological analysis; sample 2: also HBV positive; sample 3: without serological analysis but with an increased level of transaminases. indicating liver disease: sample 4: HBV negative: sample 5: HBV positive by serological analysis: sample 6: HBV negative (-) negative control: (+) positive control). Staining was done with ethidium bromide. FIGURE 25A is a mass spectrum of sample 1. which is HBV positive. The signal at 20754 Da represents the HBV related PCR product (67 nucleotides. calculated mass: 20735 Da). The mass signal at 10390 Da represents the [M+2H] 2+ signal (calculated: 10378 Da). FIGURE 25B is a mass spectrum of sample 3. which is HBV negative corresponding to PCR. serological and dot blot based assays. The PCR product is generated only in trace amounts. Nevertheless it is unambiguously detected at 20751 Da (calculated: 20735 Da). The mass signal at 10397 Da represents the [M+2H] 2τ molecule ion (calculated: 10376 Da). FIGURE 25C is a mass spectrum of sample 4. which is HBV negative, but CMV positive. As expected, no HIV specific signals could be obtained. FIGURE 26 shows a part of the E. coli lac\ gene with binding sites of the complementary oligonucleotides used in the ligase chain reaction (LCR). Here the wildtype sequence is displayed. The mutant contains a point mutation at bp 191 which is also the site of ligation (bold). The mutation is a C to T transition (G to A. respectively). This leads to a T-G mismatch with oligo A (and A-C mismatch with oligo B. respectively). FIGURE 27 is a 7.5% polyacrylamide gel stained with ethidium bromide. M: chain length standard (pUC 19 DNA. Mspl digested). Lane 1 : LCR with wildtype template. Lane 2: LCR with mutant template. Lane 3: (control) LCR without template. The ligation product (50 bp) was only generated in the positive reactive containing wildtype template. FIGURE 28 is an HPLC chromatogram of two pooled positive LCRs. FIGURE 29 shows an HPLC chromatogram the same conditions but mutant template were used. The small signal of the ligation product is due to either template-free ligation of the educts or to a ligation at a (G-T. A-C) mismatch. The 'false positive' signal is significantly lower than the signal of ligation product with wildtype template depicted in Figure 28. The analysis of ligation educts leads to 'double -peaks' because two of the oligonucleotides are 5'- phosphorylated. FIGURE 30 In a the complex signal pattern obtained by MALDI-TOF-MS analysis of Pfu DNA-ligase solution is depicted. In b a MALDI -TOF-spectrum of an unpurified LCR is shown. The mass signal 67569 Da probably represents the Pfu DNA ligase. FIGURE 31 shows a MALDI-TOF spectrum of two pooled positive LCRs (a). The signal at 7523 Da represents unligated oligo A (calculated: 7521 Da) whereas the signal at 15449 Da represents the ligation product (calculated: 15450 Da). The signal at 3774 Da is the [M+2H]- + signal of oligo A. The signals in the mass range lower than 2000 Da are due to the matrix ions. The spectrum corresponds to lane 1 in figure 2a and to the chromatogram in figure 2b. In b a spectrum of two pooled negative LCRs (mutant template) is shown. The signal at 7517 Da represents oligo A (calculated: 7521 Da). In c a spectrum of two pooled control reactions (with salmon sperm DNA as template) is displayed. The signals in the mass range around 2000 Da are due to Tween20. FIGURE 32 shows a spectrum obtained from two pooled LCRs in which only salmon sperm DNA was used as a negative control, only oligo A could be detected, as expected. FIGURE 33 shows a spectrum of two pooled positive LCRs (a). The purification was done with a combination of ultrafiltration and streptavidin DynaBeads as described in the text. The signal at 15448 Da represents the ligation product (calculated: 15450 Da). The signal at 7527 represents oligo A (calculated: 7521 Da). The signals at 3761 Da is the [M+2H] 2+ signal of oligo A. whereas the signal at 5140 Da is the [M+3H]- ~ signal of the ligation product. In b a spectrum of two pooled negative LCRs (without template) is shown. The signal at 7514 Da represents oligo A (calculated: 7521 Da). FIGURE 34 is a schematic presentation of the oligo base extension of the mutation detection primer b using ddTTP (A) or ddCTP (B) in the reaction mix. respectively. The theoretical mass calculation is given in parenthesis. The sequence shown is part of the exon 10 of the CFTR gene that bears the most common cystic fibrosis mutation ΔF508 and more rare mutations ΔI507 as well as lle506Ser. FIGURE 35 is a MALDI-TOF-MS spectra recorded directly from precipitated oligo base extended primers for mutation detection. The spectra on the top of each panel (ddTTP or ddCTP, respectively) show the annealed primer (CF508) without further extension reaction. The template of diagnosis is pointed out below each spectra and the observed expected molecular mass are written in parenthesis. FIGURE 36 shows the portion of the sequence of pRFcl DNA. which was used as template for PCR amplification of unmodified and 7-deazapurine containing 99-mer and 200-mer nucleic acids as well as the sequences of the 19-primers and the two 18-mer reverse primers. FIGURE 37 shows the portion of the nucleotide sequence of M13mpl8 RFI DNA. which was used for PCR amplification of unmodified and 7-deazapurine containing 103-mer nucleic acids. Also shown are nucleotide sequences of the 17-mer primers used in the PCR. FIGURE 38 shows the result of a polyacrylamide gel electrophoresis of PCR products purified and concentrated for MALDI-TOF MS analysis. M: chain length marker, lane 1 : 7-deazapurine containing 99-mer PCR product, lane 2: unmodified 99-mer. lane 3: 7-deazapurine containing 103-mer and lane 4: unmodified 103-mer PCR product. FIGURE 39: an autoradiogram of polyacrylamide gel electrophoresis of PCR reactions carried out with 5'-[ 32 P]-labeled primers 1 and 4. Lanes 1 and 2: unmodified and 7 -deazapurine modified 103-mer PCR product (53321 and 23520 counts), lanes 3 and 4: unmodified and 7-deazapurine modified 200-mer (71 123 and 39582 counts) and lanes 5 and 6: unmodified and 7-deazapurine modified 99-mer ( 173216 and 94400 counts). FIGURE 40 a) MALDI-TOF mass spectrum of the unmodified 103-mer PCR products (sum of twelve single shot spectra). The mean value of the masses calculated for the two single strands (31768 u and 31759 u) is 31763 u. Mass resolution: 18. b) MALDI-TOF mass spectrum of 7-deazapurine containing 103-mer PCR product (sum of three single shot spectra). The mean value of the masses calculated for the two single strands (31727 u and 31719 u) is 31723 u. Mass resolution: 67. FIGURE 41 : a) MALDI-TOF mass spectrum of the unmodified 99-mer PCR product (sum of twenty single shot spectra). Values of the masses calculated for the two single strands: 30261 u and 30794 u. b) MALDI-TOF mass spectrum of the 7-deazapurine containing 99-mer PCR product (sum of twelve single shot spectra). Values of the masses calculated for the two single strands: 30224 u and 30750 u. FIGURE 42: a) MALDI-TOF mass spectrum of the unmodified 200-mer PCR product (sum of 30 single shot spectra). The mean value of the masses calculated for the two single strands (61873 u and 61595 u) is 61734 u. Mass resolution: 28. b) MALDI-TOF mass spectrum of 7-deazapurine containing 200-mer PCR product (sum of 30 single shot spectra). The mean value of the masses calculated for the two single strands (61772 u and 61514 u) is 61643 u. Mass resolution: 39. FIGURE 43: a) MALDI-TOF mass spectrum of 7-deazapurine containing 100-mer PCR product with ribomodified primers. The mean value of the masses calculated for the two single strands (30529 u and 31095 u) is 30812 u. b) MALDI-TOF mass spectrum of the PCR-product after hydrolytic primer-cleavage. The mean value of the masses calculated for the two single strands (25104 u and 25229 u) is 25167 u. The mean value of the cleaved primers (5437 u and 5918 u) is 5677 u. FIGURE 44 A-D shows the MALDI-TOF mass spectrum of the four sequencing ladders obtained from a 39-mer template (SEQ. ID. No. 13). which was immobilized to streptavidin beads via a 3' biotinylation. A 14-mer primer (SEQ. ID. NO. 14) was used in the sequencing. FIGURE 45 shows a MALDI-TOF mass spectrum of a solid state sequencing of a 78-mer template (SEQ. ID. No. 15). which was immobilized to streptavidin beads via a 3' biotinylation. A 18-mer primer (SEQ ID No. 16) and ddGTP were used in the sequencing. FIGURE 46 shows a scheme in which duplex DNA probes with single- stranded overhang capture specific DNA templates and also serve as primers for solid state sequencing. FIGURE 47A-D shows MALDI-TOF mass spectra obtained from a 5' fluorescent labeled 23-mer (SEQ. ID. No. 19) annealed to an 3' biotinylated 18-mer (SEQ. ID. No. 20), leaving a 5-base overhang, which captured a 15-mer template (SEQ. ID. No. 21 ). FIGURE 48 shows a stacking flurogram of the same products obtained from the reaction described in FIGURE 35, but run on a conventional DNA sequencer. Detailed Description of the Invention In general, the instant invention provides mass spectrometric processes for detecting a particular nucleic acid sequence in a biological sample. As used herein, the term "biological sample" refers to any material obtained from any living source (e.g. human, animal, plant, bacteria, fungi, protist. virus). For use in the invention, the biological sample should contain a nucleic acid molecule. Examples of appropriate biological samples for use in the instant invention include: solid materials (e.g tissue, cell pellets, biopsies) and biological fluids (e.g. urine, blood, saliva, amniotic fluid, mouth wash). Nucleic acid molecules can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art. the particular isolation procedure chosen being appropriate for the particular biological sample. For example, freeze- thaw and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials: heat and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from urine: and proteinase K extraction can be used to obtain nucleic acid from blood (Rolff. A et al. PCR: Clinical Diagnostics and Research. Springer ( 1994)). To obtain an appropriate quantity of a nucleic acid molecules on which to perform mass spectrometry. amplification may be necessary. Examples of appropriate amplification procedures for use in the invention include: cloning (Sambrook et al.. Molecular Cloning : A Laboratory Manual. Cold Spring Harbor Laboratory Press. 1989). polymerase chain reaction (PCR) (C.R. Newton and A. Graham. PCR. BIOS Publishers. 1994). ligase chain reaction (LCR) (Wiedmann. M.. et. al.. (1994) PCR Methods Appl. Vol. 3, Pp. 57-64; F. Barany Proc. Natl. Acad. Sci USA 88. 189-93 (1991 ). strand displacement amplification (SDA) (G. Terrance Walker et al.. Nucleic Acids Res. 22. 2670-77 (1994)) and variations such as RT-PCR (Higuchi. et al.. Bio/Technology 77: 1026-1030 ( 1993)). allele- specific amplification (ASA) and transcription based processes. To facilitate mass spectrometric analysis, a nucleic acid molecule containing a nucleic acid sequence to be detected can be immobilized to a solid support. Examples of appropriate solid supports include beads (e.g. silica gel. controlled pore glass, magnetic. Sephadex/Sepharose. cellulose), flat surfaces or chips (e.g. glass fiber filters, glass surfaces. metal surfaces (steel, gold, silver, aluminum, copper and silicon), capillaries, plastic (e.g. polyethylene, polypropylene, polyamide, polyvinylidenedifluoride membranes or microtiter plates)): or pins or combs made from similar materials comprising beads or flat surfaces or beads placed into pits in flat surfaces such as wafers (e.g. silicon wafers). Immobilization can be accomplished, for example, based on hybridization between a capture nucleic acid sequence, which has already been immobilized to the support and a complementary nucleic acid sequence, which is also contained within the nucleic acid molecule containing the nucleic acid sequence to be detected (FIGURE 1A). So that hybridization between the complementary nucleic acid molecules is not hindered by the support, the capture nucleic acid can include a spacer region of at least about five nucleotides in length between the solid support and the capture nucleic acid sequence. The duplex formed will be cleaved under the influence of the laser pulse and desoφtion can be initiated. The solid support-bound base sequence can be presented through natural oligoribo- or oligodeoxyribonucleotide as well as analogs (e.g. thio-modified phosphodiester or phosphotriester backbone) or employing oligonucleotide mimetics such as PNA analogs (see e.g. Nielsen et al. Science. 254. 1497 (1991)) which render the base sequence less susceptible to enzymatic degradation and hence increases overall stability of the solid support-bound capture base sequence. Alternatively, a target detection site can be directly linked to a solid support via a reversible or irreversible bond between an appropriate functionality (L') on the target nucleic acid molecule (T) and an appropriate functionality (L) on the capture molecule (FIGURE IB). A reversible linkage can be such that it is cleaved under the conditions of mass spectrometry (i.e.. a photocleavable bond such as a charge transfer complex or a labile bond being formed between relatively stable organic radicals). Furthermore, the linkage can be formed with L' being a quaternary ammonium group, in which case, preferably, the surface of the solid support carries negative charges which repel the negatively charged nucleic acid backbone and thus facilitate the desoφtion required for analysis by a mass spectrometer. Desoφtion can occur either by the heat created by the laser pulse and/or. depending on L.' by specific absoφtion of laser energy which is in resonance with the L' chromophore. By way of example, the L-L' chemistry can be of a type of disulfide bond (chemically cleavable. for example, by mercaptoethanol or dithioerythrol), a biotin/streptavidin system, a heterobifunctional derivative of a trityl ether group (Koster et al.. "A Versatile Acid-Labile Linker for Modification of Synthetic Biomolecules," Tetrahedron Letters 3_L, 7095 ( 1990)) which can be cleaved under mildly acidic conditions as well as under conditions of mass spectrometry. a levulinyl group cleavable under almost neutral conditions with a hydrazinium/acetate buffer, an arginine-arginine or lysine-lysine bond cleavable by an endopeptidase enzyme like trypsin or a pyrophosphate bond cleavable by a pyrophosphatase. or a ribonucleotide bond in between the oligodeoxynucleotide sequence, which can be cleaved, for example, by a ribonuclease or alkali. The functionalities. L and L,' can also form a charge transfer complex and thereby form the temporary L-L' linkage. Since in many cases the "charge-transfer band" can be determined by UV/vis spectrometry (see e.g. Organic Charge Transfer Complexes by R. Foster. Academic Press, 1969), the laser energy can be tuned to the corresponding energy of the charge-transfer wavelength and. thus, a specific desoφtion off the solid support can be initiated. Those skilled in the art will recognize that several combinations can serve this piupose and that the donor functionality can be either on the solid support or coupled to the nucleic acid molecule to be detected or vice versa. In yet another approach, a reversible L-L' linkage can be generated by homolytically forming relatively stable radicals. Under the influence of the laser pulse, desoφtion (as discussed above) as well as ionization will take place at the radical position. Those skilled in the art will recognize that other organic radicals can be selected and that, in relation to the dissociation energies needed to homolytically cleave the bond between them, a corresponding laser wavelength can be selected (see e.g. Reactive Molecules by C. Wentrup. John Wiley & Sons. 1984). An anchoring function L' can also be incoφorated into a target capturing sequence (TCS) by using appropriate primers during an amplification procedure, such as PCR (FIGURE 4). LCR (FIGURE 5) or transcription amplification (FIGURE 6A). Prior to mass spectrometric analysis, it may be useful to "condition" nucleic acid molecules, for example to decrease the laser energy required for volatization and/or to minimize fragmentation. Conditioning is preferably performed while a target detection site is immobilized. An example of conditioning is modification of the phosphodiester backbone of the nucleic acid molecule (e.g. cation exchange), which can be useful for eliminating peak broadening due to a heterogeneity in the cations bound per nucleotide unit. Contacting a nucleic acid molecule with an alkylating agent such as alkyliodide. iodoacetamide. β- iodoethanol. or 2.3-epoxy-l-propanol. the monothio phosphodiester bonds of a nucleic acid molecule can be transformed into a phosphotriester bond. Likewise, phosphodiester bonds may be transformed to uncharged derivatives employing trialkylsilyl chlorides. Further conditioning involves incoφorating nucleotides which reduce sensitivity for depurination (fragmentation during MS) such as N7- or N9-deazapurine nucleotides, or RNA building blocks or using oligonucleotide triesters or incoφorating phosphorothioate functions which are alkylated or employing oligonucleotide mimetics such as PNA. For certain applications, it may be useful to simultaneously detect more than one (mutated) loci on a particular captured nucleic acid fragment (on one spot of an array) or it may be useful to perform parallel processing by using oligonucleotide or oligonucleotide mimetic arrays on various solid supports. "Multiplexing" can be achieved by several different methodologies. For example, several mutations can be simultaneously detected on one target sequence by employing corresponding detector (probe) molecules (e.g. oligonucleotides or oligonucleotide mimetics). However, the molecular weight differences between the detector oligonucleotides Dl. D2 and D3 must be large enough so that simultaneous detection (multiplexing) is possible. This can be achieved either by the sequence itself (composition or length) or by the introduction of mass-modifying functionalities M 1 - M3 into the detector oligonucleotide.(FIGURE 2) Mass modifying moieties can be attached, for instance, to either the 5'-end of the oligonucleotide (M • ). to the nucleobase (or bases) (M 2 , M ). to the phosphate backbone (M->). and to the 2'-position of the nucleoside (nucleosides) (M 4 . M >) or/and to the terminal 3'-position (M- ). Examples of mass modifying moieties include . for example, a halogen, an azido. or of the type. XR. wherein X is a linking group and R is a mass-modifying functionality. The mass-modifying functionality can thus be used to introduce defined mass increments into the oligonucleotide molecule. Here the mass-modifying moiety. M. can be attached either to the nucleobase. M- (in case of the c^-deazanucleosides also to C-7. M^). to the triphosphate group at the alpha phosphate. M^. or to the 2'-position of the sugar ring of the nucleoside triphosphate. M 4 and M^. Furthermore, the mass-modifying functionality can be added so as to affect chain termination, such as by attaching it to the 3'-position of the sugar ring in the nucleoside triphosphate. M-\ For those skilled in the art. it is clear that many combinations can serve the puφose of the invention equally well. In the same way. those skilled in the art will recognize that chain-elongating nucleoside triphosphates can also be mass-modified in a similar fashion with numerous variations and combinations in functionality and attachment positions. Without limiting the scope of the invention, the mass-modification. M. can be introduced for X in XR as well as using oligo-/polyethylene glycol derivatives for R. The mass-modifying increment in this case is 44. i.e. five different mass-modified species can be generated by just changing m from 0 to 4 thus adding mass units of 45 (m=0), 89 (m=l ). 133 (m=2). 177 (m=3) and 221 (m=4) to the nucleic acid molecule (e.g. detector oligonucleotide (D) or the nucleoside triphosphates (FIGURE 6(C)). respectively). The oligo/polyethylene glycols can also be monoalkylated by a lower alkyl such as methyl, ethyl, propyl. isopropyl. t-butyl and the like. A selection of linking functionalities. X, are also illustrated. Other chemistries can be used in the mass-modified compounds, as for example, those described recently in Oligonucleotides and Analogues. A Practical Approach. F. Eckstein, editor. IRL Press. Oxford. 1991. In yet another embodiment, various mass-modifying functionalities. R. other than oligo/polyethylene glycols. can be selected and attached via appropriate linking chemistries. X. A simple mass-modification can be achieved by substituting H for halogens like F. Cl, Br and/or I, or pseudohalogens such as SCN, NCS, or by using different alkyl, aryl or aralkyl moieties such as methyl, ethyl, propyl. isopropyl, t-butyl. hexyl, phenyl, substituted phenyl. benzyl, or functional groups such as CH2F. CHF2. CF3. Si(CH3)3, Si(CH3)2(C2H5 . Si(CH3)(C2H5)2- Si(C2H5 3 . Yet another mass-modification can be obtained by attaching homo- or heteropeptides through the nucleic acid molecule (e.g. detector (D)) or nucleoside triphosphates. One example useful in generating mass-modified species with a mass increment of 57 is the attachment of oligoglycines. e.g.. mass- modifications of 74 (r=l. m=O). 131 (r=l. m=2). 188 (r=l. m=3). 245 (r=l. m=4) are achieved. Simple oligoamides also can be used. e.g.. mass-modifications of 74 (r=l . m=0). 88 (r=2. m=0). 102 (r=3. m=0). 1 16 (r=4. m=0). etc. are obtainable. For those skilled in the art. it will be obvious that there are numerous possibilities in addition to those mentioned above. As used herein, the superscript 0-i designates i -^ 1 mass differentiated nucleotides. primers or tags. In some instances, the superscript 0 can designate an unmodified species of a particular reactant. and the superscript i can designate the i-th mass- modified species of that reactant. If. for example, more than one species of nucleic acids are to be concurrently detected, then i + 1 different mass-modified detector oligonucleotides (D°. D- -...D-) can be used to distinguish each species of mass modified detector oligonucleotides (D) from the others by mass spectrometry. Different mass-modified detector oligonucleotides can be used to simultaneously detect all possible variants/mutants simultaneously (FIGURE 6B). Alternatively, all four base permutations at the site of a mutation can be detected by designing and positioning a detector oligonucleotide. so that it serves as a primer for a DNA/RNA polymerase (FIGURE 6C). For example, mass modifications also can be incoφorated during the amplification process. FIGURE 3 shows a different multiplex detection format, in which differentiation is accomplished by employing different specific capture sequences which are position-specifically immobilized on a flat surface (e.g. a 'chip array'). If different target sequences Tl - Tn are present, their target capture sites TCS1 - TCSn will specifically interact with complementary immobilized capture sequences Cl-Cn. Detection is achieved by employing appropriately mass differentiated detector oligonucleotides Dl - Dn. which are mass differentiated either by their sequences or by mass modifying functionalities Ml - Mn. Preferred mass spectrometer formats for use in the invention are matrix assisted laser desoφtion ionization (MALDI), electrospray (ES), ion cyclotron resonance (ICR) and Fourier Transform. For ES. the samples, dissolved in water or in a volatile buffer, are injected either continuously or discontinuously into an atmospheric pressure ionization interface (API) and then mass analyzed by a quadrupole. The generation of multiple ion peaks which can be obtained using ES mass spectrometry can increase the accuracy of the mass determination. Even more detailed information on the specific structure can be obtained using an MS/MS quadrupole configuration In MALDI mass spectrometry. various mass analyzers can be used. e.g.. magnetic sector/magnetic deflection instruments in single or triple quadrupole mode (MS/MS). Fourier transform and time-of-flight (TOF) configurations as is known in the art of mass spectrometry. For the desoφtion/ionization process, numerous matrix/laser combinations can be used. Ion-trap and reflectron configurations can also be employed. The mass spectrometric processes described above can be used, for example, to diagnose any of the more than 3000 genetic diseases currently known (e.g hemophilias, thalassemias. Duchenne Muscular Dystrophy (DMD). Huntington's Disease (HD). Alzheimer's Disease and Cystic Fibrosis (CF)) or to be identified. The following Example 3 provides a mass spectrometer method for detecting a mutation (ΔF508) of the cystic fibrosis transmembrane conductance regulator gene (CFTR). which differs by only three base pairs (900 daltons) from the wild type of CFTR gene. As described further in Example 3, the detection is based on a single-tube, competitive oligonucleotide single base extension (COSBE) reaction using a pair of primers with the 3'- terminal base complementary to either the normal or mutant allele. Upon hybridization and addition of a polymerase and the nucleoside triphosphate one base downstream, only those primers properly annealed (i.e.. no 3'-terminal mismatch) are extended: products are resolved by molecular weight shifts as determined by matrix assisted laser desoφtion ionization time- of-flight mass spectrometry. For the cystic fibrosis ΔF508 polymoφhism. 28-mer 'normal' (N) and 30-mer 'mutant' (M) primers generate 29- and 31-mers for N and M homozygotes. respectively, and both for heterozygotes. Since primer and product molecular weights are relatively low (<10 kDa) and the mass difference between these are at least that of a single - 300 Da nucleotide unit, low resolution instrumentation is suitable for such measurements. In addition to mutated genes, which result in genetic disease, certain birth defects are the result of chromosomal abnormalities such as Trisomy 21 (Down's Syndrome). Trisomy 13 (Patau Syndrome), Trisomy 18 (Edward's Syndrome), Monosomy X (Turner's Syndrome) and other sex chromosome aneuploidies such as Klienfelter's Syndrome (XXY). Further, there is growing evidence that certain DNA sequences may predispose an individual to any of a number of diseases such as diabetes, arteriosclerosis, obesity, various autoimmune diseases and cancer (e.g. colorectal, breast, ovarian, lung); chromosomal abnormality (either prenatally or postnatally); or a predisposition to a disease or condition (e.g. obesity, artherosclerosis. cancer). Also, the detection of "DNA fingeφrints". e.g. polymoφhisms, such as "microsatellite sequences", are useful for determining identity or heredity (e.g. paternity or maternity). The following Example 4 provides a mass spectometer method for identifying any of the three different isoforms of human apolipoprotein E. which are coded by the E2. E3 and E4 alleles. Here the molecular weights of DNA fragments obtained after restriction with appropriate restriction endonucleases can be used to detect the presence of a mutation. Depending on the biological sample, the diagnosis for a genetic disease, chromosomal aneuploidy or genetic predisposition can be preformed either pre- or post¬ natally. Viruses, bacteria, fungi and other infectious organisms contain distinct nucleic acid sequences, which are different from the sequences contained in the host cell. Detecting or quantitating nucleic acid sequences that are specific to the infectious organism is important for diagnosing or monitoring infection. Examples of disease causing viruses that infect humans and animals and which may be detected by the disclosed processes include: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV. See Ratner, L. et al.. Nature. Vol. 313. Pp. 227-284 ( 1985): Wain Hobson. S. et al. Cell. Vol. 40: Pp. 9-17 (1985)): HIV-2 (See Guyader et al.. Nature. Vol. 328, Pp. 662-669 (1987); European Patent Publication No. 0 269 520: Chakraborti et al.. Nature. Vol. 328. Pp. 543-547 (1987): and European Patent Application No. 0 655 501); and other isolates, such as HIV-LP (International Publication No. WO 94/00562 entitled "A Novel Human Immunodeficiency Virus": Picornaviridae (e.g.. polio viruses, hepatitis A virus. (Gust. I.D.. et al.. Intervirology. Vol. 20. Pp. 1 -7 (1983): entero viruses, human coxsackie viruses, rhinoviruses. echoviruses): Calcivi dae (e.g.. strains that cause gastroenteritis); Togaviridae (e.g.. equine encephalitis viruses, rubella viruses); Flaviridae (e.g.. dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reovihdae (e.g.. reoviruses. orbiviurses and rotaviruses); Birnaviridae: Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses): Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2. varicella zoster virus, cytomegalovirus (CMV), heφes viruses'); Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies. the agent of delta hepatities (thought to be a defective satellite of hepatitis B virus), the agents of non-A. non-B hepatitis (class 1 = internally transmitted; class 2 = parenterally transmitted (i.e.. Hepatitis C); Norwalk and related viruses, and astroviruses). Examples of infectious bacteria include: Helicobacter pylons, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis. M. avium. M. intracellulare. M. kansaii. M. gordonae). Staphylococcus aureus. Neisseria gonorrhoeae. Neisseria memngitidis. Listeria monocytogenes. Streptococcus pyogenes (Group A Streptococcus). Streptococcus agalactiae (Group B Streptococcus). Streptococcus (viridans group). Streptococcus faecalis. Streptococcus bovis. Streptococcus (anaerobic sps.). Streptococcus pneumoniae. pathogenic Campylobacter sp.. Enterococcus sp.. Haemophilus influenzae. Bacillus antracis. corynebacterium diphtheriae. corynebacterium sp., Erysipelothrix rhusiopathiae. Clostridium perfringers, Clostridium tetani. Enterobacter aerogenes. Klebsiella pneumoniae. Pasturella multocida. Bacteroides sp.. Fusobacterium nucleatum. Streptobacillus moniliformis. Treponema pallidium. Treponema pertenue. Leptospira. and Actinomyces israelli. Examples of infectious fungi include: Cryptococcus neoformans. Histoplasma capsulatum. Coccidioides immilis, Blastomyces dermatitidis.Chlamydia trachomatis. Candida albicans. Other infectious organisms (i.e.. protists) include: Plasmodium falciparum and Toxoplasma gondii. The following Example 5 provides a nested PCR and mass spectrometer based method that was used to detect hepatitis B virus (HBV) DNA in blood samples. Similarly, other blood-borne viruses (e.g.. HIV-1. HIV-2. hepatitis C virus (HCV). hepatitis A virus (HAV) and other hepatitis viruses (e.g.. non-A-non-B hepatitis, hepatitis G. hepatits E). cytomegalovirus. and heφes simplex virus (HSV)) can be detected each alone or in combination based on the methods described herein. Since the sequence of about 16 nucleotides is specific on statistical grounds (even for a genome as large as the human genome), relatively short nucleic acid sequences can be used to detect normal and defective genes in higher organisms and to detect infectious microorganisms (e.g. bacteria, fungi, protists and yeast) and viruses. DNA sequences can even serve as a fingeφrint for detection of different individuals within the same species. (Thompson. J.S. and M.W. Thompson, eds.. Genetics in Medicine. W.B. Saunders Co.. Philadelphia. PA (1986). One process for detecting a wildtype (Dwt) and/ or a mutant (Dmut) sequence in a target (T) nucleic acid molecule is shown in Figure lC. A specific capture sequence (C) is attached to a solid support (ss) via a spacer (S). In addition, the capture sequence is chosen to specifically interact with a complementary sequence on the target sequence (T). the target capture site (TCS) to be detected through hybridization. However, if the target detection site (TDS) includes a mutation. X, which increases or decreases the molecular weight, mutated TDS can be distinguished from wildtype by mass spectrometry. For example, in the case of an adenine base (dA) insertion, the difference in molecular weights between Dw* and Dmut would be about 314 daltons. Preferably, the detector nucleic acid (D) is designed such that the mutation would be in the middle of the molecule and the flanking regions are short enough so that a stable hybrid would not be formed if the wildtype detector oligonucleotide (Dwt) is contacted with the mutated target detector sequence as a control. The mutation can also be detected if the mutated detector oligonucleotide (Dmut) with the matching base at the mutated position is used for hybridization. If a nucleic acid obtained from a biological sample is heterozygous for the particular sequence (i.e. contain both Dwt and Dmut). both Dwt and Dmut will be bound to the appropriate strand and the mass difference allows both D 1 and Dmut to be detected simultaneous! v. The process of this invention makes use of the known sequence information of the target sequence and known mutation sites. Although new mutations can also be detected. For example, as shown in FIGURE 8. transcription of a nucleic acid molecule obtained from a biological sample can be specifically digested using one or more nucieases and the fragments captured on a solid support carrying the corresponding complementary nucleic acid sequences. Detection of hybridization and the molecular weights of the captured target sequences provide information on whether and where in a gene a mutation is present. Alternatively, DNA can be cleaved by one or more specific endonucleases to form a mixture of fragments. Comparison of the molecular weights between wildtype and mutant fragment mixtures results in mutation detection. The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, published patent applications (including international patent application Publication Number WO 94/16101. entitled DNA Sequencing by Mass Spectrometry by H. Koester; and international patent application Publication Number WO 94/21822 entitled "DNA Sequencing by Mass Spectrometry Via Exonuclease Degradation" by H. Koester), and co-pending patent applications, (including U.S Patent Application Serial No. 08/406.199. entitled DNA Diagnostics Based on Mass Spectrometry by H. Koester), as cited throughout this application are hereby expressly incoφorated by reference. Example 1 MALDI-TOF desoφtion of oligonucleotides directly on solid supports 1 g CPG (Controlled Pore Glass) was functional ized with 3-(triethoxysilyl)- epoxypropan to form OH-groups on the polymer surface. A standard oligonucleotide synthesis with 13 mg of the OH-CPG on a DNA synthesizer (Milligen. Model 7500) employing β-cyanoethyl-phosphoamidites (Koster et al.. Nucleic Acids Res..12, 4539 ( 1994)) and TAC N-protecting groups (Koster et al.. Tetrahedron, 21, 362 (1981)) was performed to synthesize a 3'-T5-50mer oligonucleotide sequence in which 50 nucleotides are complementary to a "hypothetical" 50mer sequence. T5 serves as a spacer. Deprotection with saturated ammonia in methanol at room temperature for 2 hours furnished according to the determination of the DMT group CPG which contained about 10 umol 55mer/g CPG. This 55mer served as a template for hybridizations with a 26mer (with 5'-DMT group) and a 40mer (without DMT group). The reaction volume is 100 ul and contains about lnmol CPG bound 55mer as template, an equimolar amount of oligonucleotide in solution (26mer or 40mer) in 20mM Tris-HCI. pH 7.5. 10 mM MgCI and 25mM NaCI. The mixture was heated for 10' at 65°C and cooled to 37°C during 30' (annealing). The oligonucleotide which has not been hybridized to the polymer-bound template were removed by centrifugation and three subsequent washing/centrifugation steps with 100 ul each of ice-cold 50mM ammoniumcitrate. The beads were air-dried and mixed with matrix solution (3- hydroxypicolinic acid/lOmM ammonium citrate in acetonitril/water. 1 :1 ). and analyzed by MALDI-TOF mass spectrometry. The results are presented in Figures 10 and 1 1 Example 2 F.lectrosprav (ES) desoφtion and differentiation of an 1 -mer and 19-mer DNA fragments at a concentration of 50 pmole/ul in 2-propanol/10mM ammoniumcarbonate ( 1/9. v/v) were analyzed simultaneously by an electrospray mass spectrometer. The successful desoφtion and differentiation of an 18-mer and 19-mer by electrospray mass spectrometry is shown in FIGURE 12. Example 3 Detection of The Cvstic Fibrosis Mutation. ΔF508. hv single step dideoxv extension and analysis bv MALDI-TOF mass spectrometry MATERIALS AND METHODS PCR Amplification and Strand Immobilization. Amplification was carried out with exon 10 specific primers using standard PCR conditions (30 cycles: l'@95°C, l'@55°C. 2'@72°C); the reverse primer was 5' labelled with biotin and column purified (Oligopurification Cartridge, Cruachem). After amplification the PCR products were purified by column separation (Qiagen Quickspin) and immobilized on streptavidin coated magnetic beads (Dynabeads. Dynal. Norway) according to their standard protocol; DNA was denatured using 0.1M NaOH and washed with 0.1M NaOH. IxB+W buffer and TE buffer to remove the non-biotinylated sense strand. COSBE Conditions. The beads containing ligated antisense strand were resuspended in 18μl of Reaction mix 1 (2 μl 10X Taq buffer. 1 μL (1 unit) Taq Polymerase. 2 μL of 2 mM dGTP. and 13 μL H2O) and incubated at 80°C for 5' before the addition of Reaciton mix 2 (100 ng each of COSBE primers). The temperature was reduced to 60°C and the mixtures incubated for a 5' annealing/extension period: the beads were then washed in 25mM triethylammonium acetate (TEAA) followed by 50mM ammonium citrate. Primer Sequences. All primers were synthesized on a Perseptive Biosystems Expedite 8900 DNA Synthesizer using conventional phosphoramidite chemistry (Sinha et al. (1984) Nucleic Acids Res. 72:4539. COSBE primers (both containing an intentional mismatch one base before the 3 '-terminus) were those used in a previous ARMS study (Ferrie et al.. ( 1992) Am J Hum Genet 57:251 -262) with the exception that two bases were removed from the 5'-end of the normal: ExlO PCR (Forward): 5 -BIO-GCA AGT GAA TCC TGA GCG TG-3' (SEQ ID No. 1 ) Ex 10 PCR (Reverse): 5'-GTG TGA AGG GTT CAT ATG C-3' (SEQ ID No. 2) COSBE ΔF508-N 5'-ATC TAT ATT CAT CAT AGG AAA CAC CAC A-3 * (28-mer) (SEQ ID No. 3) COSBE ΔF508-M 5'-GTA TCT ATA TTC ATC ATA GGA AAC ACC ATT-3' (30-mer) (SEQ ID No. 4) Mass Spectrometry: After washing, beads were resuspended in 1 μL 18 Mohm/cm H2O. 300 nL each of matrix (Wu et al.. 1993) solution (0.7 M 3-hydroxypicolinic acid. 0.7 M dibasic ammonium citrate in 1 : 1 H2θ:CH3CN) and resuspended beads (Tang et al. ( 1995) Rapid Commun Mass Spectrom 8:121-130) were mixed on a sample target and allowed to air dry. Up to 20 samples were spotted on a probe target disk for introduction into the source region of an unmodified Thermo Bioanalysis (formerly Finnigan) Visions 2000 MALDI-TOF operated in reflectron mode with 5 and 20 kV on the target and conversion dynode. respectively. Theoretical average molecular weights (M^calc)) were calculated from atomic compositions. Vendor provided software was used to determine peak centroids using external calibration; 1.08 Da has been subtracted from these to correct for the charge carrying proton mass to yield the text M r (exp) values. Scheme. Upon annealing to the bound template, the N and M primers (8508.6 and 9148.0 Da. respectively) are presented with dGTP; only primers with proper Watson- Crick base paring at the variable (V) position are extended by the polymerase. Thus if V pairs with the 3'-terminal base of N. N is extended to a 8837.9 Da product (N+l ). Likewise, if V is properly matched to the M terminus. M is extended to a 9477.3 Da M+l product. Results Figures 14 - 18 show the representative mass spectra of COSBE reaction products. Better results were obtained when PCR products were purified before the biotinylated anti-sense strand was bound Example 4 Differentiation of Human Apolipoprotein E Isoforms hv Mass Spectrometry Apolipoprotein E (Apo E). a protein component of lipoproteins. plays an essential role in lipid metabolism. For example, it is involved with cholesterol transport. metabolism of lipoprotein particles, immunoregulation and activation of a number of lipolytic enzymes. There are three common isoforms of human Apo E (coded by E2. E3 and E4 alleles). The most common is the E3 allele. The E2 allele has been shown to decrease the cholesterol level in plasma and therefore may have a protective effect against the development of atherosclerosis. Finally, the E4 isoform has been correlated with increased levels of cholesterol, conferring predisposition to atherosclerosis. Therefore, the identity of the apo E allele of a particular individual is an important determinant of risk for the development of cardiovascular disease. As shown in Figure 19. a sample of DNA encoding apolipoprotein E can be obtained from a subject, amplified (e.g. via PCR); and the PCR product can be digested using an appropriate enzyme (e.g. Cfol). The restriction digest obtained can then be analyzed by a variety of means. As shown in Figure 20. the three isotypes of apolipoprotein E (E2. E3 and E4 have different nucleic acid sequences and therefore also have distinguishable molecular weight values. As shown in Figure 21 A-C, different Apolipoprotein E genotypes exhibit different restriction patterns in a 3.5% MetPhor Agarose Gel or 12% polyacrylamide gel. As shown in Figures 22 and 23, the various apolipoprotein E genotypes can also be accurately and rapidly determined by mass spectrometry. Example 5 Detection of hepatitis B virus in serum samples. MATERIALS AND METHODS Sample preparation Phenol/choloform extraction of viral DNA and the final ethanol precipitation was done according to standard protocols. First PCR: Each reaction was performed with 5μl of the DNA preparation from serum. 15 pmol of each primer and 2 units Taq DNA polymerase (Perkin Elmer. Weiterstadt. Germany) were used. The final concentration of each dNTP was 200μM. the final volume of the reaction was 50 μl. lOx PCR buffer (Perkin Elmer. Weiterstadt. Germany) contained 100 mM Tris-HCl . pH 8.3. 500 mM KC1. 15 mM MgCh. 0.01% gelatine (w/v). Primer sequences: Primer 1 : 5'-GCTTTGGGGCATGGACATTGACCCGTATAA- 3 ' ( SEQ ID NO . 5 ) Primer 2: 5'-CTGACTACTAATTCCCTGGATGCTGGGTCT-3 ' (SEQ ID NO . 6 ) Nested PCR: Each reaction was performed either with 1 μl of the first reaction or with a 1 : 10 dilution of the first PCR as template, respectively. 100 pmol of each primer. 2.5 u Pfu(exo-) DNA polymerase (Stratagene. Heidelberg, Germany), a final concentration of 200 μM of each dNTPs and 5 μl lOx Pfu buffer (200 mM Tris-HCl. pH 8.75. 100 mM KC1. 100 mM (NH4)2SO 20 mM MgSO 4 . 1% Triton X-100. lmg/ml BSA. (Stratagene. Heidelberg. Germany) were used in a final volume 50 μl. The reactions were performed in a thermocycler (OmniGene. MWG-Biotech. Ebersberg. Germany) using the following program: 92°C for 1 minute. 60°C for 1 minute and 72°C for 1 minute with 20 cycles. Sequence of oligodeoxynucleotides (purchased HPLC-purified at MWG-Biotech. Ebersberg. Germany): HBV13: 5'-TTGCCTGAGTGCAGTATGGT- 3 ' (SEQ ID NO . 7 ) HBV15bio: Biotin-5' -AGCTCTATATCGGGAAGCCT- 3 ' (SEQ ID NO . 8 ) Purification of PCR products: For the recording of each spectrum, one PCR, 50 μl, (performed as described above) was used. Purification was done according to the following procedure: Ultrafiltration was done using Ultrafree-MC filtration units (Millipore. Eschborn, Germany) according to the protocol of the provider with centrifugation at 8000 φm for 20 minutes. 25μl (lOμg/μl) streptavidin Dynabeads (Dynal, Hamburg. Germany) were prepared according to the instructions of the manufacturer and resuspended in 25μl of B/W buffer (10 mM Tris-HCl. pH7.5, ImM EDTA. 2 M NaCl). This suspension was added to the PCR samples still in the filtration unit and the mixture was incubated with gentle shaking for 15 minutes at ambient temperature. The suspension was transferred in a 1.5 ml Eppendorf tube and the supernatant was removed with the aid of a Magnetic Particle Collector. MPC. (Dynal. Hamburg, Germany). The beads were washed twice with 50 μl of 0.7 M ammonium citrate solution. pH 8.0 (the supernatant was removed each time using the MPC). Cleavage from the beads can be accomplished by using formamide at 90°C. The supernatant was dried in a speedvac for about an hour and resuspended in 4 μl of ultrapure water (MilliQ UF plus Millipore. Eschborn. Germany). This preparation was used for MALDI-TOF MS analysis. MΛLD1-TQF MS; Half a microliter of the sample was pipetted onto the sample holder, then immediately mixed with 0.5 μl matrix solution (0.7 M3-hydroxypicolinic acid 50% acetonitrile. 70 mM ammonium citrate). This mixture was dried at ambient temperature and introduced into the mass spectrometer. All spectra were taken in positive ion mode using a Finnigan MAT Vision 2000 (Finnigan MAT. Bremen. Germany), equipped with a reflectron (5 keV ion source. 20 keV postacceleration) and a 337 nm nitrogen laser. Calibration was done with a mixture of a 40mer and a 1 OOmer. Each sample was measured with different laser energies. In the negative samples, the PCR product was detected neither with less nor with higher laser energies. In the positive samples the PCR product was detected at different 5 places of the sample spot and also with varying laser energies. Results A nested PCR system was used for the detection of HBV DNA in blood 10 samples employing oligonucleotides complementary to the c region of the HBV genome (primer 1 : beginning at map position 1763, primer 2 beginning at map position 2032 of the complementary strand) encoding the HBV core antigen (HBVcAg). DNA was isolated from patients serum according to standard protocols. A first PCR was performed with the DNA from these preparations using a first set of primers. If HBV DNA was present in the sample a 15 DNA fragment of 269 bp was generated. In the second reaction, primers which were complementary to a region within the PCR fragment generated in the first PCR were used. If HBV related PCR products were present in the first PCR a DNA fragment of 67 bp was generated (see Fig. 25A) in this nested 0 PCR. The usage of a nested PCR system for detection provides a high sensitivity and also serves as a specificity control for the external PCR (Rolfs. A. et al.. PCR: Clinical Diagnostics and Research, Springer. Heidelberg, 1992). A further advantage is that the amount of fragments generated in the second PCR is high enough to ensure an unproblematic detection although purification losses can not be avoided. 25 The samples were purified using ultrafiltration to remove the primers prior to immobilization on streptavidin Dynabeads. This purification was done because the shorter primer fragments were immobilized in higher yield on the beads due to steric reasons. The immobilization was done directly on the ultrafiltration membrane to avoid substance losses 30 due to unspecific absoφtion on the membrane. Following immobilization, the beads were washed with ammonium citrate to perform cation exchange (Pieles. U. et al.. (1993) Nucleic Acids Res 21 :3191-3196). The immobilized DNA was cleaved from the beads using 25% ammonia which allows cleavage of DNA from the beads in a very short time, but does not result in an introduction of sodium cations. _ The nested PCRs and the MALDI TOF analysis were performed without knowing the results of serological analysis. Due to the unknown virus titer. each sample of the first PCR was used undiluted as template and in a 1 :10 dilution, respectively. Sample 1 was collected from a patient with chronic active HBV infection who was positive in HBs- and HBe-antigen tests but negative in a dot blot analysis. Sample 2 was a serum sample from a patient with an active HBV infection and a massive viremia who was HBV positive in a dot blot analysis. Sample 3 was a denatured serum sample therefore no serologicial analysis could be performed but an increased level of transaminases indicating liver disease was detected. In autoradiograph analysis (Figure 24), the first PCR of this sample was negative. Nevertheless, there was some evidence of HBV infection. This sample is of interest for MALDI-TOF anlaysis. because it demonstrates that even low-level amounts of PCR products can be detected after the purification procedure. Sample 4 was from a patient who was cured of HBV infection. Samples 5 and 6 were collected from patients with a chronic active HBV infection. Figure 24 shows the results of a PAGE analysis of the nested PCR reaction. A PCR product is clearly revealed in samples 1, 2, 3, 5 and 6. In sample 4 no PCR product was generated, it is indeed HBV negative, according to the serological analysis. Negative and positive controls are indicated by + and -. respectively. Amplification artifacts are visible in lanes 2, 5, 6 and + if non-diluted template was used. These artifacts were not generated if the template was used in a 1 : 10 dilution. In sample 3. PCR product was only detectable if the template was not diluted. The results of PAGE analysis are in agreement with the data obtained by serological analysis except for sample 3 as discussed above. Figure 25 A shows a mass spectrum of a nested PCR product from sample number 1 generated and purified as described above. The signal at 20754 Da represents the single stranded PCR product (calculated: 20735 Da, as the average mass of both strands of the PCR product cleaved from the beads). The mass difference of calculated and obtained mass is 19 Da (0.09%). As shown in Fig. 25 A. sample number 1 generated a high amount of PCR product, resulting in an unambiguous detection. Fig. 25B shows a spectrum obtained from sample number 3. As depicted in Fig. 24. the amount of PCR product generated in this section is significantly lower than that from sample number 1. Nevertheless, the PCR product is clearly revealed with a mass of 20751 Da (calculated 20735). The mass difference is 16 Da (0.08%). The spectrum depicted in Fig. 25C was obtained from sample number 4 which is HBV negative (as is also shown in Fig 24). As expected no signals corresponding to the PCR product could be detected. All samples shown in Fig. 25 were analyzed with MALDI-TOF MS. whereby PCR product was detected in all HBV positive samples, but not in the HBV negative samples. These results were reproduced in several independent experiments. Example 6 Analysis of l igase Chain Reaction Products Via MALDI-TOF Mass Spectrp ctry MATERIALS AND METHODS Oligodeoxynucleotides Except the biotinylated one and all other oligonucleotides were synthesized in a 0.2 μmol scale on a MilliGen 7500 DNA Synthesizer (Millipore. Bedford. MA. USA) using the β-cyanoethylphosphoamidite method (Sinha. N.D. et al.. (1984) Nucleic Acids Res.. Vol. 12, Pp. 4539-4577). The oligodeoxynucleotides were RP-HPLC-purified and deprotected according to standard protocols. The biotinylated oligodeoxynucleotide was purchased (HPLC-purified) from Biometra. Gottingen. Germany). Sequences and calculated masses of the oligonucleotides used: Oligodeoxynucleotide A: 5 ' -p-TTGTGCCACGCGGTTGGGAATGTA (7521 Da)(SEQ ID No. 9) Oligodeoxynucleotide B: 5 ' -p-AGCAACGACTGTTTGCCCGCCAGTTG (7948 Da) (SEQ ID No. 10) Oligodeoxynucleotide C: 5 ' -bio-TACATTCCCAACCGCGTGGCACAAC (7960 Da) (SEQ ID No. 1 1) Oligodeoxynucleotide D: 5 ' -p-AACTGGCGGGCAAACAGTCGTTGCT (7708 Da) (SEQ ID No. 12) 5'-Phosphorylation of oligonucleotides A and D This was performed with polynucleotide kinase (Boehringer. Mannheim. German) according to published procedures, the 5'-phosphorylated oligonucleotides were used unpurified for LCR. Ligase chain reaction The LCR was performed with Pfu DNA ligase and a ligase chain reaction kit (Stratagene. Heidelberg. Germany) containing two different pBluescript KII phagemids. One carrying the wildtype form of the E.coli lacl gene and the other one a mutant of this gene with a single point mutation at bp 191 of the lacl gene. The following LCR conditions were used for each reaction: 100 pg template DNA (0.74 fmol) with 500 pg sonified salmon sperm DNA as carrier. 25 ng (3.3 pmol) of each 5'-phosphorylated oligonucleotide. 20 ng (2.5 pmol) of each non-phosphorylated oligonucleotide. 4 U Pfu DNA ligase in a final volume of 20 μl buffered by Pfu DNA ligase reaction buffer (Stratagene. Heidelberg. Germany). In a model experiment a chemically synthesized ss 50-mer was used (1 fmol) as template, in this case oligo C was also biotinylated. All reactions were performed in a thermocycler (OmniGene. MWG-Biotech. Ebersberg. Germany) with the following program: 4 minutes 92°C. 2 minutes 60°C and 25 cycles of 20 seconds 92°C, 40 seconds 60°C. Except for HPLC analysis the biotinylated ligation educt C was used. In a control experiment the biotinylated and non-biotinylated oligonucleotides revealed the same gel electrophoretic results. The reactions were analyzed on 7.5% polyacrylamide gels. Ligation product 1 (oligo A and B) calculated mass: 15450 Da, ligation product 2 (oligo C and D) calculated mass: 15387 Da. SMART-HP LC Ion exchange HPLC (IE HPLC) was performed on the SMART-system (Pharmacia. Freiburg, Germany) using a Pharmacia Mono Q, PC 1.6/5 column. Eluents were buffer A (25 mM Tris-HCl, 1 mM EDTA and 0.3 M NaCl at pH 8.0) and buffer B (same as A. but 1 M NaCl). Starting with 100% A for 5 minutes at a flow rate of 50 μl/min. a gradient was applied from 0 to 70% B in 30 minutes, then increased to 100% B in 2 minutes and held at 100% B for 5 minutes. Two pooled LCR volumes (40 μl) performed with either wildtype or mutant template were injected. Sample preparation for MALD1-TOF-MS Preparation of immobilized DNA: For the recording of each spectrum two LCRs (performed as described above) were pooled and diluted 1 : 1 with 2x B/W buffer ( 10 mM Tris-HCl, pH 7.5, ImM EDTA. 2 M NaCl). To the samples 5 μl streptavidin DynaBeads (Dynal. Hamburg, Germany) were added, the mixture was allowed to bind with gentle shaking for 15 minutes at ambient temperature. The supernatant was removed using a Magnetic Particle Collector. MPC, (Dynal. Hamburg. Germany) and the beads were washed twice with 50 μl of 0.7 M ammonium citrate solution (pH 8.0) (the supernatant was removed each time using the MPC). The beads were resuspended in l μl of ultrapure water (MilliQ, Millipore. Bedford. MA. USA). This suspension was directly used for MALDI-TOF-MS analysis as described below. Combination of ultrafiltration and streptavidin DynaBeads: For the recording of spectrum two LCRs (performed as described above) were pooled, diluted 1 : 1 with 2x B/W buffer and concentrated with a 5000 NMWL Ultrafree-MC filter unit (Millipore. Eschborn. Germany) according to the instructions of the manufacturer. After concentration the samples were washed with 300 μl lx B/W buffer to streptavidin DynaBeads were added. The beads were washed once on the Ultrafree-MC filtration unit with 300 μl of lx B/W buffer and processed as described above. The beads were resuspended in 30 to 50 μl of lx B'W buffer and transferred in a 1.5 ml Eppendorf tube. The supernatant was removed and the beads were washed twice with 50 μl of 0.7 M ammonium citrate (pH 8.0). Finally, the beads were washed once with30 μl of acetone and resuspended in 1 μl of ultrapure water. The ligation mixture after immobilization on the beads was used for MALDS-TOF-MS analysis as described below. MALDI-TOF-MS A suspension of streptavi din-coated magnetic beads with the immobilized DNA was pipetted onto the sample holder, then immediately mixed with 0.5 μl matrix solution (0.7 M 3-hydroxypicolinic acid in 50% acetonitrile. 70 mM ammonium citrate). This mixture was dried at ambient temperature and introduced into the mass spectrometer. All spectra were taken in positive ion mode using a Finnigan MAT Vision 2000 (Finnigan MAT. Bremen. Germany), equipped with a reflectron (5 keV ion source. 20 keV postacceleration) and a nitrogen laser (337 run). For the analysis of Pfu DNA ligase 0.5 μl of the solution was mixed on the sample holder with 1 μl of matrix solution and prepared as described above. For the analysis of unpurified LCRs 1 μl of an LCR was mixed wiϋ 1 μl matrix solution. RESULTS AND DISCUSSION The E. coli lacl gene served as a simple model system to investigate the suitability of MALDI-TOF-MS as detection method for products generated in ligase chain reactions. This template system consists of an E.coli lacl wildtype gene in a pBluescript KII phagemid and an E. coli lacl gene carrying a single point mutation at bp 191 (C to T transition) in the same phagemid. Four different oligonucleotides were used, which were ligated only if the £. coli lacl wildtype gene was present (Figure 26). LCR conditions were optimized using Pfu DNA ligase to obtain at least 1 pmol ligation product in each positive reaction. The ligation reactions were analyzed by polyacrylamide gel electrophoresis (PAGE) and HPLC on the SMART system (Figures 27. 28 and 29). Figure 27 shows a PAGE of a positive LCR with wildtype template (lane 1 ). a negative LCR with mutant template ( 1 and 2) and a negative control which contains enzyme. oligonucleotides and no template. The gel electrophoresis clearly shows that the ligation product (50bp) was produced only in the reaction with wildtype template whereas neither the template carrying the point mutation nor the control reaction with salmon sperm DNA generated amplification products. In Figure 28. HPLC was used to analyze two pooled LCRs with wildtype template performed under the same conditions. The ligation product was clearly revealed. Figure 29 shows the results of a HPLC in which two pooled negative LCRs with mutant template were analyzed. These chromatograms confirm the data shown in Figure 27 and the results taken together clearly demonstrate, that the system generates ligation products in a significant amount only if the wildtype template is provided. Appropriate control runs were performed to determine retention times of the different compounds involved in the LCR experiments. These include the four oligonucleotides (A, B, C. and D). a synthetic ds 50-mer (with the same sequence as the ligation product), the wildtype template DNA. sonicated salmon sperm DNA and the Pfu DNA ligase in ligation buffer. In order to test which purification procedure should be used before a LCR reaction can be analyzed by MALDI-TOF-MS. aliquots of an unpurified LCR (Figure 30A) and aliquots of the enzyme stock solution (Figure 30B) were analyzed with MALDI-TOF- MS. It turned out that appropriate sample preparation is absolutely necessary since all signals in the unpurified LCR correspond to signals obtained in the MALDI-TOF-MS analysis of the Pfu DNA ligase. The calculated mass values of oligo A and the ligation product are 7521 Da and 15450 Da. respectively. The data in Figure 30 show that the enzyme solution leads to mass signals which do interfere with the expected signals of the ligation educts and products and therefore makes an unambiguous signal assignment impossible. Furthermore, the spectra showed signals of the detergent Tween20 being part of the enzyme storage buffer which influences the crystallization behavior of the analyte/matrix mixture in an unfavorable way. In one purification format streptavidin-coated magnetic beads were used. As was shown in a recent paper, the direct desoφtion of DNA immobilized by Watson-Crick base pairing to a complementary DNA fragment covalently bound to the beads is possible and the non-biotinylated strand will be desorbed exclusively (Tang. K et al., (1995) Nucleic Acids Res. 25:3126-3131). This approach in using immobilized ds DNA ensures that only the non-biotinylated strand will be desorbed. If non-immobilized ds DNA is analyzed both strands are desorbed (Tang, K. et. al., ( 1994) Rapid Comm. Mass Spectrom. 7: 183- 186) leading to broad signals depending on the mass difference of the two strands. Therefore, employing this system for LCR only the non-ligated oligonucleotide A. with a calculated mass of 7521 Da. and the ligation product from oligo A and oligo B (calculated mass: 15450 Da) will be desorbed if oligo C is biotinylated at the 5'-end and immobilized on steptavidin- coated beads. This results in a simple and unambiguous identification of the LCR educts and products. Figure 31 A shows a MALDI-TOF mass spectrum obtained from two pooled LCRs (performed as described above) purified on streptavidin DynaBeads and desorbed directly from the beads showed that the purification method used was efficient (compared with Figure 30). A signal which represents the unligated oligo A and a signal which corresponds to the ligation product could be detected. The agreement between the calculated and the experimentally found mass values is remarkable and allows an unambiguous peak assignment and accurate detection of the ligation product. In contrast, no ligation product but only oligo A could be detected in the spectrum obtained from two pooled LCRs with mutated template (Figure 3 IB). The specificity and selectivity of the LCR conditions and the sensitivity of the MALDI-TOF detection is further demonstrated when performing the ligation reaction in the absence of a specific template. Figure 32 shows a spectrum obtained from two pooled LCRs in which only salmon sperm DNA was used as a negative control, only oligo A could be detected, as expected. While the results shown in Figure 31 A can be correlated to lane 1 of the gel in Figure 27. the spectrum shown in Figure 3 IB is equivalent to lane 2 in Figure 27. and finally also the spectrum in Figure 32 corresponds to lane 3 in Figure 27. The results are in congruence with the HPLC analysis presented in Figures 28 and 29. While both gel electrophoresis (Figure 27) and HPLC (Figures 28 and 29) reveal either an excess or almost equal amounts of ligation product over ligation educts. the analysis by MALDI-TOF mass spectrometry produces a smaller signal for the ligation product (Figure 31 A). The lower intensity of the ligation product signal could be due to different desoφtion/ionization efficiencies between 24- and a 50-mer. Since the T m value of a duplex with 50 compared to 24 base pairs is significantly higher, more 24-mer could be desorbed. A reduction in signal intensity can also result from a higher degree of fragmentation in case of the longer oligonucleotides. Regardless of the purification with streptavidin DynaBeads. Figure 32 reveals traces of Tween20 in the region around 2000 Da. Substances with a viscous consistence, negatively influence the process of crystallization and therefore can be detrimental to mass spectrometer analysis. Tween20 and also glycerol which are part of enzyme storage buffers therefore should be removed entirely prior to mass spectrometer analysis. For this reason an improved purification procedure which includes an additional ultrafiltration step prior to treatment with DynaBeads was investigated. Indeed, this sample purification resulted in a significant improvement of MALDI-TOF mass spectrometric performance. Figure 33 shows spectra obtained from two pooled positive (33A) and negative (33B) LCRs. respectively. The positive reaction was performed with a chemically synthesized, single strand 50mer as template with a sequence equivalent to the ligation product of oligo C and D. Oligo C was 5'-biotinylated. Therefore the template was not detected. As expected, only the ligation product of Oligo A and B (calculated mass 15450 Da) could be desorbed from the immobilized and ligated oligo C and D. This newly generated DNA fragment is represented by the mass signal of 15448 Da in Figure 33A. Compared to Figure 32A. this spectrum clearly shows that this method of sample preparation produces signals with improved resolution and intensity. Example 7 Mutation detection bv solid phase oligo base extension of a primer and analysis hv MALDI-TOF mass spectrometry Summary The solid-phase oligo base extension method detects point mutations and small deletions as well as small insertions in amplified DNA. The method is based on the extension of a detection primer that anneals adjacent to a variable nucleotide position on an affinity-captured amplified template, using a DNA polymerase. a mixture of three dNTPs. and the missing one didesoxy nucleotide. The resulting products are evaluate and resolved by MALDI-TOF mass spectrometry without further labeling procedures. The aim of the following experiment was to determine mutant and wildtype alleles in a fast and reliable manner. Description of the experiment The method used a single detection primer followed by a oligonucleotide extension step to give products differing in length by some bases specific for mutant or wildtype alleles which can be easily resolved by MALDI-TOF mass spectrometry. The method is described by using an example the exon 10 of the CFTR-gene. Exon 10 of this gene bears the most common mutation in many ethnic groups (ΔF508) that leads in the homozygous state to the clinical phenotype of cystic fibrosis. MATERIALS AND METHODS Genomic DNA Genomic DNA were obtained from healthy individuals, individuals homozygous or heterozygous for the ΔF508 mutation, and one individual heterozygous for the 1506S mutation. The wildtype and mutant alleles were confirmed by standard Sanger sequencing. PCR amplification of exon I O of the CFTR gene The primers for PCR amplification were CFExlO-F (5- GCAAGTGAATCCTGAGCGTG-3' (SEQ ID No. 13) located in intron 9 and biotinylated) and CFExl O-R (5 -GTGTGAAGGGCGTG-3'. (SEQ ID No. 14) located in intron 10). Primers were used in a concentration of 8 pmol. Taq-polymerase including lOx buffer were purchased from Boehringer-Mannheim and dTNPs were obtained from Pharmacia. The total reaction volume was 50 μl. Cycling conditions for PCR were initially 5 min. at 95°C. followed by 1 min. at 94°C. 45 sec at 53°C. and 30 sec at 72°C for 40 cycles with a final extension tim eof 5 min at 72°C. Purification of the PCR products Amplification products were purified by using Qiagen's PCR purification kit (No. 28106) according to manufacturer's instructions. The elution of the purified products s from the column was done in 50 μl TE-buffer ( 1 OmM Tris. 1 mM EDTA. pH 7.5). Affinity-capture and denaturation of the double stranded DNA 10 μL aliquots of the purified PCR product were transferred to one well of a streptavidin-coated microtiter plate (No. 1645684 Boehringer-Mannheim or Noo. 95029262 0 Labsystems). Subsequently. 10 μl incubation buffer (80 mM sodium phosphate. 400 mM NaCl, 0.4% Tween20, pH 7.5) and 30 μl water were added. AFter incubation for 1 hour at room temperature the wells were washed three times with 200μl washing buffer (40 mM Tris. 1 mM EDTA. 50 mM NaCl. 0.1% Tween 20. pH8.8). To denaturate the double stranded DNA the wells were treated with 100 μl of a 50 mM NaOH solution for 3 min. Hence, the 5 wells were washed three times with 200 μl washing buffer. Oligo base extension reaction The annealing of 25 pmol detection primer (CF508: 5'CTATATTCATCATAGGAAACACCA-3' (SEQ ID No. 15) was performed in 50 μl 0 annealing buffer (20 mM Tris. 10 mM KC 1 , 10 mM (NH4 2SO4, 2 mM MgSO, 1 % Triton X-100, pH 8, 75) at 50°C for 10 min. The wells were washed three times with 200 μl washing buffer and oncein 200 μl TE buffer. The extension reaction was performed by using some components of the DNA sequencing kit from USB (No. 70770) and dNTPs or ddNTPs from Pharmacia. The total reaction volume was 45 μl. consisting of 21 μl water. 6 μl 5 Sequenase-buffer, 3 μl 10 mM DTT solution.4.5 μl. 0.5 mM of three dNTPs, 4.5 μl. 2 mM the missing one ddNTP. 5.5 μl glycerol enzyme diluton buffer. 0,25 μl Sequenase 2.0. and 0.25 pyrophosphatase. The reaction was pipetted on ice and then incubated for 15 min at room temperature and for 5 min at 37°C. Hence, the wells were washed three times with 200 μl washing buffer and once with 60 μl of a 70 mM NH 4 -Citrate solution. 0 Denaturation and precipitation of the extended primer The extended primer was denatured in 50 μl 10%-DMSO (dimethylsufoxide) in water at 80°C for 10 min. For precipitation. 10 μl NH4-Acetat (pH 6.5). 0.5 μl glycogen (10 mg/ml water. Sigma No. Gl 765), and 100 μl absolute ethanol were added to the 5 supernatant and incubated for 1 hour at room temperature. After centrifugation at 13.000 g for 10 min the pellet was washed in 70% ethanol and resuspended in 1 μl 18 Mohm/cm H^O water. Sample preparation and analysis on MALDI-TOF mass spectrometry Sample preparation was performed by mixing 0.3 μl of each of matrix solution (0.7 M 3-hydroxypicolinic acid. 0.07 M dibasic ammonium citrate in 1 :1 H 2 θ:CH 3 CN) and of resuspended DNA/glycogen pellet on a sample target and allowed to air dry. Up to 20 samples were spotted on a probe target disk for introduction into the source region of an unmodified Thermo Bioanalysis (formerly Finnigan) Visions 2000 MALDI-TOF operated in reflectron mode with 5 and 20 kV on the target and conversion dynode. respectively. Theoretical average molecular mass (M r (calc)) were calculated from atomic compositions: reported experimental Mr (M r (exp)) values are those of the singly-protonated form, determined using external calibration. RESULTS The aim of the experiment was to develop a fast and reliable method independent of exact stringencies for mutation detection that leads to high quality and high throughput in the diagnosis of genetic diseases. Therefore a special kind of DNA sequencing (oligo base extension of one mutation detection primer) was combined with the evaluation of the resulting mini-sequencing products by matrix-assisted laser desoφtion ionization (MALDI) mass spectrometry (MS). The time-of-flight (TOF) reflectron arrangement was chosen as a possible mass measurement system. To prove this hypothesis, the examination was performed with exon 10 of the CFTR-gene. in which some mutations could lead to the clinical phenotype of cystic fibrosis. the most common monogenetic disease in the Caucasian population. The schematic presentation as given in Figure 34 shows the expected short sequencing products with the theoretically calculated molecular mass of the wildtype and various mutations of exon 10 of the CFTR-gene. The short sequencing products were produced using either ddTTP (Figure 34A) or ddCTP (Figure 34B) to introduce a definitive sequence related stop in the nascent DNA strand. The MALDI-TOF-MS spectra of healthy, mutation heterozygous, and mutation homozygous individuals are presented in Figure 34. All samples were confirmed by standard Sanger sequencing which showed no discrepancy in comparison to the mass spec analysis. The accuracy of the experimental measurements of the various molecular masses was within a range of minus 21.8 and plus 87.1 dalton (Da) to the range expected. This is a definitive inteφretation of the results allowed in each case. A further advantage of this procedure is the unambiguous detection of the ΔI507 mutation. In the ddTTP reaction, the wildtype allele would be detected, whereas in the ddCTP reaction the three base pair deletion would be disclosed. The method described is highly suitable for the detection of single point mutations or microlesions of DNA. Careful choice of the mutation detection primers will open the window of multiplexing and lead to a high throughput including high quality in genetic diagnosis without any need for exact stringencies necessary in comparable allele- specific procedures. Because of the uniqueness of the genetic information, the oligo base extension of mutation detection primer is applicable in each disease gene or polymoφhic region in the genome like variable number of tandem repeats (VNTR) or other single nucleotide polymoφhisms (e.g.. apolipoprotein E gene). Example 8: Detection of Polymerase Chain Reaction Products Containing 7- Deazapurine Moieties with Matrix-Assisted Laser Desoφtion/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry MATERIALS AND METHODS PCR amplifications The following oligodeoxynucleotide primers were either synthesized according to standard phosphoamidite chemistry (Sinha. N.D.. et al.. (1983) Tetrahedron Let. Vol. 24. Pp. 5843-5846: Sinha. N.D.. et al., (1984) Nucleic Acids Res., Vol. 12, Pp. 4539- 4557) on a MilliGen 7500 DNA synthesizer (Millipore. Bedford. MA, USA) in 200 nmol scales or purchased from MWG-Biotech (Ebersberg, Germany, primer 3) and Biometra (Goettingen. Germany, primers 6-7). primer 1 5'-GTCACCCTCGACCTGCAG(SEQ.ID.NO.16): primer 2 5'-TTGTAAAACGACGGCCAGT(SEQ.ID.NO.17); primer 3 5'-CTTCCACCGCGATGTTGA(SEQ.ID.NO.18); primer 4 5'-CAGGAAACAGCTATGAC(SEQ.ID.NO.19); primer 5 5'-GTAAAACGACGGCCAGT(SEQ.ID.NO.20); primer 6 5'-GTCACCCTCGACCTGCAgC(g:RiboG)(SEQ.ID.NO.21): primer 7 5'-GTTGTAAAACGAGGGCCAgT(g: RiboG)(SEQ.ID.NO.22); The 99-mer and 200-mer DNA strands (modified and unmodified) as well as the ribo- and 7-deaza-modified 100-mer were amplified from pRFcl DNA (10 ng, generously supplied S. Feyerabend. University of Hamburg) in 100 μL reaction volume containing 10 mmol/L KC1. 10 mmol/L (NH4)2SO . 20 mmol/L Tris HC1 (pH = 8.8). 2 mmol/L MgSO 4 . (exo(-)Pseudococcusfuriosus (Pfu) -Buffer. Pharmacia. Freiburg. Germany). 0.2 mmol/L each dNTP (Pharmacia. Freiburg. Germany). 1 μmol/L of each primer and 1 unit of exo(-)Pfu DNA polymerase (Stratagene. Heidelberg, Germany). For the 99-mer primers 1 and 2. for the 200-mer primers 1 and 3 and for the 100-mer primers 6 and 7 were used. To obtain 7-deazapurine modified nucleic acids, during PCR-amplification dATP and dGTP were replaced with 7-deaza-dATP and 7-deaza-dGTP. The reaction was performed in a thermal cycler (OmniGene. MWG-Biotech. Ebersberg, Germany) using the cycle: denaturation at 95°C for 1 min.. annealing at 51°C for 1 min. and extension at 72°C for 1 min. For all PCRs the number of reaction cycles was 30. The reaction was allowed to extend for additional 10 min. at 72°C after the last cycle. The 103-mer DNA strands (modified and unmodified) were amplified from M13mpl8 RFI DNA (100 ng. Pharmacia. Freiburg. Germany) in 100 μL reaction volume using primers 4 and 5 all other concentrations were unchanged. The reaction was performed using the cycle: denaturation at 95°C for 1 min.. annealing at 40°C for 1 min. and extension at 72°C for 1 min. After 30 cycles for the unmodified and 40 cycles for the modified 103- mer respectively, the samples were incubated for additional 10 min. at 72°C. Synthesis of 5'- ^--P -labeled PCR-primers Primers 1 and 4 were 5'-[ 32 -P]-labeled employing T4-polynucleotidkinase (Epicentre Technologies) and (γ- 32 P)-ATP. (BLU/NGG/502A, Dupont. Germany) according to the protocols of the manufacturer. The reactions were performed substituting 10% of primer 1 and 4 in PCR with the labeled primers under otherwise unchanged reaction- conditions. The amplified DN As were separated by gel electrophoresis on a 10% polyacrylamide gel. The appropriate bands were excised and counted on a Packard TRI- CARB 460C liquid scintillation system (Packard. CT. USA). Primer-cleavage from ribo-modified PCR-product The amplified DNA was purified using Ultrafree-MC filter units (30.000 NMWL), it was then redissolved in 100 μl of 0.2 mol/L NaOH and heated at 95°C for 25 minutes. The solution was then acidified with HC1 (1 mol/L) and further purified for MALDI-TOF analysis employing Ultrafree-MC filter units ( 10.000 NMWL) as described below. Purification of PCR products All samples were purified and concentrated using Ultrafree-MC units 30000 NMWL (Millipore. Eschborn. Germany) according to the manufacturer's description. After lyophilisation. PCR products were redissolved in 5 μL (3 μL for the 200-mer) of ultrapure water. This analyte solution was directly used for MALDI-TOF measurements. MALDI-TOF MS Aliquots of 0.5 μL of analyte solution and 0.5 μL of matrix solution (0.7 mol/L 3-HPA and 0.07 mol/L ammonium citrate in acetonitrile/water (1 :1. v/v)) were mixed on a flat metallic sample support. After drying at ambient temperature the sample was introduced into the mass spectrometer for analysis. The MALDI-TOF mass spectrometer used was a Finnigan MAT Vision 2000 (Finnigan MAT. Bremen. Germany). Spectra were recorded in the positive ion reflector mode with a 5 keV ion source and 20 keV postacceleration. The instrument was equipped with a nitrogen laser (337 nm wavelength). The vacuum of the system was 3-4- 10"^ hPa in the analyzer region and 1-4* 10"? hPa in the source region. Spectra of modified and unmodified DNA samples were obtained with the same relative laser power: external calibration was performed with a mixture of synthetic oligodeoxynucleotides (7-to50-mer). RESULTS AND DISCUSSION Enzymatic synthesis of 7-deazapurine nucleotide containing nucleic acids by PCR In order to demonstrate the feasibility of MALDI-TOF MS for the rapid, gel- free analysis of short PCR products and to investigate the effect of 7-deazapurine modification of nucleic acids under MALDI-TOF conditions, two different primer-template systems were used to synthesize DNA fragments. Sequences are displayed in Figures 36 and 37. While the two single strands of the 103-mer PCR product had nearly equal masses (Δm= 8 u), the two single strands of the 99-mer differed by 526 u. Considering that 7-deaza purine nucleotide building blocks for chemical DNA synthesis are approximately 160 times more expensive than regular ones (Product Information. Glen Research Coφoration. Sterling, VA) and their application in standard β- cyano-phosphoamidite chemistry is not trivial (Product Information. Glen Research Coφoration. Sterling. VA: Schneider . K and B.T. Chait ( 1995) Nucleic Acids Res.23. 1570) the cost of 7-deaza purine modified primers would be very high. Therefore, to increase the applicability and scope of the method, all PCRs were performed using unmodified oligonucleotide primers which are routinely available. Substituting dATP and dGTP by c 7 - dATP and c 7 -dGTP in polymerase chain reaction led to products containing approximately 80% 7-deaza-purine modified nucleosides for the 99-mer and 103-meπ and about 90% for the 200-mer. respectively. Table I shows the base composition of all PCR products. TABLE I: Base composition of the 99-mer, 103-mer and 200-mer PCR amplification products (unmodified and 7-deaza purine modified) 5 1 "s" and "a" describe "sense" and "antisense" strands of the double-stranded PCR product. 2 indicates relative modification as percentage of 7-deaza purine modified nucleotides of total amount of purine nucleotides. However, it remained to be determined whether 80-90% 7-deaza-purine 10 modification is sufficient for accurate mass spectrometer detection. It was therefore important to determine whether all purine nucleotides could be substituted during the enzymatic amplification step. This was not trivial since it had been shown that c 7 -dATP cannot fully replace dATP in PCR if Taq DNA polymerase is employed (Seela. F. and A. Roelling (1992) Nucleic Acids Res.. 20.55-61). Fortunately we found that exo(-)Pfu DNA 15 polymerase indeed could accept c 7 -dATP and c 7 -dGTP in the absence of unmodified purine triphosphates. However, the incoφoration was less efficient leading to a lower yield of PCR product (Figure 38). Ethidium-bromide stains by intercalation with the stacked bases of the DNA-doublestrand. Therefore lower band intensities in the ethidium-bromide stained gel might be artifacts since the modified DNA-strands do not necessarily need to give the same 0 band intensities as the unmodified ones. To verify these results, the PCRs with [ 32 P]-labeled primers were repeated. The autoradiogram (Figure 39) clearly shows lower yields for the modified PCR-products. The bands were excised from the gel and counted. For all PCR products the yield of the -> • modified nucleic acids was about 50%. referring to the corresponding unmodified amplification product. Further experiments showed that exo(-)DeepVent and Vent DNA polymerase were able to incoφorate c 7 -dATP and c 7 -dGTP during PCR as well. The overall performance, however, turned out to be best for the exo(-)Pfu DNA polymerase giving least side products during amplification. Using all three polymerases. it was found that such PCRs employing c 7 -dATP and c 7 -dGTP instead of their isosteres showed less side-reactions giving a cleaner PCR-product. Decreased occurrence of amplification side products may be explained by a reduction of primer mismatches due to a lower stability of the complex formed from the primer and the 7-deaza-purine containing template which is synthesized during PCR. Decreased melting point for DNA duplexes containing 7-deaza-purine have been described (Mizusawa. S. et al.. ( 1986) Nucleic Acids Res. , 14. 1319- 1324). In addition to the three polymerases specified above (exo(-) Deep Vent DNA polymerase. Vent DNA polymerase and exo(-) (Pfu) DNA polymerase), it is anticipated that other polymerases. such as the Large Klenow fragment of E.coli DNA polymerase. Sequenase, Taq DNA polymerase and U AmpliTaq DNA polymerase can be used. In addition, where RNA is the template. RNA polymerases. such as the SP6 or the T7 RNA polymerase. must be used MALDI-TOF mass spectrometry of modified and unmodified PCR products. The 99-mer, 103-mer and 200-mer PCR products were analyzed by MALDI- TOF MS. Based on past experience, it was known that the degree of depurination depends on the laser energy used for desoφtion and ionization of the analyte. Since the influence of 7- deazapurine modification on fragmentation due to depurination was to be investigated, all spectra were measured at the same relative laser energy. Figures 40a and 40b show the mass spectra of the modified and unmodified 103-mer nucleic acids. In case of the modified 103-mer, fragmentation causes a broad (M+H)^ signal. The maximum of the peak is shifted to lower masses so that the assigned mass represents a mean value of (M+H) + signal and signals of fragmented ions, rather than the (M+H)^ signal itself. Although the modified 103-mer still contains about 20% A and G from the oligonucleotide primers, it shows less fragmentation which is featured by much more narrow and symmetric signals. Especially peak tailing on the lower mass side due to depurination. is substantially reduced. Hence, the difference between measured and calculated mass is strongly reduced although it is still below the expected mass. For the unmodified sample a (M+H) + signal of 31670 was observed, which is a 97 u or 0.3% difference to the calculated mass. While, in case of the modified sample this mass difference diminished to 10 u or 0.03% (31713 u found. 31723 u calculated). These observations are verified by a significant increase in mass resolution of the (M+H) + signal of the two signal strands (m/Δm = 67 as opposed to 18 for the unmodified sample with Δm = full width at half maximum, fwhm). Because of the low mass difference between the two single strands (8 u) their individual signals were not resolved. With the results of the 99 base pair DNA fragments the effects of increased mass resolution for 7-deazapurine containing DNA becomes even more evident. The two single strands in the unmodified sample were not resolved even though the mass difference between the two strands of the PCR product was very high with 526 u due to unequal distribution of purines and pyrimidines (figure 41a). In contrast to this, the modified DNA showed distinct peaks for the two single strands (figure 41 b) which makes the superiority of this approach for the determination of molecular weights to gel electrophoretic methods even more profound. Although base line resolution was not obtained the individual masses were abled to be assigned with an accuracy of 0.1%: Δm = 27 u for the lighter (calc. mass = 30224 u) and Δm = 14 u for the heavier strand (calc. mass = 30750 u). Again, it was found that the full width at half maximum was substantially decreased for the 7-deazapurine containing sample. In case of both the 99-mer and 103-mer the 7-deazapurine containing nucleic acids seem to give higher sensitivity despite the fact that they still contain about 20% unmodified purine nucleotides. To get comparable signal-to-noise ratio at similar intensities for the (M+H) + signals, the unmodified 99-mer required 20 laser shots in contrast to 12 for the modified one and the 103-mer required 12 shots for the unmodified sample as opposed to three for the 7-deazapurine nucleoside-containing PCR product. Comparing the spectra of the modified and unmodified 200-mer amplicons. improved mass resolution was again found for the 7-deazapurine containing sample as well as increased signal intensities (figures 42a and 42b). While the signal of the single strands predominates in the spectrum of the modified sample the DNA-suplex and dimers of the single strands gave the strongest signal for the unmodified sample. A complete 7-deaza purine modification of nucleic acids may be achieved either using modified primers in PCR or cleaving the unmodified primers from the partially modified PCR product. Since disadvantages are associated with modified primers, as described above, a 100-mer was synthesized using primers with a ribo-modification. The primers were cleaved hydrolytically with NaOH according to a method developed earlier in our laboratory (Koester. H. et al.. Z Physiol. Chem.. 359. 1570-1589). Figures 10a and 10b display the spectra of the PCR product before and after primer cleavage. Figure 10b shows that the hydrolysis was successful: Both hydrolyzed PCR product as well as the two released primers could be detected together with a small signal from residual uncleaved 100-mer. This procedure is especially useful for the MALDI-TOF analysis of very short PCR-products since the share of unmodified purines originating from the primer increases with decreasing length of the amplified sequence. The remarkable properties of 7-deazapurine modified nucleic acids can be explained by either more effective desoφtion and/or ionization. increased ion stability and/or a lower denaturation energy of the double stranded purine modified nucleic acid. The exchange of the N-7 for a methine group results in the loss of one acceptor for a hydrogen bond which influences the ability of the nucleic acid to form secondary structures due to non- Watson-Crick base pairing (Seela. F. and A. Kehne ( 1987) Biochemistry, 26. 2232-2238.). which should be a reason for better desoφtion during the MALDI process. In addition to this the aromatic system of 7-deazapurine has a lower electron density that weakens Watson- Crick base pairing resulting in a decreased melting point (Mizusawa. S. et al., (1986) Nucleic Acids Res., 14, 1319-1324) of the double-strand. This effect may decrease the energy needed for denaturation of the duplex in the MALDI process. These aspects as well as the loss of a site which probably will carry a positive charge on the N-7 nitrogen renders the 7- deazapurine modified nucleic acid less polar and may promote the effectiveness of desoφtion. Because of the absence of N-7 as proton acceptor and the decreased polarizaiton of the C-N bond in 7-deazapurine nucleosides depurination following the mechanisms established for hydrolysis in solution is prevented. Although a direct correlation of reactions in solution and in the gas phase is problematic, less fragmentation due to depurination of the modified nucleic acids can be expected in the MALDI process. Depurination may either be accompanied by loss of charge which decreases the total yield of charged species or it may produce charged fragmentation products which decreases the intensity of the non fragmented molecular ion signal. The observation of both increased sensitivity and decreased peak tailing of the (M+H) "r signals on the lower mass side due to decreased fragmentation of the 7-deazapurine containing samples indicate that the N-7 atom indeed is essential for the mechanism of depurination in the MALDI-TOF process. In conclusion. 7-deazapurine containing nucleic acids show distinctly increased ion-stability and sensitivity under MALDI-TOF conditions and therefore provide for higher mass accuracy and mass resolution. Example 9: Solid State Sequencing and Mass Spectrometer Detection MATERIALS AND METHODS Oligonucleotides were purchased from Operon Technologies (Alameda. CA) in an unpurified form. Sequencing reactions were performed on a solid surface using reagents from the sequencing kit for Sequenase Version 2.0 (Amersham. Arlington Heights. Illinois). Sequencing a 39-mer target Sequencing complex: 5'-TCTGGCCTGGTGCAGGGCCTATTGTAGTTGTGACGTACA-(A b ) a -3' (DNA1 1683) (SEQ. ID. No. 23) 3 CAACACTGCATGT-5' (PNA16/DNA) (SEQ. ID. No. 24) In order to perform solid-state DNA sequencing, template strand DNA 1 1683 was 3'-biotinylated by terminal deoxynucleotidyl transferase. A 30 μl reaction, containing 60 pmol of DNA 11683, 1.3 nmol of biotin 14-dATP (GIBCO BRL. Grand Island, NY), 30 units of terminal transferase (Amersham, Arlington Heights. Illinois), and lx reaction buffer (supplied with enzyme), was incubated at 37°C for 1 hour. The reaction was stopped by heat inactivation of the terminal transferase at 70°C for 10 min. The resulting product was desalted by passing through a TE-10 spin column (Clonetech). More than one molecules of biotin- 14-dATP could be added to the 3'-end of DNA1 1683. The biotinylated DNA1 1683 was incubated with 0.3 mg of Dynal streptavidin beads in 30 μl lx binding and washing buffer at ambient temperature for 30 min. The beads were washed twice with TE and redissolved in 30 μl TE. 10 μl aliquot (containing 0.1 mg of beads) was used for sequencing reactions. The 0.1 mg beads from previous step were resuspended in a 1 Oμl volume containing 2 μl of 5x Sequenase buffer (200 mM Tris-HCl. pH 7.5. 100 mM MgC12. and 250 mM NaCl) from the Sequenase kit and 5 pmol of corresponding primer PNA16/DNA. The annealing mixture was heated to 70°C and allowed to cool slowly to room temperature over a 20-30 min time period. Then 1 μl 0.1 M dithiothreitol solution. 1 μl Mn buffer (0.15 M sodium isocitrate and 0.1 M McC12). and 2 μl of diluted Sequenase (3.25 units) were added. The reaction mixture was divided into four aliquots of 3 μl each and mixed with termination mixes (each consists of 3 μl of the appropriate termination mix: 32 μM c7dATP. 32 μM dCTP. 32 μM c7dGTP. 32 μM dTTP and 3.2 μM of one of the four ddTNPs. in 50 mM NaCl). The reaction mixtures were incubated at 37°C for 2 min. After the completion of extension. the beads were precipitated and the supernatant was removed. The beads were washed twice and resuspended in TE and kept at 4°C. Sequencing a ?8-mer (qrge( Sequencing complex: 5'-AAGATCTGACCAGGGATTCGGTTAGCGTGACTGCTGCTGCTGCTGCTGCTGC TGGATGATCCGACGCATCAGATCTGG-(A b ) n -3 (SEQ. ID. NO. 25) (TNR.PLASM2) 3'-CTACTAGGCTGCGTAGTC-5' (CM 1 ) (SEQ. ID. NO. 26) The target TNR.PLASM2 was biotinylated and sequenced using procedures similar to those described in previous section (sequencing a 39-mer target). Sequencing a 15-mer target with partially duplex probe Sequencing complex: 5 '-F-GATGATCCGACGCATCACAGCTC 3 ' (SEQ. ID. No. 27) 3 '-b-CTACTAGGCTGCGTAGTGTCGAGAACCTTGGCT- 3 ' (SEQ. ID. No. 28) CM1B3B was immobilized on Dynabeads M280 with streptavidin (Dynal. Norway) by incubating 60 pmol of CM1B3B with 0.3 magnetic beads in 30 μl 1M NaCl and TE ( 1 x binding and washing buffer) at room temperature for 30 min. The beads were washed twice with TE and redissolved in 30 μl TE, 10 or 20 μl aliquot (containing 0.1 or 0.2 mg of beads respectively) was used for sequencing reactions. The duplex was formed by annealing corresponding aliquot of beads from previous step with 10 pmol of DF1 la5F (or 20 pmol of DF1 la5F for 0.2 mg of beads) in a 9 μl volume containing 2 μl of 5x Sequenase buffer (200 mM Tris-HCl, pH 7.5. 100 mM MgCll . and 250 mM NaCl) from the Sequenase kit. The annealing mixture was heated to 65 °C and allowed to cool slowly to 37°C over a 20-30 min time period. The duplex primer was then mixed with 10 pmol of TSlo (20 pmol of TS10 for 0.2 mg of beads) in 1 μl volume, and the resulting mixture was further incubated at 37°C for 5 min. room temperature for 5-10 min. Then 1 μl 0.1 M dithiothreitol solution. 1 μl Mn buffer (0.15 M sodium isocitrate and 0.1 M MnCl 2 ). and 2 μl of diluted Sequenase (3.25 units) were added. The reaction mixture was divided into four aliquots of 3 μl each and mixed with termination mixes (each consists of 4 μl of the appropriate termination mix: 16 μM dATP. 16 μM dCTP. 16 μM dGTP. 16 μ M dTTP and 1.6 μM of one of the four ddNTPs. in 50 mM NaCl). The reaction mixtures were incubated at room temperature for 5 min. and 37°C for 5 min. After the completion of extension, the beads were precipitated and the supernatant was removed. The beads were resuspended in 20 μl TE and kept at 4°C. An aliquot of 2 μl (out of 20 μl) from each tube was taken and mixed with 8 μl of formamide. the resulting samples were denatured at 90-95° C for 5 min and 2 μl (out of 10 μl total) was applied to an ALF DNA sequencer (Pharmacia. Piscataway. NJ) using a 10% polyacrylamide gel containing 7 M urea and 0.6x TBE. The remaining aliquot was used for MALDI-TOFMS analysis. MALDI sample preparation and instrumentation Before MALDI analysis, the sequencing ladder loaded magnetic beads were washed twice using 50 mM ammonium citrate and resuspended in 0.5 μl pure water. The suspension was then loaded onto the sample target of the mass spectrometer and 0.5 μl of saturated matrix solution (3-hydropicolinic acid (HPA): ammonium citrate = 10: 1 mole ratio in 50% acetonitrile) was added. The mixture was allowed to dry prior to mass spectometer analysis. The reflectron TOFMS mass spectrometer (Vision 2000, Finnigan MAT, Bremen, Germany) was used for analysis. 5 kV was applied in the ion source and 20 kV was applied for postacceleration. All spectra were taken in the positive ion mode and a nitrogen laser was used. Normally, each spectrum was averaged for more than 100 shots and a standard 25-point smoothing was applied. RESULTS AND DISCUSSIONS Conventional solid-state sequencing In conventional sequencing methods, a primer is directly annealed to the template and then extended and terminated in a Sanger dideoxy sequencing. Normally, a biotinylated primer is used and the sequencing ladders are captured by streptavidin-coated magnetic beads. After washing, the products are eluted from the beads using EDTA and formamide. However, our previous findings indicated that only the annealed strand of a duplex is desorbed and the immobilized strand remains on the beads. Therefore, it is advantageous to immobilize the template and anneal the primer. After the sequencing reaction and washing, the beads with the immobilized template and annealed sequencing ladder can be loaded directly onto the mass spectrometer target and mix with matrix. In MALDI. only the annealed sequencing ladder will be desorbed and ionized, and the immobilized template will remain on the target. A 39-mer template (SEQ. ID. No. 23) was first biotinylated at the 3' end by adding biotin- 14-dATP with terminal transferase. More than one biotin- 14-dATP molecule could be added by the enzyme. However, since the template was immobilized and remained on the beads during MALDI. the number of biotin- 14-dATP would not affect the mass spectra. A 14-mer primer (SEQ. ID. No. 29) was used for the solid-state sequencing. MALDI-TOF mass spectra of the four sequencing ladders are shown in Figure 34 and the expected theoretical values are shown in Table II. Table II A-reaction C-reaction G-reaciioπ T-reaction 5 , -TCTGGCCTGGTGCAGGGCCTATTGTAG7TGTGACGTACA-(A b ) n -3' 3'-TCAACACTGCATGT-5' 4223.8 4223.8 4223.8 4223.8 S'-ATCAACACTGCATGT-S' 4521.0 3 -CATCAACACTGCATGT-5' 4810.2 3'-ACATCAACACTGCATGT-5' 5123.4 3 , -AACATCAACACTGCATGT-5' 5436.6 S'-TAACATCAACACTGCATGT-S' 5740.8 3'-ATAACATCAACACTGCATGT-5' 6054.0 S'-GATAACATCAACACTGCATGT-S' 6383.2 3 , -GGATAACATCAACACTGCATGT-5 , 6712.4 3 -CGGATAACATCAACACTGCATGT-5' 7001.6 S'-CCGGATAACATCAACACTGCATGT-S' 7290.8 3 -CCCGGATAACATCAACACTGCATGT-5' 7580.0 S'-TCCCGGATAACATCAACACTGCATGT-S' 7884.2 3 -GTCCCGGATAACATCAACACTGCATGT-5' 8213.4 3'-CGTCCCGGATAACATCAACACTGCATGT-5' 8502.6 3'-ACGTCCCGGATAACATCAACACTGCATGT-5' 8815.8 3 -CACGTCCCGGATAACATCAACACTGCATGT-5' 105.0 3 -CCACGTCCCGGATAACATCAACACTGCATGT-5 1 9394.2 3 -ACCACGTCCCGGATAACATCAACACTGCATGT-5' 9707.4 3'-GACCACGTCCCGGATAACATCAACACTGCATGT-5' 10036.6 3--GGACCACGTCCCGGATAACATCAACACTGCATGT-5' 10365.8 3 -CGGACCACGTCCCGGATAACATCAACACTGCATGT-5' 10655.0 3 -CCGGACCACGTCCCGGATAACATCAACACTGCATGT-5' 10944.2 3 -ACCGGACCACGTCCCGGATAACATCAACACTGCATGT-5' 1 1257.4 3 -GACCGGACCACGTCCCGGATAACATCAACACTGCATGT-5' 1 1586.6 3 -AGACCGGACCACGTCCCGGATAACATCAACACTGCATGT-5' 1 1899.8 The sequencing reaction produced a relatively homogenous ladder, and the full-length sequence was determined easily. One peak around 5150 appeared in all reactions are not identified. A possible explanation is that a small portion of the template formed some kind of secondary structure, such as a loop, which hindered sequenase extension. Mis- incoφoration is of minor importance, since the intensity of these peaks were much lower than that of the sequencing ladders. Although 7-deaza purines were used in the sequencing reaction, which could stabilize the N-glycosidic bond and prevent depurination. minor base losses were still observed since the primer was not substituted by 7-deazapurines. The full length ladder, with a ddA at the 3' end. appeared in the A reaction with an apparent mass of 1 1899.8. However, a more intense peak of 122 appeared in all four reactions and is likely due to an addition of an extra nucleotide by the Sequenase enzyme. The same technique could be used to sequence longer DNA fragments. A 78- mer template containing a CTG repeat (SEQ. ID. No. 25) was 3'-biotinylated by adding biotin- 14-dATP with terminal transferase. An 18-mer primer (SEQ. ID. No. 26) was annealed right outside the CTG repeat so that the repeat could be sequenced immediately after primer extension. The four reactions were washed and analyzed by MALDI-TOFMS as usual. An example of the G-reaction is shown in Figure 35 and the expected sequencing ladder is shown in Table III with theoretical mass values for each ladder component. All sequencing peaks were well resolved except the last component (theoretical value 20577.4) was indistinguishable from the background. Two neighboring sequencing peaks (a 62-mer and a 63-mer) were also separated indicating that such sequencing analysis could be applicable to longer templates. Again, an addition of an extra nucleotide by the Sequenase enzyme was observed in this spectrum. This addition is not template specific and appeared in all four reactions which makes it easy to be identified. Compared to the primer peak, the sequencing peaks were at much lower intensity in the long template case. Further optimization of the sequencing reaction may be required. TABLE ddATP ddCTP ddGTP ddTTP 5' AAGATCTGACCAGGGATTCGGTTAGCGTGACTGCTGCTGCTGCTGCTGGATGATCCGACGCATCAGATCTGG- (A b ) n -3 ' 3' -CTACTAGGCTGCGTAGTC-5' 5491 6 5491 6 5491.6 5491 6 3' -CCTACTAGGCTGCGTAGTC-5' 5764 8 3• -ACCTACTAGGCTGCGTAGTC-5' 6078 0 3' -GACCTACTAGGCTGCGTAGTC-5 ■ 6 07.2 3' -CGACCTACTΛGGCTGCGTAGTC-5' 6696 4 3' -ACGACCTACTAGGCTGCGTAGTC-5' 7009 6 3 ' -GACGACCTACTAGGCTGCGTAGTC-5' 7338.8 3' -CGACGACCTACTAGGCTGCGTAGTC-5 ' 7628 0 3 ■ -ACGACGACCTACTAGGCTGCGTAGTC-5' 7941 2 3 ' -GACGACGACCTACTAGGCTGCGTAGTC-5' 8270 4 3 '-CGACGACGACCTACTAGGCTGCGTAGTC-5' 8559 6 ' -ACGACGACGACCTACTAGGCTGCGTAGTC-5' 8872 8 3 • -GACGACGACGACCTACTAGGCTGCGTAGTC-5 ■ 9202.0 3 • -CGACGACGACGACCTACTAGGCTGCGTAGTC-5' 9491 2 3' -ACGACGACGACGACCTACTAGGCTGCGTAGTC-5 • 9804 4 ' -GACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 10133 6 ' -CGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 10422 8 ' -ACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 10736.0 I 3 ' -GACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 • 11065.2 3' -CGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 11354.4 ' 3 • -ACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 11667.6 3 ' -GACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 11996.8 3 • -CGACGACGACGACGACGA-GACCTACTAGGCTGCGTAGTC-5' 12286.0 3 ' -ACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 125 9.2 3' -GACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 12928.4 3 ■ -TGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 13232.6 3' -CTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 13521.8 • -ACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 13835.0 3' -CACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 14124.2 3 • -GCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 1 453.4 3 ' -CGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 ■ 14742.6 3 • -TCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 15046. 3 ' -ATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 15360 0 3 ' -AATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 15673.2 3' -CAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 15962.4 3' -CCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 16251.6 3' -GCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 16580.8 3 ' -AGCCAATCGCACTGACGACGACGA-GACGACGACGACCTACTAGGCTGCGTAGTC-5' 16894 0 • ' -AAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5' 17207.2 3 ' -TAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 • 1 511.i 3 ' -CTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 ' 17800.6 3 " -CCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 ■ 18089.8 3 • -CCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 ' 18379.0 3 ' -TCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 ' 18683.2 3 ' -GTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 ' 19012.4 3 ' -GGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 ' 19341.6 3 ' -TGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 ' 19645.8 3 ' -CTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 ' 19935.0 3 ' -ACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 ' 20248.2 3 ' -GACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 ' 20577.4 3 ' -AGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 ' 20890.6 3 ' -TAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 ' 21194.8 3 ' -CTAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 ' 21484.0 3 ' -TCTAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 • 21788.2 3 • -TTCTAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5 ' 22092.4 Sequencing using duplex DNA probes for capturing and priming Duplex DNA probes with single-stranded overhang have been demonstrated to be able to capture specific DNA templates and also serve as primers for solid-state sequencing. The scheme is shown in Figure 46. Stacking interactions between a duplex probe and a single-stranded template allow only 5-base overhand to be sufficient for capturing. Based on this format, a 5' fluorescent-labeled 23-mer (5'-GAT GAT CCG ACG CAT CAC AGC TC) (SEQ. ID. No. 29) was annealed to a 3 '-biotinylated 18-mer (5'-GTG ATG CGT CGG ATC ATC) (SEQ. ID. No. 30). leaving a 5-base overhang. A 15-mer template (5'-TCG GTT CCA AGA GCT) (SEQ ID. No. 31) was captured by the duplex and sequencing reactions were performed by extension of the 5-base overhang. MALDI-TOF mass spectra of the reactions are shown in Figure 47A-D. All sequencing peaks were resolved although at relatively low intensities. The last peak in each reaction is due to unspecific addition of one nucleotide to the full length extension product by the Sequenase enzyme. For comparison, the same products were run on a conventional DNA sequencer and a stacking fluorogram of the results is shown in Figure 48. As can be seen from the Figure, the mass spectra had the same pattern as the fluorogram with sequencing peaks at much lower intensity compared to the 23-mer primer. Improvements of MALDI-TOF mβSS spectrometry as a detection technique Sample distribution can be made more homogenous and signal intensity could potentially be increased by implementing the picoliter vial technique. In practice, the samples can be loaded on small pits with square openings of 100 um size. The beads used in the solid-state sequencing is less than 10 um in diameter, so they should fit well in the microliter vials. Microcrystals of matrix and DNA containing "sweet spots" will be confined in the vial. Since the laser spot size is about 100 μm in diameter, it will cover the entire opening of the vial. Therefore, searching for sweet spots will be unnecessary and high repetition-rate laser (e.g. > 1 OHz) can be used for acquiring spectra. An earlier report has shown that this device is capable of increasing the detection sensitivity of peptides and proteins by several orders of magnitude compared to conventional MALDI sample preparation technique. Resolution of MALDI on DNA needs to be further improved in order to extend the sequencing range beyond 100 bases. Currently, using 3-HP A/ammonium citrate as matrix and a reflectron TOF mass spectrometer with 5kV ion source and 20 kV postacceleration. the resolution of the run-through peak in Figure 33 (73-mer) is greater than 200 (FWHM) which is enough for sequence determination in this case. This resolution is also the highest reported for MALDI desorbed DNA ions above the 70-mer range. Use of the delayed extraction technique may further enhance resolution. All of the above-cited references and publications are hereby incoφorated by reference. Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims: Claims 1. A process for detecting a target nucleic acid sequence present in a biological sample, comprising the steps of: a) obtaining a nucleic acid molecule from a biological sample; b) immobilizing the nucleic acid molecule onto a solid support, to produce an immobilized nucleic acid molecule; c) hybridizing a detector oligonucleotide with the immobilized nucleic acid molecule and removing unhybridized detector oligonucleotide: d) ionizing and volatizing the product of step c); and e) detecting the detector oligonucleotide by mass spectrometry. wherein detection of the detector oligonucleotide indicates the presence of the target nucleic acid sequence in the biological sample. 2. A process of claim 1. wherein step b), immobilization is accomplished by hybridization between a complementary capture nucleic acid molecule, which has been previously immobilized to a solid support, and a complementary specific sequence on the target nucleic acid sequence. 3. A process of claim 1, wherein step b). immobilization is accomplished via direct bonding of the target nucleic acid sequence to a solid support. 4. A process of claim 1. wherein prior to step b). the target nucleic acid sequence is amplified. 5. A process of claim 4. wherein the target nucleic acid sequence is.amplified by an amplification procedure selected from the group consisting of: cloning, transcription based amplification, the polymerase chain reaction (PCR). the ligase chain reaction (LCR). and strand displacement amplification (SDA). 6. A process of claim 1. wherein the solid support is selected from the group consisting of: beads, flat surfaces, pins, combs and wafers. 7. A process of claim 6. wherein step b). immobilization is accomplished by hybridization between an array of complementary capture nucleic acid molecules, which have been previously immobilized to a solid support, and a portion of the nucleic acid molecule, which is distinct from the target nucleic acid sequence. 8. A process of claim 7. wherein the complementary capture nucleic acid molecules are oligonucleotides or oligonucleotide mimetics. 9. A process of claim 1. wherein the immobilization is reversible. 10. A process of claim 1 wherein the mass spectrometer is selected from the group consisting of: Matrix- Assisted Laser Desoφtion/Ionization Time-of-Flight (MALDI- TOF). Electrospray (ES). Ion Cyclotron Resonance (ICR). Fourier Transform and combinations thereof. 1 1. A process of claim 1. wherein prior to step d). the sample is conditioned. 12. A process of claim 1 1. wherein the sample is conditioned by mass differentiating at least two detector oligonucleotides or oligonucleotide mimetics to detect and distinguish at least two target nucleic acid sequences simultaneously. 13. A process of claim 12, wherein the mass differentiation is achieved by differences in the length or sequence of the at least two oligonucleotides. 14. A process of claim 12. wherein the mass differentiation is achieved by the introduction of mass modifying functionalities in the base, sugar or phosphate moiety of the detector oligonucleotides. 15. A process of claim 12. wherein the mass differentiation is achieved by exchange of cations or removal of the charge at the phosphodiester bond. 16. A process of claim 1. wherein the nucleic acid molecule obtained from a biological sample is replicated into DNA using mass modified deoxynucleoside triphosphates and RNA dependent DNA polymerase prior to mass spectrometric detection. 17. A process of claim 1. wherein the nucleic acid molecule obtained from a biological sample is replicated into RNA using mass modified ribonucleoside triphosphates and DNA dependent RNA polymerase prior to mass spectrometric detection. 18. A process of claim 1 wherein the target nucleic acid sequence is a DNA fingeφrint or is implicated in a disease or condition selected from the group consisting of a genetic disease, a chromosomal abnormality, a genetic predisposition, a viral infection, a fungal infection, a bacterial infection and a protist infection. 19. A process for detecting a target nucleic acid sequence present in a biological sample, comprising the steps of: a) obtaining a nucleic acid molecule containing a target nucleic acid sequence from a biological sample: b) amplifying the target nucleic acid sequence using an appropriate amplification procedure, thereby obtaining an amplified target nucleic acid sequence. c) hybridizing a detector oligonucleotide with the nucleic acid molecule and removing unhybridized detector oligonucleotide: d) ionizing and volatizing the product of step c); and e) detecting the detector oligonucleotide by mass spectrometry. wherein detection of the detector oligonucleotide indicates the presence of the target nucleic acid sequence in the biological sample. 20. A process of claim 19, wherein the target nucleic acid is amplified by an amplification procedure selected from the group consisting of: cloning, transcription based amplification, the polymerase chain reaction (PCR). the ligase chain reaction (LCR), and strand displacement amplification (SDA). 21. A process of claim 19. wherein the mass spectrometer is selected from the group consisting of: Matrix-Assisted Laser Desoφtion/Ionization. Time-of-Flight (MALDI- TOF), Electrospray (ES). Ion Cyclotron Resonance (ICR). Fourier Transform and combinations thereof. 22. A process of claim 19. wherein prior to step d), the sample is conditioned. 23. A process of claim 22. wherein the sample is conditioned by mass differentiation. 24. A process of claim 23. wherein the mass differentiation is achieved by mass modifying functionalities attached to primers used for amplification. 25. A process of claim 23. wherein the mass differentiation is achieved by exchange of cations or removal of the charge at the phosphodiester bond. 26. A process of claim 19. wherein the nucleic acid molecule is DNA. 27. A process of claim 19. wherein the nucleic acid molecule is RNA. 28. A process of claim 19. wherein prior to step d). amplified target nucleic acid sequences are immobilized onto a solid support to produce immobilized target nucleic acid sequences. 29. A process of claim 28. wherein immobilization is accomplished by hybridization between a complementary capture nucleic acid molecule, which has been previously immobilized to a solid support, and the target nucleic acid sequence. 30. A process of claim 28. wherein the solid support is selected from the group consisting of: beads, flat surfaces, pins, combs and wafers. 31. A process of claim 28. wherein the immobilization is reversible. 32. A process of claim 19 wherein the target nucleic acid sequence is a DNA fingeφrint or is a disease or condition selected from the group consisting of a genetic disease. a chromosomal abnormality, a genetic predisposition, a viral infection, a fungal infection, a bacterial infection and a protist infection. 33. A process for detecting a target nucleic acid sequence present in a biological sample, comprising the steps of: a) obtaining a target nucleic acid sequence from a biological sample: b) replicating the target nucleic acid sequence, thereby producing a replicated nucleic acid molecule: c) specifically digesting the replicated nucleic acid molecule using at least one appropriate nuclease. thereby producing digested fragments: d) immobilizing the digested fragments onto a solid support containing complementary capture nucleic acid sequences to produce immobilized fragments: and e) analysing the immobilized fragments by mass spectrometry. wherein hybridization and the determination of the molecular weights of the immobilized fragments provide information on the target nucleic acid sequence. 34. A process of claim 33. wherein the solid support is selected from the group consisting of: beads, flat surfaces, pins, combs and wafers. 35. A process of claim 33. wherein the complementary capture nucleic acid sequences are oligonucleotides or oligonucleotide mimetics. 36. A process of claim 33. wherein the immobilization is reversible. 37. A process of claim 33 wherein the mass spectrometer is selected from the group consisting of: Matrix- Assisted Laser Desoφtion/Ionization Time-of-Flight (MALDI- TOF). Electrospray (ES). Ion Cyclotron Resonance (ICR). Fourier Transform and combinations thereof. 38. A process of claim 33. wherein prior to step e). the sample is conditioned. 39. A process of claim 38, wherein the sample is conditioned by mass differentiation. 40. A process of claim 38. wherein the mass differentiation is achieved by the introduction of mass modifying functionalities in the base, sugar or phosphate moiety of the detector oligonucleotides. 41. A process of claim 39, wherein the mass differentiation is achieved by exchange of cations or removal of the charge at the phosphodiester bond. 42. A process of claim 33. wherein after step a), the target nucleic acid sequence is replicated into DNA using mass modified deoxynucleoside and/or dideoxynucleoside triphosphates and RNA dependent DNA polymerase. 43. A process of claim 33. wherein after step a), the target nucleic acid sequence is replicated into RNA using mass modified ribonucleoside and/or 3'- deoxynucleoside triphosphates and DNA dependent RNA polymerase. 44. A process of claim 33. wherein after step a), the target nucleic acid is replicated into DNA using mass modified deoxynucleoside and/or dideoxynucleoside triphosphates and a DNA dependent DNA polymerase. 45. A process of claim 33 wherein the target nucleic acid sequence is a DNA fingeφrint or a disease or condition selected from the group consisting of a genetic disease, a chromosomal abnormality, a genetic predisposition, a viral infection, a fungal infection, a bacterial infection or a protist infection. 46. A process for detecting a target nucleic acid sequence present in a biological sample, comprising the steps of: a) obtaining a nucleic acid molecule containing a target nucleic acid sequence from a biological sample: b) contacting the target nucleic acid sequence with at least one primer, said primer having 3' terminal base complementarity to the target nucleic acid sequence: c) contacting the product of step b) with an appropriate polymerase enzyme and sequentially with one of the four nucleoside triphosphates; d) ionizing and volatizing the product of step c); and e) detecting the product of step d) by mass spectrometry, wherein the molecular weight of the product indicates the presence or absenceof a mutation next to the 3' end of the primer in the target nucleic acid sequence. 47. A process for detecting a target nucleotide present in a biological sample. comprising the steps of: a) obtaining a nucleic acid molecule that contains a target nucleotide: b) immobilizing the nucleic acid molecule onto a solid support, to produce an immobilized nucleic acid molecule: c) hybridizing the immobilized nucleic acid molecule with a primer oligonucleotide that is complementary to the nucleic acid molecule at a site immediately 5' of the target nucleotide: d) contacting the product of step c) with a complete set of dideoxynucleosides or 3 '-deoxynucleoside triphosphates and a DNA dependent DNA polymerase. so that only the dideoxynucleoside or 3 '-deoxynucleoside triphosphate that is complementary to the target nucleotide is extended onto the primer: e l ionizing and volatizing the product of step d); and f) detecting the primer by mass spectrometry. to determine the identity of the target nucleotide. 48. A process for detecting a mutation in a nucleic acid molecule, comprising the steps of: a) obtaining a nucleic acid molecule: b) hybridizing the nucleic acid molecule with an oligonucleotide probe, thereby forming a mismatch at the site of a mutation: c) contacting the product of step b) with a single strand specific endonuclease: d) ionizing and volatizing the product of step c): and e) detecting the products obtained by mass spectrometn.'. wherein the presence of more than one fragment, indicates that the nucleic acid molecule contains a mutation. 49. A process for detecting a target nucleic acid sequence present in a biological sample, comprising the steps of: a) obtaining a nucleic acid containing a target nucleic acid sequence from a biological sample; b) performing at least one hybridization of the target nucleic acid sequence with a set of ligation educts and a thermostable DNA ligase. thereby forming a ligation product; c) ionizing and volatizing the product of step b): and d) detecting the ligation product by mass spectrometry and comparing the value obtained with a known value to determine the target nucleic acid sequence.

Inventors: KOESTER HUBERT, KOESTER, HUBERT
Applicants: SEQUENOM INC, SEQUENOM, INC.
Application year: 1996
Main classification: C12Q1
IPC codes: C12N15, C12Q1, G01N27, G01N33, G01N35