MXPA98005955A - Methods and compositions to analyze nucleic acid molecules by using classification techniques - Google Patents

Abstract

The present invention relates to labels and linkers specifically designed for a wide variety of nucleic acid reactions, which are suitable for a wide variety of nucleic acid reactions where separation of nucleic acid molecules based on the size is required.

Description

METHODS AND COMPOSITIONS FOR ANALYZING NUCLEIC ACID MOLECULES BY USING CLASSIFICATION TECHNIQUES TECHNICAL FIELD The present invention relates generally to methods and compositions for analyzing nucleic acid molecules, and more specifically to labels that can be used in a wide variety of nucleic acid reactions, where separation of nucleic acid molecules with base is required. in size BACKGROUND OF THE INVENTION The detection and analysis of nucleic acid molecules are among the most important techniques in biology. These techniques are at the heart of molecular biology and play a rapid expansion role in the rest of biology.

Generally, a type of nucleic acid reaction analysis involves the separation of nucleic acid molecules based on length. For example, a widely used technique, polymerase chain reaction (PCR) (see, Patents of E. U.A.

Nos. 4,683, 195, 4, 683,202, and 4, 800, 159) has become a technique widely used to identify sequences present in a sample and to synthesize DNA molecules for further manipulation.

Briefly, in PCR, DNA sequences are amplified by an enzymatic reaction that synthesizes new DNA strands geometrically or linearly. After amplification, the DNA sequences must be detected and identified. Due to non-specific amplifications, which would otherwise confuse the analysis, or the need for purity, the PCR reaction products are generally subjected to separation before detection. Separation based on the size (ie, length) of the products provides the most useful information. The method that gives the highest resolution of nucleic acid molecules is electrophoretic separation. In this method, each individual PCR reaction is applied to an appropriate gel and subjected to a potential voltage. The number of samples that can be processed is limited by the number of wells in the gel. In most gel devices, approximately 10 to 64 samples can be separated into a single gel. In this way, the processing of large sample numbers is labor intensive and material intensive. The electrophoretic separation must be coupled with some detection system in order to obtain data. Nucleic acid detection systems commonly and almost exclusively use an intercalation dye or radioactive label, and less frequently, a non-radioactive label. Intercalation dyes, such as ethidium bromide, are simple to use. The dye is included in the gel matrix during electrophoresis or, after electrophoresis, the gel is rinsed in a solution containing dye. The dye can be visualized directly in some cases, but more often, and for ethidium bromide in particular, it is excited by light (eg, UV) to fluorescence. Despite this apparent ease of use, such dyes must have notable disadvantages. First, the dyes are insensitive and there must be a large mass amount of nucleic acid molecules in order to visualize the products. Second, the dyes are typically mutagenic or carcinogenic. A more sensitive detection technique than colorants uses a radioactive (or non-radioactive) label. Typically, a radiolabeled nucleotide or a radiolabeled primer is included in the PCR reaction. After separation, the radiolabel is "visualized" by autoradiography. Although more sensitive, the detection suffers from film limitations, such as reciprocity failure and lack of linear characteristic. These limitations can be overcome by detecting the mark by phosphor image analysis. However, radios have security requirements, increase the use of resources and need specialized equipment and personnel training. For these reasons, the use of non-radioactive brands has increased in popularity. In such systems, the nucleotides contain a label, such as a fluorophore, biotin or digoxin, which can be detected by an antibody or another molecule (eg, another member of a pair of ligands) that is labeled with an enzyme reactive with a chromogenic substrate. These systems do not have the security problems as described above, but they use components that are often unstable and can produce non-specific reactions, resulting in high antecedent (ie, low signal-to-noise ratio). The present invention provides novel compositions and methods that can be used in a wide variety of nucleic acid reactions, and that also provides other related advantages.

BRIEF DESCRIPTION OF THE INVENTION In brief, the present invention provides compositions and methods that can be used in a wide variety of ligand pair reactions where separation of molecules of interest, such as nucleic acid molecules, is required based on size. Representative examples of methods that can be improved with the description provided herein include PCR, differential display, RNA finger print, PCR-SSCP, oligo-lysis assays, nuclease digestion methods (eg, exo-based assays). and endo-nuclease), and imprint of disesoxy footprint. The methods described herein can be used in a broad array of brands, for example, in the development of clinical or research-based diagnostics, the determination of polymorphisms, and the development of genetic maps.

Within one aspect of the present invention, methods are provided for determining the identity of a nucleic acid molecule comprising the steps of (a) generating labeled nucleic acid molecules from one or more selected target nucleic acid molecules, in where a tag correlates with a particular nucleic acid fragment and that can be detected by non-fluorescent spectrometry or potentiometry, (b) separate the fragments marked by size, (c) cut the marks of the marked fragments, and (d) detect marks by non-fluorescent spectrometry or potentiometry, and determine the identity of the nucleic acid molecules. Within a related aspect of the invention, methods are provided for detecting a selected nucleic acid molecule, comprising the steps of (a) combining labeled nucleic acid probes with target nucleic acid molecules under conditions and for a sufficient time to allowing hybridization of a labeled nucleic acid probe to a complementary selected target nucleic acid sequence, wherein a labeled nucleic acid probe can be detected by non-fluorescent spectrometry or potentiometry, (b) altering the size of hybrid labeled probes, probes non-hybrid or target molecules, or the probe: target hybrids, (c) separate the probes marked by size, (d) cut marks from the labeled probes, and (e) detect the marks by non-fluorescent spectrometry or potentiometry, and detect the selected nucleic acid molecule. Within other aspects, methods for genotyping a selected organism are provided, comprising the steps of (a) generating labeled nucleic acid molecules from a selected target molecule, wherein a tag is correlated with a > particular fragment and can be detected by non-fluorescent spectrometry or potentiometry, (b) separate the labeled molecules by length in sequence, (c) cut the label of the target molecule, and (d) detect the mark by non-fluorescent spectrometry or potentiometry , and determine the genotype of the organism. Within another aspect, methods are provided for genotyping a selected organism, comprising the steps of (a) combining a labeled nucleic acid molecule with a selected target molecule under conditions and for a time or enough to allow hybridization of the labeled molecule to the target molecule, wherein a tag correlates with a particular fragment and can be detected by non-fluorescent spectrometry or potentiometry, (b) separating the tagged fragments by length in sequence, (c) cutting the labeled fragment tag, and ( d) detect the mark by non-fluorescent spectrometry or potentiometry, and determine the genotype of the organism. Within the context of the present invention it should be understood that "biological samples" include not only samples obtained from living organisms (e.g., mammals, fish, bacteria, parasites, viruses, fungi, and the like) or from the environment (e.g. air, water or solid samples), but biological materials that can be artificially or synthetically produced (for example, phage libraries, libraries of organic molecules, collections of genomic clones, cDNA clones, RNA clones or the like). Representative examples of biological samples include biological fluids (e.g., blood, semen, cerebral spinal fluid, urine), biological cells (e.g., stem cells, B or T cells, liver cells, fibroblasts, and the like), and biological tissues. . Finally, representative examples of organisms that can be genotyped include almost any unicellular or multicellular organism, such as warm-blooded animals, mammals or vertebrates (eg, humans, chimpanzees, macaques, horses, cows, pigs, sheep, dogs, cats, rats and mice, as well as cells of any of these), bacteria, parasites, viruses, fungi and plants. Within various embodiments of the methods described above, the nucleic acid probes and / or molecules of the present invention can be generated, for example, by ligation, cutting or extension reaction (e.g., PCR). Within other related aspects, the probes or nucleic acid molecules can be labeled by non-3'-labeled oligonucleotide primers (eg, 5'-labeled oligonucleotide primers) or dideoxynucleotide terminators. Within other embodiments of the invention, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 250, 300, 350 may be used. , 400, 450, or more than 500 different and unique labeled molecules within a given reaction simultaneously, wherein each label is unique to a selected nucleic acid molecule or fragment, or probe, and can be identified separately. Within other embodiments of the invention, the tag (s) can be detected by fluorometry, mass spectrometry, infrared spectrometry, ultraviolet spectrometry, or potentiostatic amperometry (for example, by using colorimetric or amperometric detectors). Representative examples of suitable spectrometric techniques include time-of-flight mass spectrometry, quad-mass spectrometry, magnetic sector mass spectrometry and electrical sector mass spectrometry. The specific modalities of these techniques include iron trap mass spectrometry, electroaspersion ionisation mass spectrometry, ion spray mass spectrometry, liquid ionization mass spectrometry, atmospheric pressure ionization mass spectrometry, electronic ionization mass, fast atomic bombardment ionization mass spectrometry, MALDI mass spectrometry, photo-ionization time-of-flight mass spectrometry, laser drop mass spectrometry, MALDI-TOF mass spectrometry, APCI mass spectrometry , nano-spray mass spectrometry, cloudy spray ionization mass spectrometry, chemical ionization mass spectrometry, resonance ionization mass spectrometry, secondary ionization mass spectrometry and thermospray mass spectrometry.

Within still other embodiments of the invention, target molecules, hybrid labeled probes, non-hybrid probes or target molecules, probe-targeting hybrids, or labeled probes or nucleic acid molecules can be separated from other molecules by using methods that discriminate between the size of the molecules (either real linear size, or three-dimensional size) Representative examples of such methods include gel electrophoresis, capillary electrophoresis, micro-channel electrophoresis, HPLC, size exclusion chromatography, filtration, polyacrylamide gel electrophoresis, liquid chromatography, reversed-phase liquid chromatography, field-to-pulse electrophoresis, field inversion electrophoresis, dialysis, and fluorescence-activated liquid drop classification. Alternatively, target molecules, hybrid labeled probes, non-hybrid target probes or molecules, target probe hybrids or labeled probes or nucleic acid molecules can be attached to a solid support (eg, hollow fibers (Amicon Corporation, Danvers, Mass.), Globules (Polysciences, Warrington, Pa.), Magnetic beads (Robbin Scientific, Mountain View, Calif.), Plates, plates, and jars (Corning Glass Works, Corning, NY), meshes (Becton Dickinson, Mountain View , Calif.), Sieves and solid fibers (see Edelman and other US Patent No. 3,843,324, see also Kuroda et al., US Patent No. 4,416,777), membranes (Millipore Corp., Bedford, Mass.), And toothpicks) . If the first or second member, or exposed nucleic acids bind to a solid support, within certain embodiments of the invention, the methods described herein may further comprise the step of washing the solid support of unbound material. Within other embodiments, the labeled nucleic acid probes or molecules can be cut by methods such as chemical methods, oxidation, reduction, unstable acidic, base unstable, enzymatic, electrochemical, heat and fofoinaseable methods. Within more modalities, the steps of separating, cutting and detecting can be done continuously, for example, in a single device that can be automatic. Within certain embodiments of the invention, the size of the hybrid-labeled probes, probes or non-hybrid target molecules, or probe-target hybrids are altered by a method selected from the group consisting of polymerase extension, ligation, digestion of exonuclease, endonuclease digestion, restriction enzyme digestion, site-specific recombinase digestion, ligation, nuclease digestion of specific inequality, specific methylation nuclease digestion, covalent attachment of probe to target and hybridization. The methods and compositions described herein can be used in a wide variety of applications, including, for example, identifying PCR amplicons, RNA fingerprinting, differential display, detection of single-strand polymorphism, fingerprint printing dideoxy , restriction maps and restriction fragment length polymorphisms, i DNA fingerprint compression, genotype, mutation detection, oligonucleotide ligation assay, specific sequence amplifications, for diagnostics, forensics, identification, developmental biology, biology, molecular medicine, toxicology, animal husbandry. These and other aspects of the present invention will be apparent with reference to the following detailed description and accompanying drawings. In addition, several references are set forth below that describe in detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated herein by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the flow chart for the synthesis of pentafluorophenyl esters from mass spectroscopy labels that can be chemically cut, to release labels with carboxyl amide terminals, Figure 2 illustrates the flow chart for the synthesis of pentafluorophenyl esters of mass spectroscopy labels that can be chemically cut, to release labels with carboxyl amide terminals. Figures 3-6 and 8 illustrate the flow chart for the synthesis of tetrafluorophenyl esters from a series of 36 mass spectroscopy marks that can be photochemically cut. Figure 7 illustrates the flow chart for the synthesis of a series of 36 mass spectroscopy marks that can be photochemically cut to finished amine. Figure 9 illustrates the synthesis of 36 photochemically labeled mass spectroscopy-labeled oligonucleotide oligonucleotides made from the corresponding series of 36 tetrafluorophenyl esters of mass spectroscopy brand acids that can be photochemically cut. only. Figure 1 illustrates the synthesis of 36 olyotropic mass spectroscopy nucleotides that can be photochemically cut from the corresponding series of 36 mass spectroscopy marks that can be photochemically cut to finished amine. Figure 11 illustrates the simultaneous detection of multiple tags by mass spectrometry. Figure 12 shows the mass spectrogram of the alpha-cyano matrix alone. Figure 13 illustrates a labeled nucleic acid fragment constructed modularly.

DETAILED DESCRIPTION OF THE INVENTION As noted above, the present invention provides compositions and methods for analyzing nucleic acid molecules, wherein the separation of nucleic acid molecules based on size is required. The present methods allow the simultaneous detection of molecules of interest, which include nucleic acids and fragments, proteins, peptides, etc. In short, in one aspect, the present invention provides compounds wherein a molecule of interest, or precursor thereto, is linked via an unstable bond (or unstable linkages) to a tag. In this manner, the compounds of the invention can be observed as having the general formula: TLX where T is a mark component, L is the linker component that is, or contains, an unstable bond, and X is the molecule component of interest (MOI) or a functional group component (Lh) through which the MOI can join TL. Therefore, the compounds of the invention can be represented by the more specific general formulas: T-L-MOI and T-L-Lh For reasons described in detail below, series of T-L-MOI compounds can be subjected to conditions that cause the unstable bond to split, thus releasing a brand portion from the remainder of the compound. The brand portion is then characterized by one or more analytical techniques, in order to provide direct information about the structure of the brand portion, and (more importantly) indirect information about the identity of the corresponding MOI. As a simple illustrative example of a representative compound of the invention wherein L is a direct bond, reference is made to the following structure (i): Structure (i) Nucleic acid fragment) linker (L) Brand Component Molecule Component of Interest In structure (i), T is a polycyclic aromatic portion containing nitrogen bonded to a carbonyl group, X is an MOI (and specifically a nucleic acid fragment terminating in an amine group), and L is the bond that forms a amide group. The amide bond is unstable with respect to the T bonds since, as recognized in the art, an amide bond can be chemically cut (divided) by acid or base conditions leaving the bonds within the brand component unchanged . In this way, a branded portion (i.e., the cutting product containing T) can be released as shown below: Structure (i) eico nucleic Brand portion Rest of the compound However, the linker L can be more than just a direct link, as shown in the following illustrative example, where reference is made to another representative compound of the invention having the structure (ii) shown below: Structure (ii) It is well known that compounds having a portion of ortho-nitrobenzylamine (see box atoms within structure (ii) are photolytically unstable, in that the exposure of said compounds to actinic radiation of a specific wavelength will cause Selective cutting of the benzylamine bond (see link denoted with heavy line in structure (ii).) Thus, structure (ii) has the same T and MOI groups as structure (i), however, the linker group contains multiple atoms and bonds in where there is a particularly unstable bond The photolysis of the structure (ii) in this way releases a brand portion (portion containing T) from the rest of the compound as shown below.

Structure (ii) Brand portion The compound in this way provides compounds which, during exposure to appropriate cutting conditions, undergo a shear reaction to release a brand portion from the remainder of the compound. The compounds of the invention can be described in terms of the marl portion, the MOI (or precursor to it)., Lh), and the unstable link that unites the two groups together. Alternatively, the compounds of the invention can be described in terms of the components of which they are formed. In this manner, the compounds can be described as the reaction product of a brand reagent, a linker reagent and an MOI reagent, as follows. The brand reagent consists of a chemical handle (Th) and a variable component (Tvc), so that it is observed that the brand reagent has the general structure: T c-T | - To illustrate this nomenclature, reference is made to structure (iii), which shows a brand reagent that can be used to prepare the compound of structure (ii). The brand reagent that has the structure (iii) contains a variable brand component and a brand handle, as shown below: Structure (iii) Component Handle brand variable brand In structure (iii), the marking handle (-C (= O) -A) simply provides an avenue for reacting the label reagent with the interlacing reagent to form a portion of T-L. The group "A" in structure (ii) indicates that the carboxyl group is in a chemically active state, so that it is ready to be coupled with other handles. "A" can be, for example, a hydroxyl group or pentafluorophenoxy, among many other possibilities. The invention provides for a large number of possible brand handles that can be linked to a variable brand component, as discussed in detail below. The brand variable component in this way is part of "T" in the formula T-L-X, and will also be part of the brand portion that is formed from the reaction that cuts L.

Also as discussed below, the variable component of the mark is so called since in preparing series of compounds according to the invention, it is desired that the members of a series have unique variable components, so that the individual members can be distinguished each other by an analytical technique. As an example, the variable component of the structure mark (ii) can be a member of the following series, where the members of the series can be distinguished by their UV or mass spectrum: Also, the interlacing reagent can be described in terms of its chemical handles (that there are necessarily at least two, each can be distinguished as Lh) that bypass an unstable interlacing component, where the unstable interlacing component consists of the required unstable portion (L2). ) and optional unstable portions (L1 and L3), wherein the optional unstable portions effectively serve to separate L2 from the handles Lh, and the required unstable portion serves to provide an unstable link within the unstable interleaver component. In this way, it can be seen that the interlacing reagent has the general formula: Lh-L1-L2-L The nomenclature used to describe the interlacing reagent can be illustrated in view of structure (iv), which once again extracts the compound of structure (ii): Structure (iv) Linker handle Linker handle As structure (iv) illustrates, atoms can serve in more than one functional role. Thus, in the structure (v), the benzyl nitrogen functions as a chemical handle to allow the interlacing reagent to bind the labeling reagent by means of an amide-forming reaction, and subsequently also serves as a necessary part of the reaction. structure of the unstable portion L2 in that the benzene carbon nitrogen bond is particularly susceptible to photolytic cutting. The structure (? V) also illustrates that an interlacing reagent may have an L3 group (in this case, a methylene group), although it does not have an L1 group. Also, the linker reagents may have a group L1 but not a group L3, or they can have groups L1 and L3, or they can have neither groups L1 nor L3. In structure (iv), the presence of the group 'P' together with the carbonyl group indicates that the carbonyl group is protected from reaction, given this configuration, the activated carboxyl group of the brand reagent (II) can react cleanly with the amine group of the interlacing reagent (iv) to form an amide bond and to give a compound of the formula TL-Lh The MOI reagent is a suitable reactive form of a molecule of interest wherein the molecule of interest is an acid fragment nucleic acid, a suitable MOI reagent is a fragment of nucleic acid linked through its 5 'hydroxyl group to a phosphodiester group and then to an alkylene chain ending in an amino group.This amino group can then be reacted with the carbonyl group of the structure (iv), (then, of course, deprotecting the carbonyl group, and preferably after subsequently activating the carbonyl group to the reaction with the amine group) in order to bind the MO I to the interlayer. When viewed in chronological order, the invention is observed to take a branded reagent (which has a chemical marking handle and a variable branded component), an interlacing reagent (which has two chemical interlacing handles, a portion unstable required and 0-2 optional unstable portions) and an MOI reagent (which has a molecule component of interest and a molecule chemical interest handle) to form TL-MOI. In this way, to form TL-MO I, the brand reagent and the interlacing reagent are first reacted together to provide TL-Lh, and then the reactant of MO I is reacted with TL-Lh to provide TL- MO I, or (less preferable) the interlacing reagent and the MO I reagent are first reacted together to provide Lh-L-MO I, and then Lh-L-MO I is reacted with the brand reagent to provide TL-MO I. For purposes of convenience, compounds having the formula T-L-MOI will be described in terms of the brand reagent, the interlacing reagent and the MOI reagent that can be used to form said compounds. Of course, the same compounds of the formula T-L-MOI can be prepared by other (typically, more laborious) methods, and still fall within the scope of the T-L-MOI compounds of the invention. In any case, the invention provides a T-L-MOI compound that can be subjected to cutting conditions, so that a brand portion is released from the remainder of the compound. The brand portion will comprise at least the brand variable compound, and typically will also comprise some or all of the brand handle atoms, some or all of the atoms of the interlacing handle that was used to join the brand reagent to the interlacing reagent. , the optional unstable portion L1 if this group was present in TL-MOI, and perhaps it will contain some part of the required unstable portion L2 depending on the precise structure of L2 and the nature of the cutting chemistry. For convenience, the brand portion can be referred to as the portion containing T since T will typically constitute the main portion (in terms of mass) of the brand portion. Given this introduction to one aspect of the present invention, the different components T, L and X will be described in detail. This description begins with the following definitions of certain terms, which will be used later to describe T, L and X.

As used herein, the term "nucleic acid fragment" means a molecule that is complementary to a target nucleic acid molecules selected (ie, complementary to all or a portion thereof), and may be derived from molecules natural or synthetically produced recombinantly, including molecules that occur unnaturally, and can be double or single chain as appropriate; and includes an oligonucleotide (e.g., RNA or DNA). a primer, a probe, a nucleic acid analogue (e.g., APN), an oligonucleotide that extends in a 5 'to 3' direction by a polymerase, a nucleic acid that is cut chemically or enzymatically, a nucleic acid that ends with a dideoxy terminator or capped on end 3! or 5 'with a compound that prevents polymerization at the 5' or 3 'end, and combinations thereof. The complementary aspect of a nucleic acid fragment to a selected target nucleic acid molecule generally means the display of at least about 70% of specific base pair over the entire length of the fragment. Preferably, the nucleic acid fragment exhibits at least about 80% specific base pair; and very preferably at least about 90%. Trials to determine the percent inequality (and thus the specific base pair percent) are well known in the art and are based on percent inequality as a function of the Tm when referenced for torque control of total base.

As used herein, the term "alkyl", alone or in combination, refers to a straight or branched chain, saturated hydrocarbon radical containing from 1 to 10, preferably from 1 to 6 and more preferably from 1 to 4, carbon atoms. Examples of such radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, decyl, and the like. The term "alkylene" refers to a diradical! straight or branched chain hydrocarbon containing from 1 to 10, preferably from 1 to 6 and more preferably from 1 to 4, carbon atoms. Examples of such diradicals include, but are not limited to, methylene, ethylene, (-CH2-CH2-), propylene and the like.

The term "alkenyl", alone or in combination, refers to a straight or branched chain hydrocarbon radical having at least one carbon to carbon double bond in a total of 2 to 10, preferably 2 to 6, and more preferably from 2 to 4, carbon atoms. Examples of such radicals include, but are not limited to, ethenyl, E- and Z-propenyl, isopropenyl, E- and Z-butenyl, E- and Z-isobutenyl, E- and Z-pentenyl, decenyl and the like. The term "alkenylene" refers to a straight or branched chain hydrocarbon diradical having at least one carbon to carbon double bond in a total of 2 to 10, preferably 2 to 6 and more preferably 2 to 4. , carbon atoms. Examples of such diradicals include, but are not limited to, methylidene (= CH2), ethydedene (-CH = CH), propylidene (-CH2-CH = CH-) and the like. The term "alkynyl", alone or in combination, refers to a. straight or branched chain hydrocarbon radical having at least one triple carbon to carbon bond in a total of 2 to 10, preferably 2 to 6 and more preferably 2 to 4, carbon atoms. Examples of such radicals include, but are not limited to, ethynyl (acetylenyl), propynyl (propargyl), butynyl, hexynyl, decynyl, and the like. The term "alkynylene", alone or in combination, refers to a straight or branched chain hydrocarbon diradical having at least one triple carbon to carbon bond in a total of 2 to 10, preferably 2 to 6, and more preferably from 2 to 4, carbon atoms. Examples of such radicals include, but are not limited to, ethynylene (-C = C-), propynylene (-CH2-CsC-) and the like. The term "cycloalkyl", alone or in combination, refers to a saturated cyclic arrangement of carbon atoms with a number of 3 to 8 and preferably 3 to 6, carbon atoms. Examples of said cycloalkyl radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The term "cycloalkylene" refers to a diradical form of a cycloalkyl. The term "cycloalkenyl", alone or in combination, refers to a cyclic carbocycle containing from 4 to 8, preferably 5 or 6, carbon atoms and one or more double bonds. Examples of said cycloalkenyl radicals include, but are not limited to, cyclopentenyl, cyclohexenyl, cyclopentadienyl and the like. The term "cycloalkenylene" refers to a diradical form of a cycloalkenyl. The term "aryl" refers to a carbocyclic aromatic group (consisting entirely of carbon and hydrogen) selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, azulenyl, fluorenyl and anthracenyl; or selected from the group aromatic heterocyclic group consisting of furyl, thienyl, pyridyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl, 1, 2,3-oxadiazolyl, 1,2 , 3-triazolyl, 1,3,4-thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazinyl, 1, 3,5-trithianyl, indolizinyl, indolyl, isoindolyl, 3H-indolyl, indolinyl, benzofblfuranilo, 2 , 3-dihydrobenzofuranyl, benzofibythiophenyl, 1H-indazoly !, benzimidazolyl, benzthiazolyl, purinyl, 4H-quinicinyl, quinolinyl, isoquinolinyl, cinolinyl, phthalazinyl, quinazolinyl, quinoxaiinyl, 1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl , and phenoxazinyl. The "aryl" groups, as defined in this application, may independently contain one to four substituents that are independently selected from the group consisting of hydrogen, halogen, hydroxyl, amino, nitro, trifluoromethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl , cyano, carboxy, carboalkoxy, 1, 2-dioxyethylene, alkoxy, alkenoxy or alkynoxy, alkylamino, alkenylamino, alkynylamino, aliphatic or aromatic acyl, alkoxy-carbonylamino, alkylsulfonylamino, morpholinocarbonylamino, tiomorfoünocarbonilamino, N-alky! guanidino, aralkylaminosulfonyl; aralkoxyalkyl; N-aralkoxyurea; N-hydroxylurea; N-alkenylurea; N, N- (alkyl hydroxy) urea; heterocyclyl; thioaryloxy-substituted aryl; N, N- (aryl, alkyl) hydrazino; Ar'-substituted sulfonylheterocyclyl; substituted aralkyl heterocyclyl; cycloalkyl and cycloalkenyl-substituted heterocyclyl; cycloalkyl-fused aryl; aliphatic acyl substituted aliphatic or aromatic, ar'-substituted ammocarbonyloxy; ar'-Ar'-disubstituted aryl, acyl-substituted acyl aliphatic or aromatic, cycloalkylcarbonylalkyl, amino-cycloalkyl-substituted aryloxy-carbonyl-alkyl; acid or ester fosforodiamidilo, "Ar" is a carbocyclic aplo or heterocyclic group as defined above having one to three substituents selected from the group consisting of hydrogen, halogen, hydroxyl, amino, nitro, tpfluorometilo, tpfluorometoxi substituents, alkyl, alkenyl, alqumilo, 1, 2-d? ox? met? leno, 1, 2-d? ox? et? leno, alkoxy, alkenoxy, alqumoxi, alkylamino, alkenylamino or alquimlammo, alkylcarbonyloxy, aliphatic or aromatic acyl, alkylcarbonylamino, alcoxicarbonilammo , alkylsulfonylamine, N-aikel or N, N-dialkylurea The term "alkoxy", alone or in combination, refers to an alkyl ether radical wherein the term "alkyl" is as defined above. Suitable alkyl ether include, but are not limited to, methoxy, ethoxy, n-propo ?? ? so-propox ?, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy and the like The term "alkenoxy", alone or in combination refers to a radical of the formula aiquen? iO-, where the term "Alkenyl" is as defined above with the proviso that the radical is not an ether of ene! Examples of suitable alkenoxy radicals include, but are not limited to, allyloxy, E- and Z-3-met? L-2-propenox? and the like The term "alkyloxy", alone or in combination, refers to a radical of the alkenyl-O- formula, wherein the term "alkylo" is as defined above provided that the radical is not an ether Examples of suitable alkyloxy radicals include, but are not limited to, propargyloxy, 2-butynyloxy and the like The term "thioalkoxy" refers to a thioether radical of the formula alkyl-S-, wherein alkyl is The term "alkylamino", alone or in combination, refers to a mono- or di-alkyl-substituted amino radical (i.e., a radical of the formula a) qui-o-NH or (alkyl) 2-N-), wherein the term "alkyl" is as defined above Examples of suitable alkylamino radicals include, but are not limited to, methylamino, ethylene, propylamine, isopropylamino, t-butylamino, N, N-diethylamino and the like. The term "alkenylamino", alone or in combination, refers to a radical of the foa a! Queni! Or-NH- or (alkenyl) 2N-, wherein the term "alkenyl" is as defined above, with the proviso that the radical is not enamine. An example of said alkenylamino radicals is the allylamino radical. The term "alkynylamino", alone or in combination, refers to a radical of the foa alkynyl-NH or (alkynyl) 2N-, wherein the term "alkynyl" is as defined above, with the proviso that the radical does not be inamine. An example of said alkynylamino radicals is the propargyl amino radical. The term "amide" refers to -N (R1) -C (= O) - or -C (= O) -N (R1) - wherein R1 is defined herein to include hydrogen as well as other groups. The term "substituted amide" refers to the situation where R1 is not hydrogen, while the term "unsubstituted amide" refers to the situation where R1 is hydrogen.

The term "aryloxy", alone or in combination, refers to a radical of the formula ary! O-O-, wherein aryl is as defined above. Examples of aryloxy radicals include, but are not limited to, phenoxy, naphthoxy, pyridyloxy and the like. AND! term "arylamino", alone or in combination, refers to a radica! of the formula ary! -NH-, wherein aryl is as defined above. Examples of arylamino radicals include, but are not limited to, phenylamino (anuido), naphthalimino, 2-, 3- and 4-pyridylamino and the like. AND! The term "aryl-fused cycloalkyl", alone or in combination, refers to a cycloalkyl radical that shares two adjacent atoms with a radical! of arüo, where the terms "Cycloalkyl" and "aryl" are as defined above. An example of a radica! of aryl-fused cycloalkyl is the benzofused cyclobutyl radical. The term "alkylcarbonylamino", alone or in combination, refers to a radical! of the formula alkyl-CON H, wherein the term "alkyl" is as defined above. The term "" alkoxycarbonylamino ", alone or in combination, refers to a radical! of the formula alkylo-S02N H-, where e! The term "alkyl" is as defined above. The term "arylsulfonylamino", alone or in combination, refers to a radical of the formula ## STR2 ## wherein the term "aryl" is as defined above.

The term "N-alkylurea", alone or in combination, refers to a radical of the formula alkyl-NH-CO-NH-, wherein the term "alkyl" is as defined above. The term "N-aplurea", alone or in combination, it refers to a radical of the formula ary! -NH-CO-NH-, wherein the term "aryl" is as defined above E! term "halogen" means fluorine, chlorine, bromine and iodine The term "hydrocarbon radical" refers to an arrangement of carbon and hydrogen atoms that only need a single hydrogen atom as an independent stable molecule. Thus, a radical ! of hydrocarbon has a valence site open in a carbon atom, through! which is the root! of hydrocarbon may be attached to another atom (s) Alkyl, alkenyl, cycloalkyl, etc., are examples of hydrocarbon radicals The term "hydrocarbon hydrocarbon" refers to an arrangement of carbon and hydrogen atoms that need two hydrogen atoms to be an independent stable molecule In this way, a hydrocarbon radical has two valence sites open on one or more carbon atoms, through which the radica! of hydrocarbon can be attached to other atom (s) Alkylene, alkenylene, alkyne, cycloalkylene, etc., are examples of hydrocarbon diradicals The term "hydrocarbyl" refers to any arrangement consisting entirely of carbon and hydrogen having a single valence site to which it is attached to another portion, and thus includes radicals known as alkyl alkenyl alkynyl cycloalkyl cycloalkenyl, aryl (without incorporation of heteroatom in the aryl ring), arylalkyl, alkylaryl and the like. The hydrocarbon radical is another name for hydrocarbyl. The term "hydrocarbylene" refers to any stable arrangement consisting entirely of carbon and hydrogen having two valence sites to which it is attached to other portions, and thus includes alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, arylene ( without incorporation of heteroatom in the arylene ring), arylalkylene, alkylarylene and the like. AND! hydrocarbon diradical is another name for hydrocarbylene. The term "hydrocarbyl-O-hydrocarbylene" refers to a hydrocarbyl group attached to an oxygen atom, wherein the oxygen atom also joins a hydrocarbylene group at one of the two valence sites where the hydrocarbylene group is attached to other portions. The terms "hydrocarbyl-S-hydrocarbylene", "hydrocarbyl-NH-hydrocarbylene" and "hydrocarbyl-amide-hydrocarbylene" have equivalent meanings, where oxygen has been replaced with sulfur, -NH- or an amide group, respectively. The term N- (hydrocarbyl) hydrocarbon refers to a hydrocarbylene group wherein one of the two valence sites is attached to a nitrogen atom, and this atom of nitrogen is not simultaneously a hydrogen group and a hydrocarbon. i lo. The term N, N-di (hydrocarbyl) hydrocarbylene refers to a hydrocarbylene group in which one of the two valence sites is attached to a "nitrogen * atom, and that nitrogen atom joins two groups simultaneously. The term "hydrocarbyl-hydrocarbylene" refers to a hydrocarbon group attached through an acyl group (C (-O) -) to one of the hydrocarbyl groups. two valence sites of a hydrocarbylene group. The terms "heterocyclylhydrocarbyl" and "heterocyclyl" refer to a cyclic arrangement of atoms that include carbon atoms and up to four atoms (referred to as heteroatoms) selected from oxygen, nitrogen, phosphorus and sulfur. The cyclic arrangement may be in the form of a monocyclic ring of 3-7 atoms, or a bicyclic ring of 8-11 atoms. The rings may be saturated or unsaturated (including aromatic rings), and may optionally be benzofused. The nitrogen and sulfur atoms in the ring can be in any oxidized form, including the quaternized form. A heterocyclylhydrocarbyl can be attached to any endocyclic carbon or heteroatom that results in W ^ - 'the creation of a stable structure. Preferred heterocyclylhydrocarbyls include 5-7 membered monocyclic heterocycles containing one or two nitrogen heteroatoms. A substituted heterocyclylhydrocarbyl refers to a heterocyclylhydrocarbyl as defined above, e? wherein at least one ring atom thereof is attached to an indicated substituent extending from the ring. When referring to hydrocarbyl and hydrocarbylene groups, the The term "derivatives of any of the foregoing in which one or more hydrogens is replaced with an equal number of fluors" refers to molecules containing carbon, hydrogen and fluorine atoms, but not other atoms. The term "activated ester" is an ester that contains a "leaving group" 8 which readily moves by a nucleophile, tat as an amine, and alcohol or a thiol nucleophile, said leaving groups are well known and include, without limitation, N- hydroxysuccinimide, N-hydroxybenzotriazole, halogen (halides), alkoxy including tetrafiuorophenolates, thioalkoxy and the like The term "protected ester" refers to an ester group that is masked or otherwise unreactive, see, for example, Greene, " Protecting Groups In Organic Synthesis. "In view of the above definitions, other chemical terms used in this application may be readily understood by those skilled in the art.The terms may be used alone or in combination thereof. Most preferred radicals apply for all such combinations.

A. GENERATION OF MARKED NUCLEIC ACID FRAGMENTS As noted above, one aspect of the present invention provides a general scheme for DNA sequence that allows the use of more than 16 labels in each line; With continuous detection, the marks can be detected and the sequence can be read as the separation of size occurs, just as with the sequence based on conventional fluorescence. This scheme applies to any of the DNA sequence techniques based on the size separation of labeled molecules. Brands and linkers suitable for use within the present invention, as well as methods for sequencing nucleic acids, are discussed in detail below. 1 - . 1 - Trademarks "Trademark", as used herein, generally refers to a chemical moiety that is used to identify only a "molecule of interest", and more specifically to the variable component of a trademark as well as to a trademark. anything that can be more closely linked to it in any brand reagent, brand component and brand portion. A mark that is useful in the present invention possesses several attributes: 1) It is capable of distinguishing itself from all other marks. This discrimination of other chemical portions can be based on the chromatographic behavior of the label (in particular after the cutting reaction), its spectroscopic or potentiometric properties, or some combination thereof. The spectroscopic methods by which the marks are usefully distinguished include mass spectrometry (MS), infrared (IR), ultraviolet (UV), and fluorescence, where MS, IR and UV are preferred, and MS is the most preferred spectroscopic methods. Potentiometric amperometry is a preferred potentiometric method. 2) The mark is capable of being detected when present at 10"22 to 10" 6 moles. 3) The brand has a chemical handle through which you can set the MOI that the brand intends to identify only. The fixation can be made directly to the MOI, or indirectly through an "interlacing" group. 4) The mark is chemically stable towards all fas manipulations that it undergoes, including fixing and cutting from MOI, and any manipulation of the MOI while the mark is fixed to it. d) The mark does not interfere in an important way with the manipulations carried out by the MOI while the mark is fixed to it. For example, if the label is fixed to an oligonucleotide, the label should not interfere significantly with any hybridization or enzymatic reactions (eg, reactions in sequence by PCR) performed in the opiogonucleotide. Similarly, if the label is bound to an antibody, it should not interfere significantly with the recognition of antigen by the antibody. A portion of the mark that must be detected by a spectroscopic or potentiometric method must have properties that improve the sensitivity and specificity of detection by that method. Typically, the brand portion will have those properties as they have been designed in the variable brand component, which would typically constitute the main portion of the brand portion. In the following discussion, the use of the word "trademark" typically refers to the trademark portion (ie, the cut product containing the variable trademark component), however, it may also be considered to refer to the variable component. brand itself since it is the portion of the brand portion that is typically responsible for providing the properties that can be detected only. In compounds of formula T-L-X, the "T" portion will contain the variable component of the label. Where the variable component of the mark has been designed to be characterized, for example, by mass spectrometry, the "T" portion of T-L-X must be referred to as Tm *. Likewise, the cutting product of T-L-X containing T can be referred to as the portion containing Tm *. The following spectroscopic and potentiometric methods can be used to characterize portions that contain Tw *. to. MS Marees characteristics Where a mark can be analyzed by mass spectrometry (ie, it is a mark that can be read by MS, also referred to herein as a MS mark or "portion containing Tms"), the essential characteristic of the brand is that it is capable of ionizing. In this way, it is a preferred element in the design of data that can be read by MS to incorporate in them a chemical functionality that can carry a positive or negative charge under ionization conditions in the MS. This feature confers improved ion formation efficiency and overall detection sensitivity, in particular in electro-spray ionization. The chemical functionality that supports an ionized charge can be derived from Tms or L or both. Factors that can increase the relative sensitivity of an analyte being detected by mass spectrometry are discussed in, for example, Sunner, J., et al., Anaf. Chem. 60: 1300-1307 (1988). A preferred functionality for facilitating the charging of a negative charge is an organic acid, such as phenolic hydroxyl, carboxylic acid, phosphonate, phosphate, trezole, sulfonylurea, perfluoroalcohol and sulfonic acid. The preferred functionality for facilitating the charging of a positive charge under ionization conditions are aliphatic or aromatic amines. Examples of functional amine groups that give improved detection ability of MS labels include quaternary amines (ie, amines having four bonds, each to carbon atoms, see Aebersold, US Pat. No. 5,240,859) and tertiary amines (eg. say, amines that have three bonds, each to carbon atoms, which includes C "= NC groups as they are present in pyridine, see Hess et al., Anal. Biochem. 224: 373, 1995; Sures and others, Anaf. Biochem. 224: 364, 1995). The hindered tertiary amines are particularly preferred. The tertiary and quaternary amines can be alkyl or aryl. A portion that contains Tmß must carry at least one species that can be ionized, but it can have more than one species that can be ionized. The preferred charge state is a single ionized species per brand. Accordingly, it is preferred that each portion containing TMS. { and each brand variable component) contains only one hindered amine group or organic acid.

Suitable amine-containing radicals which can form part of the Tms-containing portion include the following: - (Cj-CI0) -N (C; -C10) 2 - (C, - CoV-N Identification of a tag by spectrometry mass is preferably based on its ratio of molecular mass to charge (m / z) .The preferred molecular mass scale of MS marks is around 100 to 2,000 daltons, and preferably the portion containing T s has a mass of at least about 250 daltons, more preferably at least about 300 daltons, and still more preferably at least about 350 daltons.It is generally difficult for mass spectrometers to distinguish between portions having ions origin below about 200- 250 daltons (depending on the precise instrument), and thus the preferred Tms-containing portions of the invention have masses on that scale.As explained above, the portion containing Tms may contain different from those present in the variable brand component, and in fact others different from those present in Tms by itself. Accordingly, the mass of Tms by itself can be less than about 250 daltons, as long as the portion containing Tms has a mass of at least about 250 daltons. In this manner, the mass of Tms can vary from 15 (ie, a methyl radical) to about 10,000 daltons, and preferably ranges from 100 to about 5,000 daltons, and more preferably ranges from about 200 to about 1,000 daltons. It is relatively difficult to distinguish marks by mass spectrometry when those marks incorporate atoms that have more than one isotope in significant abundance. Accordingly, the preferred T groups which are intended for spectroscopic identification (Tms groups), contain carbon atoms, at least one of hydrogen and fluorine, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine. Although other atoms may be present in the T s, their presence may make the analysis of the mass spectral data somewhat more difficult. Preferably, the Tms groups have only carbon, nitrogen and oxygen atoms, in addition to hydrogen and / or fluorine. Fluorine is an optional but preferred atom to have in a Tms group. In comparison with hydrogen, fluorine, of course, is heavier. In this way, the presence of fluorine atoms instead of hydrogen atoms leads to groups T s of higher mass, thus allowing the group T s to reach and exceed a mass of more than 250 daltons, which is desired as explained before . In addition, the replacement of hydrogen with fluorine confers greater volatility in the portion containing Tms, and greater analyte volatility improves sensitivity when mass spectrometry is used as the detection method. The molecular formula of Tms falls within the range of C? -5ooNo-? OoOo-? OoSo-? OPo-? OHaFß! S where the sum of a, ß and d is sufficient to satisfy the otherwise dissatisfied valences of the atoms C, N, O, S and P. The designation C? .5ooN0-? OoOo-? OoSo-? OPo-? OHaFßld means that Tms contains at least one, and can contain any number from 1 to 500 carbon atoms, in addition to optionally containing as much as 100 nitrogen atoms ("N0-" means that Tms does not need to contain any nitrogen atoms), and as many as 100 oxygen atoms, and as many as 10 sulfur atoms and as many as 10 phosphorus atoms. The symbols a, ß and d represent the number of atoms of hydrogen, fluorine and iodine in Tms, where any two of these numbers can be zero, and where the sum of these numbers is equal to tota! of the otherwise dissatisfied valences of the atoms of C, N, O, S and P. preferably, Tms has a molecular formula that falls within the range of C? .5oNo-? oOo-? oHaF? where the sum of a ß is equal to the number of hydrogen and fluorine atoms, respectively, present in the portion. b. Characteristics of IR Marks There are two primary forms of IR detection of organic chemical groups: Raman IR scan and IR absorption. The Raman IR scintillation spectra and IR absorption are complementary spectroscopic methods. In general, Raman excitation depends on bias polarization changes while IR absorption depends on dipole junction moment changes. The weak IR absorption lines become strong Raman lines and vice versa. The wave number is the characteristic unit for IR spectra. There are three spectral regions for IR marks that have separate applications: close to IR at 12500 to 4000 cm "1, to half IR to 4000 to 600 cm" 1. away from IR at 600 to 30 cm. "1 For the uses described herein wherein a compound serves as a mark to identify an MOI, probe or primer, the half-spectral regions should be preferred. carbonyl (1850 to 1750 cm "1) would be measured for carboxylic acids, carboxylic esters and amides, and alkyl and aryl carbonates, carbamates and ketones. The bending of N-H (1750 to 160 cm "1) would be used to identify amines, ammonium ions and amides, at 1400 to 1250 cm" 1, the bending of R-OH is detected as well as the stretching of C-N in amides. The aromatic substitution patterns are detected at 900 to 690 cm "1 (doubling of CH, doubling of NH for ArNH2), saturated CH, olefins, aromatic rings, double and triple bonds, esters, acetals, ketals, ammonium salts, compounds of NO such as oximes, nitro, N-oxides, and nitrates, azo, hydrazones, quinones, carboxylic acids, amides, and lactams all possess infrared correlation data of vibration (see Pretsch et al., Spectral Data for Structure Determination of Organic Compounds , Springer-Verlag, New York, 1989) The preferred compounds would include an aromatic nitrile exhibiting a very strong nitrile stretching vibration at 2230 to 2210 cm "1. Other useful types of compounds are aromatic alkynes which have a strong stretching vibration which gives rise to an absorption band between 2140 and 2100 cm.l A third type of compound is the aromatic azides which exhibit strong absorption band in the region of 2160 to 2120 cm "1. Thiocyanates are representative of compounds that have a strong absorption at 2275 to 2263 cm. "1 Characteristics of UV Markings A compilation of organic chromophoric types and their respective UV-visible properties is given in Scott (Interpretation of the UV Spectra of Natural Products, Permagon Press, New York, 1962) A chromophore is an atom or group of atoms or electrons that are responsible for the absorption of particular light.There are empirical rules for the maximum% ap * in conjugate systems (see Pretsch and others, Spectral Data for Structure Determination of Organic Compounds, pp. B65 and B70, Springer-Verlag, New York, 1989.) Preferred compounds (with conjugated systems) would possess transitions nap * and pap * .These compounds are exemplified by acid violet. 7, acridine orange, acridine yellow G, bright blue G, congo red, crystal violet, malachite green oxalate, methanil yellow, methylene blue, methyl orange, violet of methyl B, naphthol green B, oil blue N, oil red O, 4-phenylazophenol, safranie O, green solvent 3, and orange sweat G, of which all are commercially available (Aldrich, Milwaukee, Wl ). Other suitable compounds are listed in, for example, Jane, I., et al., J. Chrom. 323: 191-225 (1985). d. Feature of a Fluorescent Mark Fluorescent probes are identified and quantified more directly by their wavelengths and intensities of absorption and fluorescence emission. The emission spectra (fluorescence and phosphorescence) are much more sensitive and allow more specific measurements than the absorption spectra. Other photophysical characteristics such as excited state life and fluorescence anisotropy are used less. The most useful intensity parameters are generally the molar extinction coefficient (e) for absorption and the yield (QY) for fluorescence. The value of e is specified at a single wavelength (usually the maximum absorption of the probe), whereas QY is a measure of the total emission photo over the entire spectral fluorescence profile. A narrow optical wave amplitude (<20 nm) is usually used for fluorescence excitation (by means of absorption), while the fluorescence detection band amplitude is much more variable, varying from full spectrum to maximum sensitivity to narrow band (-20 nm) for resolution maximum. The fluorescence intensity per probe molecule is proportional to the product of e and QY. The scale of these parameters between fluorophores of current practical importance is approximately 10,000 to 100,000 cm "M" 1 for e and 0.1 to 1.0 for QY. The compounds that can serve as fluorescent labels are the following: fluorescein, rhodamine, blue lambda 470, green lambda, red lambda 664, red lambda 665, acridine orange, and propidium iodide, which are commercially available from Lambda Fluorescence Co. (Pleasant Gap, PA). Fluorescent compounds such as Nile Red, Texas Red, Lissamine ™, BODIPY ™ are available from Molecular Probes (Eugene, OR). and. Characteristics of Potentiometric Marks The principle of electrochemical detection (ECD) is based on the oxidation or reduction of the compounds that at certain voltages applied, electrons are donated or accepted thus producing a current that can be measured. When certain compounds are subjected to a power difference, the molecules go through a molecular rearrangement on the surface of the working electrodes with the loss (oxidation) or gain (reduction) of electrons, said compounds are said to be electronic and go through electrochemical reactions. The EC detectors apply a voltage on an electrode surface on which the liquid leaving HPLC flows. The electroactive compounds that leave the column donate electrons (oxidize) or acquire electrons (reduce) generating a maximum current in real time. Importantly, the amount of current generated depends on the concentration of the analyte and the applied voltage, with each compound having a specific voltage where it begins to oxidize or reduce. The most popular electrochemical detector at present is the amperometric detector where the potential remains constant and the current produced from the electrochemical reaction is then measured. This type of spectrometry is currently called "potencytostatic amperometry". Commercial ammeters are available from ESA, Inc., Chelmford, MA. When the detection efficiency is 100%, the specialized detectors are called "colométricos". Colometric detectors are sensitive that have a number of practical advantages with respect to the selectivity and sensitivity that make these types of detectors useful in an arrangement. In colometric detectors, for a given analyte concentration, the signal current is plotted as a function of the applied potential (voltage) to the working electrode. The resulting sigmoidal graph is called the current-voltage curve or hydrodynamic votamagrama (HDV). The HDV allows the best choice of potential applied to the working electrode that allows one to maximize the observed signal. A major disadvantage of ECD is its inherent sensitivity with detection current levels at the subfemtomolar scale. Numerous chemicals and compounds are electrochemically active including many biochemicals, pharmaceuticals and pesticides. Chromatographically coeluted compounds can be effectively resolved even if their average wave potentials (the maximum potential of the average signal) differ by only 30-60 mV. The newly developed colometric sensors provide selectivity, identification and resolution of co-elution compounds when used as detectors in separations based on liquid chromatography. Therefore, these arranged detectors add another series of separations achieved in the detector itself. the current instruments have 16 channels that in principle are limited by the speed at which the data is acquired. The number of compounds that can be resolved in the EC arrangement is limited chromatographically (ie, limited plate count). However, if two or more compounds co-eluting chromatographically have a difference in mean wave potentials of 30-60 mV, the arrangement is able to distinguish the compounds. The ability of a compound to be electrochemically active depends on the possession of an active group EC (ie, -OH, -O, -N, -S).

Compounds that have been successfully detected using detectors colorimetric include 5-hydroxytryptamine, 3-methoxy-4-hydroxyphenyl-glycol, homogentisic acid, dopamine, metanofrina, 3-hidroxikynureninr, acetominophen, 3-hydroxytryptophol, 5-hydroxyindoleacetic acid, octanesulfonic acid , phenol, o-cresol, pyrogallol, 2-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol, 4,6-dinitrocresol, 3-methyl-2-nitrophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, 2 , 4,5-trichlorophenol, 4-chloro-3-methylphenol, 5-methylphenol, 4-methyl-2-nitrophenol, 2-hydroxyaniline, 4-hydroxyaniline, 1,2-phenylenediamine, benzocatechin, buturon, clortoluron, diuron, isoproturon , linuron, metobromuron, metoxuron, monolinuron, monuron, methionine, tryptophan, tyrosine, 4-aminobenzoic acid, 4-hidroxicumárico, 7-methoxycoumarin, baicalein apigenin, caffeic acid, catechin, centaureína, chlorogenic acid, daidzein, datiscetin, diosmetin , epicatechin gallate, epigal catechin, epigote catechin gallate alo, eugenol, eupatorin, ferulic acid, fisetin, galangin, gallic acid, gardenina, genistein, gentisic acid, hesperidin, irigenina, kaemferol, leucocyanidin, luteolin, mangostin, morin, myricetin, naringin, nariutina, pelargondina, peonidin, phloretin, pratenseína , 6-hydroxydopamine protocatechuic acid, rametina, quercetin, sakuranetina, escutelareína, scopoletin, syringaldehyde, syringic acid, tangeritin, troxerutin, umbelliferone, vanillic acid, 1, 3-dimethyl tegrahidroisoquinolina, r-salsolinol, N-methyl-r-salsolinol .tetrahydroisoquinoline, amitriptyline, apomorphine, capsaicin, chlordiazepoxide, chlorpromazine, daunorubicin, desipramine, doxepin, fluoxetine, flurazepam, imipramine, isoproterenol, methoxamine, morphine, morphine-3-glucuronide, nortriptyline, oxazepam, phenylephrine, trimipramine, ascorbic acid, N- acetyl serotonin, 3,4-dihydroxybenzylamine, 3,4-dihydroxymandelic acid (DOMA), 3,4-dihydroxyphenylacetic acid (DOPAC), 3 , 4-dihydroxyphenylalanine (L-DOPA), 3,4-dihydroxyphenylglycol (DHPG), 3-hydroxyanthranilic acid, 2-hydroxyphenylacetic acid (2HPAC), 4-hydroxybenzoic acid (4HBAC), 5-hydroxyindole-3-acetic acid (5HIAA ), 3-hidroxikynurenina acid, 3-hydroxymandelic (5HTP), 5-hydroxytryptophol (5HTOL), 5-hydroxytryptamine (5HT), suifato 5-hydroxytryptamine, 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-methoxytryptamine, 5 -metoxitriptofan, 5-metoxitriptofol, 3-methoxytyramine (3MT), 3-metoxitirosina (3-OM-DOPA), 5-methylcysteine, 3-methylguanine, bufotenin, dopamine, dopamine-3-glucuronide, dopamine-3-sulfate, dopamine -4-sulfate, epinephrine, epinin, folic acid, glutathione (reduced), guanine, guanosine, homogentisic acid (HGA), homovanillic acid (HVA), homovanilyl alcohol (HVOL), homoveratic acid, hva sulfate, hypoxanthine, indole , indole-3-acetic acid, indole-3-lactic acid, kynurenine, melatonin, methanofrine, N-methyltriptamine, N-methylthiramine, N, N-dimethyltryptamine, N , N-dimethyltyramine, norpinephrine, normetanofrine, octopamine, pyridoxal, pyridoxal phosphate, pyridoxamine, synephrine, triptofol, tryptamine, tyramine, uric acid, vanillylmandelic acid (vma), xanthine and xanthosine. Other suitable compounds are set forth in, for example, Jane, I., et al., J. Chrom. 323: 191-225 (1985) and Musch, G., et al., J. Chrom. 348: 97-110 (1985). These compounds can be incorporated into compounds of the formula T-L-X by methods known in the art. For example, compounds having a carboxylic acid group can react with amine, hydroxyl, etc., to form amide, ester and other ligatures between T and L. In addition to the above properties, and regardless of the detection method intended, he prefers that the brand has a modular chemical structure. This helps in the construction of large numbers of structurally related marks by using combinatorial chemistry techniques. For example, the Tms group desirably has several properties. Desirably, it contains a functional group that supports a single ionized charge state when the Tms-containing portion is subjected to mass spectrometry (referred to more simply as a "mass spectrum sensitivity enhancer" or MSSE group). As well, desirably it can serve as a member in a family of portions containing T s, wherein the members of the family have a different mass / charge ratio, however they have approximately the same sensitivity in the mass spectrometer. In this way, the members of the family desirably have the same MSSE. In order to allow the creation of families of compounds, it has been found convenient to generate brand reagents by means of a modular synthesis scheme, so that the brand components can be observed as comprising modules. In a preferred modular proposal for the structure of the group T s, T s has the formula T2- (J-T3-) n- where T2 is an organic portion formed of carbon and one or more of hydrogen, fluorine, iodine, oxygen , nitrogen, sulfur and phosphorus, having a mass scale of 15 to 500 daltons; T3 is an organic portion formed of carbon and one or more of hydrogen, fluorine, iodine, oxygen, nitrogen, sulfur and phosphorus, having a mass scale of 50 to 1000 daltons; J is a direct bond or a functional group such as amide, ester, amine, sulfur, ether, thioester, disulfide, thioether, urea, thiourea, carbamate, thiocarbamate, Schiff's base, reduced Schiff's base, imine, oxime, hydrazone, phosphate, phosphonate, phosphoramide, phosphonamide, sulfonate, sulfonamide or carbon-carbon bond; and n is an integer ranging from 1 to 50, so that when n is greater than 1, each T3 and J is independently selected. The modular structure T2- (J-T3) n- provides a convenient entry to families of compounds T-L-X, where each member of the family has a different T group. For example, when T is Tms, and each family member desirably has the same MSS E, one of the T3 groups can provide that MSSE structure. In order to provide variability among members of a family in terms of mass of Tms, group T2 may vary among family members. For example, one family member may have T2 = methyl, while another has T2 = ethyl, and another has T = propyl, etc. In order to provide "gross" or mass jumps, a group T3 can be designed that adds significantly (for example, one or several hundred) units of mass to T-L-X. Said group T3 can be referred to as a molecular weight scale adjusting group ("WRA"). A WRA is quite useful if one works with a single series of T2 groups, which would have masses that extend over a limited scale. A single series of T2 groups can be used to create Tms groups that have a broad mass scale simply by incorporating one or more T3 groups of WRA into the Tms. In this way, when using a simple example, if a series of T2 groups can have a mass scale of 350-340 daltons, for the Tms, the addition of a single WRA, which has, as an example number, 100 daltons , as a group T3 provides access to the mass scale of 350-440 daltons at the same time using the same series of groups T3. Similarly, the addition of two MWA groups of 100 daltons (each as a T3 group) provides access to the mass scale of 450-540 daltons, where this incremental addition of WRA groups can continue to provide access to a very large mass scale for the Tms group. Preferred compounds of the formula T2- (J-T3-) nLX have the formula RVWC- (RW A) W- R MS S E-LX where VWC is a group "T2", and each of the groups WRA and MSSE are "T3" groups. This structure is illustrated in Figure 12, and represents a modular proposal to the preparation of Tms. In the formula T2- (J-T3-) n-, T2 and T3 are preferably selected from hydrocarbyl, hydrocarbyl-O-hydrocarbylene, hydrocarbyl-S-hydrocarbylene, hydrocarbyl-N-hydrocarbylene, rock id rb il -measure-hydrocarbylene, N- (hydrocarbyl) hydrocarbylene, N, N-di (hydrocarbyl) hydrocarbylene, hydrocarbyl-hydrocarbylene .. heterocyclylhydrocarbyl wherein the heteroatom is selected from oxygen, nitrogen, sulfur and phosphorus, substituted heterocyclylhydrocarbyl wherein the heteroatom is selected from oxygen, nitrogen, sulfur and phosphorus and the substituents are selected from hydrocarbyl, hydrocarbyl-O- hydrocarbylene, id rocarbyl-NH-hydrocarbylene, hydrocarbyl-S-hydrocarbylene, N- (hydrocarbyl) hydrocarbylene, N, N-di (hydrocarbyl) hydrocarbylene and hydrocarbyl-hydrocarbylene. In addition, T2 and / or T3 may be a derivative of any of the potential T / T3 groups previously listed, so that one or more hydrogens are replaced fluorines. Also with respect to the formula T2- (J-T3-) n-, a preferred T3 has the formula -G (R2) -, where G is alkylene chain of C? .s having a single substituent R2. Thus, if G is ethylene (-CH2-CH2-) one of the two ethylene carbons can have a substituent R2, and R2 is selected from alkyl, alkenyl. alkynyl, cycloalkyl, aryl-fused cycloalkyl, cycloalkenyl. aryl, aralkyl, alkenyl or aryl-substituted alkynyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted cycloalkyl. biaryl, alkoxy, alkenoxy, alkyloxy, aralkoxy, alkenoxy or aryl-substituted alkyloxy, alkylamino, alkenylamino or alkynylamino. aryl-substituted alkylamino, alkenylamino or aryl-substituted alkylamino, aryloxy, arylamino, N-alkyl-substituted alkyl. N-substituted alkylurea, alkylcarbonylamino substituted alkyl, substituted aminocarbonyl alkyl, heterocyclyl, substituted heterocyclyl alkyl, substituted heterocyclyl amino, substituted carboxyalkyl aralkyl, loo fused aryl oxocarbocyclic and heterocyclic alkyl; cycloalkenyl, aryl-substituted alkyl, and, aralkyl, hydroxy-substituted alkyl, alkoxy-substituted alkyl, aralkoxy-substituted alkyl, alkoxy-substituted alkyl, aralkoxy-substituted alkyl, amino-substituted alkyl, alkyl (aryl-substituted alkyloxycarbonylamino) -substituted , thiol-substituted alkyl, alkylsulphonyl-substituted alkyl, alkyl (hydroxy-substituted alkylthio) -substituted, thioalkoxy-substituted alkylate, unsubstituted alkylcarboxylalkyl, alkyl heterocycle, and the substituted alkylamineunsubstituted hydrocarbyl-substituted-heterocyclylaxylamine, alkylsulfonylamino-substituted alkyl, arylsulphonylamino-substituted alkyl, lino-alkyl mole, thiomorph or lino-to-alkyl, alkyl-substituted morpholino-carbon, unsubstituted-alkyl-thiomorpholinocarbon, alkyl [N - (alkyl, alkenyl or alkynyl) - or N, N- [dialkyl, dialkenyl, dialkynyl or (alkyl, alkenyl) -aminojcarbonyl-substituted, heterocyclylaminocarbonyl, heterocyclyl-alkyleneaminocarbonyl, heterocyclylaminocarbonyl-substituted alkyl, alkyl heterocarboxylaminocarbonyl- substituted, N, N- [dialkyl!] alkyleneaminocarbonyl, N, N- [dialkyl] alkylene-aminocarbonyl-substituted alkyl, substituted-alkyl heterocycliccarbonyl, heterocycliccarbonyl-substituted-alkyl, substituted-carboxyl-alkyl, unsubstituted-acylamino-alkyl-dialkylamide and chains amino acid sides selected from arginine, asparagine, glutamine, S-methyl cysteine, methionine and corresponding sulfoxide and sulfone derivatives thereof, glycine, leucine, isoleucine, allo-isoleucine, ter-leucine, norleucine, phenylalanine, tyrosine, tryptophan, proline, alanine, ornithine, histidine, glutamine, valine, threonine, serine, acid aspartic acid, beta-cyanoalanine, and allotreonin; alkynyl and heterocyclylcarbonyl, aminocarbonyl. amido, mono- or dialkylaminocarbonyl, mono- or diarylamino) (carbonyl, alkylarylaminocarbonyl, diarylaminocarbonyl, mono- or diacylaminocarbonyl, aromatic or aliphatic acyl, alkyl optionally substituted by substityents selected from amino, carboxy, hydroxy, mercapto, mono- or dialkylamino, mono- or diarylamino, alkylarylamino, diarylamino, mono- or di-acylamino, alkoxy, alkenoxy, aryloxy, thioalkoxy, thioal Iquotenoxy, thioalkynoxy, thioaryloxy and heterocyclyl.

A preferred compound of the formula T2- - ((JJ-TT3: L-X has structure: r I wherein G is (CHa) l.β so that a hydrogen in one and only one of the CH2 groups represented by a single "G" is replaced with - (CH2) C-Am, da-T < , T2 and t < they are organic portions of (a formula C. 2sN0-9O9-9HaF () so that the sum of a and ß is sufficient to satisfy the otherwise dissatisfied valences of the atoms of C, N, and O; amide is O O II II - N- C- or - C- N-; R1 R1 wherein R1 is hydrogen or C?.? 0 alkyl; c is an integer that varies from 0 to 4; and n is an integer ranging from 1 to 50 so that when n is greater than 1, G, c, amide, R1 and T4 are independently selected. In another preferred embodiment, a compound of the formula T - (J-T3-) n-L-X has the structure: Amida P where T5 is an organic portion of the formula C? .2sN0-9? o-9HaF3 so that the sum of a and ß is sufficient to satisfy the otherwise dissatisfied valences of the atoms of C, N, and O. T5 includes a tertiary or quaternary amine or an organic acid, m is an integer ranging from 0-49, and T2, T4, R1, L and X have been previously defined.

Another preferred compound having the formula T2- (J-T3-) n-L-X has the particular structure: where T5 is an organic portion of the formula so that the sum of a and ß is sufficient to satisfy the otherwise dissatisfied valences of the atoms of C, N, and O. and T5 includes a tertiary or quaternary amine or an acid organic, m is an integer that varies from 0-49, and T2, T4, c, R1, "Amide", L and X have been previously defined. In the above structures having a T5 group, -Amida-t is preferably one of the following, which is conveniently made by reacting organic acids with free amine groups extending from "G".

Where the above compounds have a T5 group, and the "G" group has a free carboxyl group (or reactive equivalent thereof), then the following group -Amida-T5 is preferred, which can be conveniently prepared by reacting the appropriate organic amine with a free carboxyl group extending from a "G" group: CNH- - (C2-C10) -N (C C10) 2; - CNH- (C- C10) - N rx! • II II \ '"0 0 In three preferred embodiments of the invention, T-L-MO I has the structure: or the structure: r i or the structure: wherein T2 and T4 are organic portions of the formula C? -2SNo-9? 0. 9S0-3P0-3HaFpl6 so that the sum of y ß is sufficient to satisfy the otherwise dissatisfied valences of the atoms of C, N, O, S and P; G is (CH2)? - 6 wherein one and only one hydrogen in the CH2 groups represented by each G is replaced with - (CH) C-Amide-t4; Amida is O O II II - N- C- or - C- N-; R1 R1 wherein R1 is hydrogen or C? -10 alkyl; c is an integer that varies from 0 to 4; "C2-C10" represents a hydrocarbylene group having from 2 to 10 carbon atoms; "ODN-3'-OH" represents a fragment of nucleic acid having a 3 'terminal hydroxyl group (i.e., a fragment of nucleic acid linked to (C 1 -C 10) in another one different from the 3' end of the acid fragment nucleic); and n is an integer ranging from 1 to 50 so that when n is greater than 1, then G, c, Amide, R1 and T4 are independently selected. Preferably, there are no heteroatoms attached to a single carbon atom. In structures as set forth above that contain a T2-C (= O) -N (R1) group, this group can be formed by reacting an amine of the formula HN (R1) - with an organic acid selected from the following , which are examples only and should not constitute an exhausted list of potential organic acids; formic acid, acetic acid, propiolic acid, propionic acid, fluoroacetic acid, 2-butinoic acid, cyclopropanecarboxylic acid, butyric acid, methoxyacetic acid, difluoroacetic acid, 4-pentynoic acid, cyclobutanecarboxylic acid, 3,3-dimethylacrylic acid, acid valeric, N, N-dimethylglycine, N-formyl-gly-OH, ethoxyacetic acid, (methylthio) acetic acid, prirrol-2-carboxylic acid, 3-furoic acid, isoxazole-5-carboxylic acid, trans-3-hexenoic acid, trifluoroacetic acid, hexanoic acid, Ac-Gly-OH, 2-hydroxy-2-methylbutyric acid, benzoic acid, nicotinic acid, 2-pyrazinecarboxylic acid, 1-methyl-2-arylcarboxylic acid. 2-cyclopentene-1-acetic acid, cichlorpentylacetic acid, (S) - (-) - 2-? - Ridolidone-5-carboxylic acid, N-methyl-L-proline, heptanoic acid, Ac-b-Ala-OH, acid 2-ethy! -2-hydroxybutyric acid, 2- (2-methoxyethoxy) acetic acid, p-toluic acid, 6-methynicotinic acid, 5-methyl-2-pyrazinecarboxylic acid, 2,5-dimethyl-urea-3-carboxylic acid , 4-fluorobenzoic acid, 3,5-dimethylisoxazole-4-carboxylic acid, 3-cyclopentylpropionic acid, octanoic acid, N, N-Dimethylsuccinic acid, phenylpropiolic acid, cinnamic acid, 4-ethylbenzoic acid, p-anisic acid, acid 1, 2,5-trimethylpyrrole-3-carboxylic acid, 3-fluoro-4-methylbenzoic acid, Ac-DL-propargylglycine, 3- (trifluoromethyl) butyric acid, 1-piperidinpropionic acid, N-acetylproline, 3,5-difluorobenzoic acid , Ac-L-Va! -OH, indole-2-carboxylic acid, 2-benzofurancarboxylic acid, benzotriazole-5-carboxylic acid, 4-n-propylbenzoic acid, 3-dimethylaminobenzoic acid, 4-ethoxybenzoic acid, 4- (Methylthio) benzoic acid, N- (2-furoyl) glycine, 2- (methylthio) nicotinic acid, 3-fluoro-4-methoxybenzoic acid, Tfa-Gly-OH, 2-naphthoic acid, quinaldic acid, Ac- L-lle-OH, 3-methylidene-2-carboxylic acid, 2-quinoxalincarboxylic acid, 1-methyltin-ol-2-carboxylic acid, 2,3,6-trifluorobenzoic acid, N-formyl-L-Met-OH, acid 2- [2- (2-methoxy-ethoxy) ethoxy] acetic acid, 4-n-butylbenzoic acid, N-benzoglycine, 5-fluoroindole-2-carboxylic acid, 4-n-propoxybenzoic acid, 4-acetyl-3 acid, 5-dimethyl-2-pyrrolcarboxylic acid, 3,5-dimethoxybenzoic acid, 2,6-dimethoxynicotinic acid, cyclohexane pentanoic acid, 2-naphthylacetic acid, 4- (1 H-pyrrol-1-yl) benzoic acid, indole-3 acid -propionic, m-trifluoromethylbenzoic acid. 5-methoxyindole-2-carboxylic acid, 4-pentylbenzoic acid, Bz-b-Ala-OH, 4-diethylaminobenzic acid, 4-butoxybenzoic acid, 3-methyl-5-CF3-isoxazole-4-carboxylic acid , (3,4-dimethoxy-nyl) acetic acid, 4-biphenylcarboxylic acid, Pivaloyl-Pro-OH, Octanoyl-Gly-OH, (2-naphthoxy) -acetic acid, indole-3-butyric acid, 4- (trifluoromethyl) acid phenylacetic acid, 5-methoxyindole-3-acetic acid, 4- (trifluoromethoxy) benzoic acid, Ac-L-Phe-OH, 4-pentyloxybenzoic acid, Z-Gly-OH, 4-carboxy-N- (fur-2-methyl) ) pyrroidin-2-one, 3,4-diethoxybenzoic acid, 2,4-dimethyl-5-CO2-t-pyrrole-3-carboxylic acid, N- (2-fluorophene) succinamic acid, 3,4,5-trimethoxybenzoic acid , N-phenylanthranic acid, 3-phenoxybenzoic acid, Nonanoil-Gly-OH, 2-phenoxypyridine-3-carboxylic acid, 2,5-dimethyl-1-phenylpyrrole-3-carboxylic acid, trans-4- (trifluoromethyl) cinnamic acid , (5-methyl-2-phenyloxazo! -4-yl) acetic acid, 4- (2-cyclohexenyloxy) benzoic acid, 5-methoxy-2-m acid ethylindof-3-acetic, trans-4-cotinicarboxylic acid, Bz-5-aminovaleric acid, 4-hexyloxybenzoic acid, N- (3-methoxyphenyl) succinamic acid, Z-Sar-O H, acid 4- (3, 4-dimethoxyphenyl) butyric acid, Ac-o-fluoro-DL-Phe-OH, N- (4-fluorophenyl) glutaramic acid, 4'-ethyl-l-4-biphenyl carboxylic acid, acid 1, 2,3,4- tetrahydroacridinecarboxylic acid, 3-phenoxyphenylacetic acid, N- (2,4-difluorophenyl) succinamic acid, N-decanoyl-G ly-O H, (+) - 6-methoxy-a-methyl-2-naphtha! - (trifluoromethoxy) cinnamic N-formyl-DL-Trp-OH, (R) - (+) - α-methoxy-a- (trifluoromethyl) phenylacetic acid, Bz-DL-Leu-OH, 4- (trifluoromethoxy) acid phenoxyacetic acid, 4-heptyloxybenzoic acid, 2, 3,4-trimethoxycinnamic acid, 2,6-dimethoxybenzoyl-Gly-OH, 3- (3, 4, 5-trimethoxyphenyl) propionic acid, acid 2, 3,4,5,6 -pentafluorophenoxyacetic. N- (2,4-difluorophenyl) glutaramic acid, N-undecanoyl-Gly-O H, 2- (4-fluorobenzoyl) benzoic acid, 5-trifluoromethoxyindole-2-carboxylic acid, N- (2,4-difluorophenyl) acid diglycolamic, Ac-L-Trp-OH, Tfa-L-phenylglycine-OH, 3-iodobenzoic acid, 3- (4-n-pentylbenzoyl) propionic acid, 2-phenyl-4-quinolinecarboxylic acid, 4-octyloxybenzoic acid, Bz-L-Met-OH, 3,4,5-triethoxybenzoic acid, N -lauroyl-Gly-OH, 3,5-bi (trifluoromethyl) benzoic acid, Ac-5-methyl-DL-Trp-OH, 2-iodophenylacetic acid, 3-iodo-4-methylbenzoic acid, 3- (4-) acid n-hexylbenzoyl) propionic, N-hexanoyl-L-Phe-OH, 4-nonyloxybenzoic acid, 4 '- (trifluoromethyl) -2-biphenylcarboxylic acid, Bz-L-Phe-OH, N-tridecanoyl-GIy-OH, acid 3,5-bi (tri fluoro methyl) fe ni (acetic, 3- (4-n-hepty! Benzoyl) propionic acid, N-heptanoyl-L-Phe-OH, 4-decyloxybenzoic acid, N- (a, a, α-trifluoro-m-tolyl) anthranilic acid, niflumic acid, 4- (2-hydroxyhexafluoroisopropyl) benzoic acid, N-myristoyl-Gly-OH, 3- (4-n-octylbenzoyl) propionic acid, N-octanoyl-L -Phe-OH, 4-undecyloxybenzoic acid, 3- (3,4,5-trimethoxyphenyl) propionyl-Gly-OH, 8-iodaproic acid, N-pentadecanoyl-Gly-OH, 4-dodecyloxybenzoic acid, N-palmitoyl-Gly -OH, and N-estearo il-Gly-OH These organic acids are available from one or more of Advanced ChemTech, Louisville, KY; Bachem Bioscience Inc., Torrance, CA; Calbiochem-Novabiochem Corp., San Diego, CA; Farchan Laboratories Inc., Gainesville FL; Lancaster Synthesis, Windham NH; and MayBridge Chemical Company (c / o Ryan Scientific), Columbia, SC. The catalogs of these companies use the abbreviations that were used before to identify the acids.

F. Combinatorial Chemistry as a Medium to Prepare Trademarks Combinatorial chemistry is a type of synthetic strategy that leads to the production of large chemical collections (see, for example, PCT Application Publication No. WO 94/08051). These combinatorial collections can be used as marks for the identification of molecules of interest (MOIs). Combinatorial chemistry must be defined as the systematic and repetitive, covalent connection of a series of different "building blocks" of structures that vary among themselves to produce a large array of molecular entities. The building blocks can take many forms, both naturally occurring and synthetic, such as nucleophiles, electrophiles, dienes, alkylating or acylating agents, diamines, nucleotides, amino acids, sugars, lipids, organic monomers, synthons, and combinations of the previous ones. The chemical reactions used to connect the building blocks may involve killing, acylation, oxidation, reduction, hydrolysis, substitution, elimination, addition, cyclization, condensation and the like. This process can produce collections of compounds that are oligomeric, non-oligomeric, or combinations thereof. If they are oligomeric, the compounds can be branched, unbranched or cyclic. Examples of oligomeric structures that can be prepared by combinatorial methods include oligopeptides, oligonucleotides, oligosaccharides, polylipids, polyesters, polyamides, polyurethanes, polyureas, polyethers, polyphosphorus derivatives, for example, phosphates, phosphonates, phosphoramides, phosphonamides, phosphites, phosphinamides, etc. , and polyazulfur derivatives, for example, sulfones, sulfonates, sulfonamides, sulfenamides, etc. A common type of combinatorial oligomeric collection is the combinatorial collection of peptides. Recent innovations in peptide chemistry and molecular biology have allowed collections consisting of billions of different peptide sequences to be prepared and used. These collections can be divided into three broad categories. A category of collections involves the chemical synthesis of collections of bound peptide without soluble support (eg, Houghten et al., Nature 354: 84, 1991). A second category involves the chemical synthesis of assembled peptide collections with support, presented as solid supports such as plastic pins, resin globules, or cotton (Geysen et al., Mol.Immunol., 23: 709, 1986; Lam et al. , Nature 354: 82, 1991; Eichler and Houghten, Biochemistry 32: 1 1035, 1993). In these first two categories, the building blocks are typically L-amino acids, D-am inoacids, non-natural amino acids, or some mixture or combination thereof. A third caterogy uses biology proposals to prepare peptides or proteins on the surface of phage particles with filament or plasmids (Scott and Craig, Curr, Biotech Opinion, 5:30, 1994). The collections of peptides joined without support. soluble ones seem to be suitable for a number of applications, including use as trademarks. The available repertoire of chemical diversities in peptide collections can be expanded by steps such as permethylation (Ostresh et al., Prc-Nat. Acad. Sci., US 91: 11188, 1994). Numerous variants of combinatorial collections of peptides are possible where the base structure of peptides is modified, and / or the amide bonds have been replaced by mimetic groups. Amide mimetic groups that can be used include ureas, urethanes, and carbonylmethylene groups. The restructuring of the base structure so that the side chains emanate from the amide nitrogens of each amino acid, instead of the aliacarbons, gives collections of compounds known as peptides (Simón et al., Proc. Nati. Acad. Sci., USA 89: 9367, 1992). Another common type of oligomeric combinatorial collection is the combinatorial collection of oligonucleotide, wherein the building blocks are some form of nucleotide derivatives or polysaccharides that occur naturally or unnaturally, including where various organic and inorganic groups can be substituted for the phosphate ligation, and nitrogen or sulfur can be replaced by oxygen in an ether ligation (Schneider et al., Biochem 34: 9599, 1995; Frier et al., J. Med. Chem. 38: 344, 1995; Frank, J. Biotechnology 41: 259, 1995; Schneider et al., PCT Published WO 942052; Ecker et al., Nucleic Acids Res. 21: 1853, 1993). More recently, the combinatorial production of collections of non-oligomeric small molecule compounds has been described (DeWitt et al., Proc. Nati, Acad. Sci., USA 90: 690, 1993, Bunin et al., Proc. Nati, Acad. Sci., USA 91: 4708, 1994). Suitable structures for processing in small molecule collections encompass a wide variety of organic molecules, eg heterocyclic, aromatic, alicyclic, aliphatic, steroid, antibiotic, enzyme inhibitor, ligand, hormone, drug, alkaloid, opioid, terpene, porphyrin. , toxins, catalysts, as well as combinations thereof. g. Specific Methods for Combinatorial Synthesis of Marks Two methods are delineated for the preparation and use of a diverse series of MS brands containing amine. In both methods, solid phase synthesis is used to allow simultaneous parallel synthesis of a large number of labeled linkers, using combinatorial chemistry techniques. In the first method, the final cut of the label from the oligonucleotide results in the release of a carboxyl amide. In the second method, the cut of the brand produces a carboxylic acid. The chemical components and linkers used in these methods are abbreviated as follows; R = FMOC resin = Fluorenylmethoxycarbonyl protecting group All = Allyl protecting group CO2H = carboxylic acid group CONH2 = carboxylic amide group N H2 = amino group OH = hydroxyl group WITH H = amide bond COO = ester bond NH2 - Rink - C02H = 4 - [(a-amino ) -2,4-dimethoxybenzyl] phenoxybutyric (Rink interlayer) OH - 1MeO - CO2H = (4-Hydroxymethyl) phenoxybutyric acid OH - 2MeO - CO2H = (4-hydroxymethyl! -3-methoxy) phenoxyacetic acid NH2-A-COOH = Amino acid with affinity or aromatic amine side chain functionality X2 ... Xn-COOH- = Series of n various carboxylic acids with unique molecular weights oligol ... ouego (n) = Series of n oligonucleotides HBTU = O-benzotriazole hexafluorophosphate- 1-yl N, N, N ', N'-tetramethyluronium The sequence of steps in method 1 is as follows: OH-2MeO-CONH-R i FMOC-NH-Rink-C02H; dock (eg HBTU) FMOC - NH - Rink - COO - 2MeO - CONH - R i piperidine (remove FMOC) NH 2 - Rink - COO - 2MeO - CONH - R i FMOC - NH - A - COOH; coupling (eg, HBTU) FMOC - NH - A - CONH - Rink - COO - 2MeO - CONH - R i - piperidine (eliminating FMOC) NH2 - A - CONH - Rink - COO - 2MeO - CONH - R l dividing n aliquots 4 Í - coupling with different acids X1 ... Xn - COOH X1 ... Xn - CONH - A - CONH - Rink - COO - 2MeO - CONH - R - - '' '' cut labeled resin binders with 1% TFA X1 ... Xn - CONH - A - CONH - Rink - CO2H > I coupling oligos (oligol ... oligo (n)) (eg Vía esres de Pfp) X1 ... Xn - CONH - A - CONH - Rink - CONH - oligol ... oligo (n) i extract oligos marked 4- perform reaction in sequence 4- separate fragments of different length from reaction in sequence (eg, via HPLC or CE) - cut linker marks with 25% -100% TFA X1 ... Xn = CONH - A - CONH l analyze by mass spectrometry The sequence of steps in method 2 is as follows: OH - 1MeO - C02 - All FMOC - NH - A - C02H; coupling (eg, HBTU) FMOC - NH - A - COO - 1MeO - C02 - All i palladium (remove allyl) FMOC - NH - A - COO - 1MeO - C02H 4 OH - 2MeO - CONH - R; dock (eg, HBTU) FMOC - NH - A - COO - 1MeO - COO - 2MeO - CONH - R - piperidine (remove FMOC) NH2 - A - COO - 1MeO - COO - 2MeO - CONH - R 4- divide into n aliquots 4.4444 - couple in n different acids X1 ... Xn - CO2H X1 ... Xn - CONH - A - COO - 1MeO - COO - 2MeO - CONH - R 44444 Cut labeled resin binders with 1% TFA X1 ... Xn - CONH - A - COO - 1MeO - C02H 44-4 -44 coupling oligos (oligol ... oligo (n)) (eg via Pfp esters) X1 ... Xn - CONH - A - COO - 1MeO - CONH - oligol ... oligo (n) 4 remove oligos marked 4 perform reaction in sequence 4- separate fragments of different length from reaction in sequence (eg, via HPLC OR CE) 4- cut linker labels with 25% -100% TFA X1 ... Xn - CONH - A - C02H 4 analyze by mass spectrometry 2. Linkers U n "interlacing" component (or L), as used herein, means a direct covalent bond or an organic chemical group that is used to connect a "tag" (or T) to a "molecule of interest" (or MO I) through covalent chemical bonds. . In addition, the direct link by itself, of one or more links within the interleaver component can be cut under conditions that allow T (in other words, cut) to be released from the rest of the TLX compound (including the MOI component). . The variable component of the mark that is present within T must be stable to the cutting conditions. Preferably, cutting can be achieved quickly; in a few minutes and preferably in approximately 15 seconds or less. In general, an interleaver is used to connect each of a large series of marks to each of a similarly large series of MOIs. Typically, a single mark-interleaver combination is fixed to each MOI (to give several TL-MOIs), but in some cases, more than one mark-interleaver combination can be set to each individual MOI (to give several (TL- ) n-MO I). In another embodiment of the present invention, two or more tags are joined to a single interleaver through multiple independent sites in the interleaver, and this multiple tag-interleaver combination is then attached to an individual MOI (to give several (T) nL-MOI). After several manipulations of the series of labeled MOIs, special chemical and / or physical conditions are used to cut one or more covalent bonds in the interleaver, resulting in the release of the marks from the MOIs. The link that can be cut may or may not be from some of the links that were formed when the brand, interleaver, and MOI were connected together. The design of the interlacer, in large part, will determine the conditions under which the cut can be achieved. Accordingly, linkers can be identified by the cutting conditions to which they are also particularly susceptible. When an interlacer is photo-stable (ie, prone to cutting by exposure to actinic radiation), the enlacer may obtain the designation Lhu. Also, the designations | acid, Lbase, L [0], L [R], Lepz, Lelc, LA and Lss can be used to refer to linkers that are particularly susceptible to acid, base, chemical oxidation, chemical reduction, the catalytic activity of an enzyme (more simply "enzyme"), oxidation or electrochemical reduction, high temperature ("thermal") and exchange of thio !, respectively. Certain types of interleaver are unstable to a single type of cutting condition, while others are unstable to several types of cutting conditions. In addition, in linkers that are capable of joining multiple tags (to give structures of the type of (T) n-L-MOI), each of the tag attachment sites may be unstable at different cutting conditions. For example, in an interleaver that has two marks attached to it, one of the marks may be unstable only to the base, and the other may be unstable only to photolysis.

An interleaver that is useful in the present invention has several attributes: 1) The interleaver has a chemical handle (Lh) through which an MO can be set! 2) The interleaver has a second separate chemical handle (Lh) through which the ma is fixed to the interleaver. If multiple marks are fixed to a single interleaver (structures of the type of (T) n-L-MOI), then there is a separate handle for each mark. 3) The interleaver is stable towards all manipulations to which it is subjected, with the exception of conditions that allow cutting so that a portion containing T is freed from! rest of the compound, including MOI. In this way, the interlacer is stable during the fixing of the mark to the interlacer, fixing of! interleaver to the MOI, and any manipulation of the MOI while the mark and interleaver (T-L) are attached to it. 4) The interleaver does not interfere significantly with the manipulations performed in the MOI while the T-L is fixed to it. For example, if the T-L is attached to an oligonucleotide, the T-L should not interfere significantly with any hybridization or enzymatic reactions (eg, PCR) performed on the oligonucleotide. Similarly, if e! T-L is bound to an antibody, it must not interfere significantly with the recognition of antigen by the antibody.

) The cut of the brand of the rest of the compound occurs in a highly controlled manner, by using physical or chemical processes that do not adversely affect the detection of the brand. For any given interleaver, it is preferred that the interleaver can be set to a wide variety of MOIs, and that a wide variety of marks can be set to the interleaver. Said flexibility is advantageous since it allows a collection of T-L conjugates, once prepared, to be used with several series of different MOIs. As explained above, a preferred interleaver has the formula Lh-L1-L2-L3-Lh wherein each Lh is a reactive handle that can be used to interlace the interleaver to a brand reagent and a molecule reagent of interest. L2 is an essential part of! interleaver, since L2 imparts instability to the interleaver. L1 and L3 are optional groups that effectively serve to separate L2 from the handles Lh. L1 (which, by definition, is closer to T than L3), if rve to 'separate T from the required portion L2 i nestable. This separation can be useful when the shear reaction generates particularly reactive species (eg, free radicals) that can cause random changes in the structure of the portion containing T. As the cutting site is further separated from the portion that contains T, there is a reduced probability that the reactive species formed at the cutting site will distort the structure of the portion containing T. as well, since the atoms in L1 will typically be present in the portion containing T, these atoms L1 can impart a desirable quality to the portion containing T. For example, where the portion containing T is a portion containing Tms, a hindered amine desirably appears as part of the structure of the portion containing Tms (to serve , for example, as an MSSE), the hindered amine may be present in the unstable portion of L1. In other cases, L1 and / or L3 may be present in an interlacing component simply because the commercial supplier of an interleaver chooses to sell the interleaver in a form having said group L1 and / or L3. In that case, there is no harm in using linkers that have L1 and / or L3 groups, (as long as these groups do not inhibit the shear reaction) although they may fail to contribute to any particular performance for the compounds they incorporate. In this way, the present invention allows groups L1 and / or L3 to be present in the interlacing component. The L and / or L3 groups can be a direct bond (in which case the group is not present effectively), a hydrocarbylene group (for example, alkylene, arylene, cycloalkylene, etc.), -O-hydrocarbylene (for example, -O-CH2-, O-CH2CH (CH3) -, etc.), or hydrocarbylene- (O-hydrocarbon) w- wherein w is an integer ranging from 1 to about 10 (e.g., -CH2- 0-Ar-, CH2- (0-CH2-CH2) 4-, etc.).

With the advent of solid phase synthesis, a large body of literature has been developed with respect to linkers that are unstable for specific reaction conditions. In typical solid phase synthesis, a solid support is attached through an unstable interleaver to a reactive site, and a molecule to be synthesized is generated at the reactive site. When the molecule has been completely synthesized, the solid support-molecule-interlayer construction is subjected to cutting conditions that release the molecule from the solid support. Unstable linkers that have been developed for use in this context (or that can be used in this context) can also be used as the crosslinking reagent in the present invention. Lloyd-Williams, P., et al., "Convergent Soüd-Phase Peptide Synthesis", Tetrahedron Report No. 347, 49: 481 1065- 1 1 133 (1993) provides an extensive discussion of linkers that are unstable to actinic radiation (ie, say, photolysis), as well as acid, base and other cutting conditions. Other sources of information about unstable linkers can be obtained. As described above, different linker designs will confer cut ("instability") under different specific physical or chemical conditions. Examples of conditions serving to cut various interlacing designs include acid, base, oxidation, reduction, fluorine, thiol exchange, photolysis, and enzyme conditions. Examples of linkers that can be cut that satisfy the general criteria for linkers listed above will be well known to those skilled in the art and include those found in the available catalog of Pierce (Rockford, IL). Examples include "• Gl? Cobi (succinim? Dilsuccinate) ethylene (EGS), an amine reactive interlayer reagent that can be cut by hydroxylamine (1 M at 37 ° C for 3-6 hours); • Disucchymidyl tartarate. (DST) and sulfo-DST, which are amine reactive linker reagents, which can be cut by 0.015 M sodium pepodate, • Bi [2- (succinimidylox? Carbonyloxy) ethyl] sulfone (BSOCOES) and sulfo-BSOCOES, which are amine reactive linker reagents, which can be cut per base (pH 11.6), • 1,4-di- [3 '- (2'-p? r? d? lt? o (prop? onamido)) butane (DPDPB ), a pipdilditiol interlayer that can be cut by thiol exchange or reduction, • N- [4- (p-azidosalicylamido) -butyl] -3 '- (2'-pyrididithio) propionamide (APDP), a pyridyldithiol interlayer that can be cut by exchange of til or reduction; • Bi- [beta-4- (azido!? cilamido) ethyl] -disulfide, a photoreactive interlayer that can be cut by thiol exchange or reduction; • N-succ? nimidil - (4-az? Dofen il) -1, 3'-d? thiopropionate (SADP), a photoreactive interlayer that can be cut by thiol exchange or reduction; • Sufosuccinimidi! -2- (7-azido-4-methylcumarin-3-acetamide) ethyl-1,3'-dithiopropionate (SA ED), a photoreactive interlayer that can be cut by thiol exchange or reduction; • Sulfosuccinimidyl-2- (m-azido-o-nitrobenzamido) -ethyl-1,3-dithiopropionate (SAN D), a photoreactive interlayer that can be cut by thiol exchange or reduction. Other examples of binders that can be cut and the cutting conditions that can be used to release marks are as follows. A silyl crosslinking group can be cut by fluorine or under acidic conditions. A 3-, 4-, 5- or 6-substituted-2-nitrobenzyloxy or 2-, 3-, 5-, or 6-substituted-4-nitrobenzyloxy group can be cut by a source of photons (photolysis). A 3-, 4-, 5- or 6-substituted-2-alkoxyphenoxy or 2-, 3-, 5- or 6-substituted-4-alkoxyphenoxy group can be cut by Ce (N H4) 2 (N03) and (oxidation). An NC02 interlayer (urethane) can be cut by hydroxide (base), acid, or LiAIH (reduction). A 3-pentyl, 2-butenyl or 1-butenyl interlacing group can be cut by 03, 0S0 / I04", or KMn04 (oxidation) A 3- [3-, 4- or 5-substituted-furyl interlacing group ] oxy can be cut by 02 l Br2, MeOH, or acid The conditions for cutting other unstable linking groups include: t-alkyloxy linkers groups that can be cut by acid; methyl (dialkyl) methoxy or 4-substituted-2-alkyl-1,3-dioxlan-2-yl linkers which can be cleaved by H30 +; 2-silylethoxy linker groups which can be cut by fluorine or acid; linker groups 2- (X) -ethoxy (wherein X = keto, ester, amide, cyano, N02, sulphide, sulfoxide, sulfone) which can be cut under alkaline conditions; linker groups 2-, 3-, 4-, 5- or 6-substituted-benzyloxy which can be cut by acid or under reducing conditions; 2-butenyloxy linkers which can be cleaved by (Ph3P) 3RhCl (H), 3-, 4-, 5- or 6-substituted-2-bromophenoxy linkers which can be cleaved by Li, Mg, or BuLi; methylthiomethoxy linker groups which can be cleaved by Hg2 +; linker groups 2- (X) -ethyloxy (wherein X = a halogen) which can be cut by Zn or Mg; 2-hydroxyethyloxy linker groups that can be cut by oxidation (for example, with Pb (OAc) 4). Preferred linkers are those that are cut by acid or photolysis. Several of the acid-labile binders that have been developed for solid-phase peptide synthesis are useful for interlayer markings at MOIs. Some of these linkers are described in a recent review by Lloyd-Williams and others (Tetrahedron 49: 1 1065-1,1333, 1993). A useful type of interlayer is based on p-alkoxybenzyl alcohols, of which two, 4-hydroxymethylphenoxyacetic acid and 4- (4-hydroxymethyl-3-methoxyphenoxy) butyric acid, are available commercially from Advanced ChemTech (Louisville, KY ). Both linkers can be attached to a label via an ester linkage to the benzylalcohol, and to an amine containing MOI via an amide linkage to the carboxylic acid. The tags interlaced by these molecules are released from the MOI with varying concentrations of trifluoroacetic acid. The cutting of these entrßlazadors results in the release of a carboxylic acid in the brand. Acidic cleavage of labels bound through related linkers, such as 2,4-dimethoxy-4 '- (carboxymethyloxy) -benchidplamine (available from Advanced ChemTech in FMOC-protected form), results in the release of an amide carboxylic in the released brand. The photoinstable binders useful for this application have also been largely developed for solid phase peptide synthesis (see review by Lloyd-Williams). These linkers are usually based on 2-nitrobenzyl esters or 2-nitrobenzylamides. Two examples of photoinstable linkers that have recently been recorded in the literature are 4- (4- (1-Fmoc-am ino) ethyl) -2-methoxy-5-nitrophenoxy) butanoic acid (Holmes and Jones, J. Org. 60: 2318-2319, 1995) and 3- (Fmoc-amino) -3- (2-nitropheni!) Propionic acid (Brown et al., Molecular Diversity 1: 4-12, 1995). Both linkers can be fixed via the carboxylic acid to an amine in the MOI. The fixation of the mark to the interleaver is made by forming an amide between a carboxylic acid in the label and the amine in the interleaver. The cutting of photoinstable linkers is usually carried out with UV light of 350 nm wavelength at intensities and times known in the art. The cutting of the linkers results in the release of a primary amide in the label. Examples of photoinstable linkers include nitrophenyl glycine esters, exo- and endo-2-benzonorborneyl chlorides and methane sulfonates, and 3-amino-3 (2-nitropheni!) Propionic acid. Examples of enzymatic cleavage include stearases that will cut ester bonds, nucleases that will cut phosphodiester bonds, proteases that will cut peptide bonds, etc.

A preferred crosslinker component has an ortho-nitrobenzyl structure as shown below: d wherein a carbon atom at positions a, b, c, d or e is substituted with -L3-X, and L1 (which is preferably a direct bond) is present to the left of N (R1) in the above structure. Said interlacing component is susceptible to selective photo-induced cleavage of the bond between the carbon labeled "a" and N (R1). The identity of R1 is not typically critical to the shear reaction, however R1 is preferably selected from hydrogen and hydrocarbyl. The present invention provides that in the above structure, -N (R 1) - can be replaced with -O-. Also in the above structure, one or more positions b, c, d or e can optionally be substituted with alkyl, alkoxy, fluorine, chloride, hydroxyl, carboxylate or amide, wherein these substituents are independently selected in each occurrence. Another preferred interlacing component with a chemical handle Lh has the following structure: wherein one or more positions b, c, doe is substituted with hydrogen, alkyl, alkoxy, fluorine, chloride, hydroxyl, carboxylate or amide, R 1 is hydrogen or hydrocarbyl, and R 2 is -OH or a group that protects or activates a carboxylic acid to couple with another portion. The fluorocarbon and hydrofluorocarbon groups are preferred groups that activate a carboxylic acid toward coupling with another portion. 3. Molecule of Interest (MOI) Examples of MOIs include nucleic acids or nucleic acid analogs (e.g., APN), fragments of nucleic acids) (i.e., nucleic acid fragments), nucleic acids or synthetic fragments, oligonucleotides (e.g. example, DNA or RNA), proteins, peptides, antibodies or antibody fragments, receptors, receptor ligands, members of a pair of ligands, cytokines, hormones, oligosaccharides, synthetic organic molecules, drugs, and combinations thereof. Preferred MOIs include nucleic acid fragments. Preferred nucleic acid fragments are primer sequences that are complementary to sequences present in vectors, wherein the vectors are used for base sequence. Preferably, a fragment of nucleic acid binds directly or indirectly to a tag in another one different from the 3 'end of the fragment; and very preferable at the 5 'end of! fragment. Nucleic acid fragments can be acquired or prepared based on genetic databases (eg, Dib et al., Nature 380: 152-154, 1996 and CEPH Genotype Datábase, http: // www. Cephb. Fr) and commercial vendors (for example, Promega, Madison, Wl). As used herein, MOI includes derivatives of an MO! which contains useful functionality to join the MO I to a compound of T-L-Lh. For example, a nucleic acid fragment having a phosphodiester at the 5 'end, wherein the phosphodiester is also attached to an alkyleneamine, is an MOI. Said MO I is described in, for example, Patent of E. U.A. 4, 762,779, which is incorporated herein by reference. A fragment of nucleic acid with an internal modification is also an MOI. An example internal modification of a nucleic acid fragment is where the base (eg, adenine, guanine, cytosine, thymidine, uracil) has been modified to add a reactive functional group. Such internally modified nucleic acid fragments are commercially available from, for example, Glen Research, Herndon, VA. Another exemplary internal modification of a nucleic acid fragment is where an abasic phosphoramidate is used to synthesize a modified phosphodiester that is interposed between a sugar and phosphate group of a nucleic acid fragment. AND! Abasic phosphoramidate contains a reactive group that allows a nucleic acid fragment containing this portion derived from postforamidate to bind to another portion, for example, a compound of T-L-Lh. Said abasic phosphoramidates are commercially available from, for example, Clonetech Laboratories, Inc., Palo Alto, CA. 4. Physical Manichas (Lh) A chemical handle is a stable but reactive atomic disposition present as part of a first molecule, where the handle can pass through chemical reaction with a complementary chemical handle present as part of a second molecule, in order to form a covalent bond between the two molecules. For example, the chemical handle can be a hydroxyl group, and the complementary chemical handle can be a carboxylic acid group (or an activated derivative thereof, for example a hydrofluoroaryl ester), wherein the reaction between these two handles it forms a covalent bond (specifically, an ester group) that binds the two molecules together.

Chemical handles can be used in a large number of reactions that form covalent bonds that are suitable for fixing labels to linkers, and linkers to MOIs. Such reactions include alkylation (e.g., to form ethers, thioethers), acylation (e.g., to form esters, amides, carbamates, ureas, thioureas), phosphorylation (e.g., to form phosphates, phosphonates, phosphoramides, phosphonamides), sulfonylation (for example, to form sulfonates, sulfonamides), condensation (for example, to form mines, oximes, hydrazones), sweating, disulfide formation, and generation of reactive intermediates, such as nitrenes or carbenes, by photolysis. In general, handles and link-forming reactions that are suitable for setting tags to linkers are also suitable for attaching linkers to MOIs, and vice versa. In some cases, the MOI may undergo prior modification or derivatization to provide the handle necessary to fix the interleaver. One type of link especially useful for linking linkers to MOIs is the disulfide bond. Its formation requires the presence of a thiol group ("handling") in the linker, and another thiol group in the MOI. Then the mild oxidation conditions may be sufficient for the binding of the two thiols as a disulfide. The formation can also be induced using an excess of an appropriate disulfide exchange reagent, for example, pyridyl disulfides. Since the disulfide formation is easily reversible, the disulfide can also be used as the separable bond to release the label, if desired. This is commonly achieved under mild conditions in a similar manner, using an excess of an appropriate thiol exchange reagent, for example, dithiothreitol. The formation of amide bonds is of particular interest for linking tags (or tags with linkers) with oligonucleotides. Primary aliphatic amine handling can be easily introduced into oligonucleotides with phosphoramidite (available from Glenn Research, Sterling, VA). The amines found in natural nucleotides such as adenosine and guanosine are virtually inactive when compared to the introduced primary amine. This difference in reactivity forms the basis of the ability to selectively form amides and related linking groups (eg, ureas, thioureas, sulfonamides) with the introduced primary amine, and not the aucleotide amines.

As listed in the Molecular Test catalog (Eugene, OR), a partial enumeration of reactive amine functional groups includes activated carboxylic esters, isocyanates, isothiocyanates, sulfonyl halides and dichlorotriazenes. The active esters are excellent reactants for amine modification since the amide products formed are very stable. Also, these reagents have a good reactivity with aliphatic amines and low reactivity with nucleotide amines of oligonucleotides. Examples of active esters include N-hydroxysuccinimide esters, pentafluorophenyl esters, tetrafluorophenyl esters and p-nitrophenyl esters. Active esters are useful since they can be made virtually from any carboxylic acid-containing molecule. Methods to make active esters are listed in Bodansky (Principies of Peptide Chemistry (2nd ed.), Springer Verlag, London, 1993). 5. Linker Union Commonly, an individual type of linker is used to connect a particular set or family of marks to a particular set or family of MOIs. In a preferred embodiment of the invention, a simple uniform procedure can be followed to create all the various T-L-MOI structures. This is especially advantageous when the set of structures T-L-MO I is large, since it allows the assembly to be prepared using the combination chemistry methods or other parallel processing technology. Similarly, the use of an individual type of linker allows a simple and uniform procedure to be used to separate all the various T-L-MO I structures. Again, this is advantageous for a large set of T-L-MOI structures, since the set can be processed in a parallel, repetitive and / or automatic manner. However, there is another embodiment of the present invention, in which two or more types of linker are used to connect different subsets of tags with corresponding subsets of MOIs. In this case, the selective separation conditions can be used to separate each one in the linkers independently, without separating the linkers present in other subsets of MOIs. A large number of reactions that form a covalent bond are suitable for linking tags with linkers, and linkers with MOIs. Such reactions include alkylation (e.g., to form ethers, thioethers), acylation (e.g., to form esters, amides, carbamates, ureas, thioureas), phosphorylation (e.g., to form sulfonates, sulfonamides), condensation (e.g. to form mines, oximes, hydrazones), sylation, disulfide formation and generation of reactive intermediates, such as nitrenes or carbenes, by means of photolysis. In general, handling and link-forming reactions that are suitable for joining tags with linkers are also useful for linking linkers with MOIs and vice versa. In some cases, the MOI may undergo modification or prior derivation to provide the necessary handling to join the linker. One type of link especially useful for linking linkers with MOIs is the disulfide bond. Its formation requires the presence of a thiol group ("handling") in the linker; and another thiol group in the MOI. The mild oxidation conditions are then sufficient to bind the two thiols together as a disulfide. The disulfide formation can also be induced using an excess of an appropriate disulfide exchange reagent, for example, pyridyl disulfides. Because the disulfide formation is easily reversible, the disulfide can also be used as the separable bond to release the label, if desired. This is commonly achieved under similarly mild conditions, using an extension of an appropriate thiol exchange reagent, for example, dithiothreitol.

The formation of amide bonds is of particular interest for linking tags with oligonucleotides. The handling of primary aliphatic amine can be easily introduced into synthetic oligonucleotides with phosphoramidites such as 6-monomethoxytritylyl hexylcyanoethyl-N, N-diisopropyl phosphoramidite (available from Glenn Research, Sterling, VA). The amines found in natural nucleotides such as adenosine and guanosine are virtually nonreactive when compared to the introduced primary amine. This difference in reactivity forms the basis of the ability to selectively form amides and related linking groups (eg, ureas, thioureas, sulfonamides) with the introduced primary amine and not the nucleotide amines. As listed in the Molecular Test catalog (Eugene, OR), a partial enumeration of reactive amine functional groups includes activated carboxylic esters, isocyanates, isothiocyanates, sulfonyl halides and dichlorotriazenes. The active esters are excellent reactants for amine modification since the amide products formed are very stable. Also, these reagents have a good reactivity with aliphatic amines and low reactivity with nucleotide amines of oligonucleotides. Examples of active esters include N-hydroxysuccinimide esters, pentafiuorophenyl esters, tetrafluorophenyl esters and p-nitrophenyl esters. Active esters are useful since they can be made virtually from any carboxylic acid-containing molecule. Methods for making active esters are listed in Bodansky (Principies of Peptide Chemistry (2nd ed.), Springer Verlag, London, 1993). There are numerous commercial crosslinking reagents that can serve as linkers (for example, see Pierce Crosslinkers, Pierce Chemical Co., Rockford, I L). Among these are the homobifunctional amine reactant crosslinking reagents which are exemplified by homobifunctional imidoesters and N-hydroxysuccinimidyl (N HS) esters. There are also heterobifunctional interlacing reagents that have two or more different reagent groups that allow sequential reactions. The imidoesters react rapidly with amines at an alkaline pH. Esters of N HS give stable products when they react with primary or secondary amines. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyl disulfides are reactive to thiol. The maleimides are specific for thiol (sulfhydryl) groups on the pH scale of 6.5 to 7.5, and at an alkaline pH they can be reacted to amine. The thioether bond is stable under physiological conditions. The alpha-haloacetyl entanglement reagents contain the iodoacetyl group and are reactive towards sulfhydryls. Imidazoles can react with the iodoacetyl moiety, but the reaction is very slow. The pyridyl disulfides react with thiol groups to form a disulfide bond. The carbodiimides couple carboxyls with primary amines of hydrazides which give rise to the formation of an acyl-hydrazine bond. Arilazides are photoaffinity reagents that are chemically inert until they are exposed to ultraviolet or visible light. Reactive aryl nitrene is relatively non-specific. The glyoxal are reactive toward the guanidinyl portion of arginine. In a typical embodiment of the present invention, a tag is first linked to a linker, then the tag-linker combination is linked to a MOI, to create the T-L-MOI structure. Alternatively, the same structure is formed by first linking a linker to an MOI, and then linking the linker and MOI combination with a tag. An example is where MOI is a DNA primer or oligonucleotide. In that case, the tag is commonly linked to a linker, then T-L is linked to a DNA primer or oligonucleotide, which is later used, for example, in a sequence reaction. A useful form in which a tag can be reversibly linked to an MOI (eg, an oligonucleotide or DNA sequence primer) is through a chemically unstable linker. A preferred design for the linker allows the linker to be removed when exposed to a volatile organic acid, for example, trifluoroacetic acid (TFA). In particular, TFA is compatible with most MS ionization methods, including electrospray. As described in more detail below, the invention provides methodology for forming genotypes. A composition that is useful in the method of forming genotypes comprises a plurality of compounds of the formula: Tms-L-MOI wherein, Tms is an organic group that can be detected by mass spectrometry, comprising carbon, at least one of hydrogen and fluorine, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine. In the formula, L is an organic group that allows a portion containing Tms to be separated from the remainder of the compound, wherein the portion containing Tms comprises a functional group that supports an individual ionized charge state when the compound is subjected to mass spectrometry and it is a fragment of nucleic acid in which L is conjugated with MO I in a different location from the 3 'end of MOI. In the composition, at least two compounds have the same Tms, but the MOI groups of those molecules do not have identical nucleotide lengths. Another composition that is useful in the genotype forming method comprises a plurality of compounds of the formula: Tms-L-MOI wherein T s is an organic group that can be detected by means of mass spectrometry, comprising carbon, by at least one of hydrogen and fluorine, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine. In the formula, L is an organic group that allows a portion containing Tms to be separated from the remainder of the compound, wherein the portion containing Tms comprises a functional group that supports an individual ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid. In the formula, MOI is a fragment of nucleic acid in which L is conjugated with MOI at a location different from that at the 3 'end of MOI. In the composition, at least two compounds have the same Tms but those compounds do not have identical elution times by column chromatography. Another composition that can be used in the genotype-forming method comprises a plurality of compounds of the formula: Tms-L-MO! wherein Tms is an organic group that can be detected by mass spectrometry, comprising carbon, at least one of hydrogen and fluorine, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine. In the formula, L is an organic group that allows a portion containing Tms to be separated from the remainder of the compound, wherein the portion containing T s comprises a functional group that supports an individual ionized charge state when the compound is it is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid. In the formula, MOI is a fragment of nucleic acid in which L is conjugated with MOI at a location different from that at the 3 'end of MOI. In the composition, the two compounds that have the same nucleotide length of MOI do not have the same Tms. In the above composition, preferably the plurality is greater than 2, and preferably greater than 4. Also, the nucleic acid fragment in the MOI has a sequence complementary to a portion of a vector, wherein the fragment is capable of initiating the synthesis of polynucleotide. Preferably, the Tms groups of members of the plurality differ by at least 2 amu, and may differ by at least 4 amu. The invention also provides a composition comprising a plurality of sets of compounds, each set of compounds having the formula: Tms-L-MOI wherein Tms is an organic group that can be detected by mass spectrometry, comprising carbon, at least one of hydrogen and fluorine, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine. In the formula, L is an organic group that allows a portion containing Tms to be separated from the remainder of the compound, wherein the portion containing T s comprises a functional group that supports an individual ionized charge state when the compound is undergoes mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid. Also, in the formula, MOI is a nucleic acid fragment in which L is conjugated with MOI at a location other than the 3 'end of MOI. In the composition, the members within the first set of compounds have identical Tms groups, however they have non-identical MOI groups with numbers that differ from the nucleotides in MOI and there are at least ten m members within the first set, where between sets , the T s groups differ by at least 2 amu. The plurality preferably is at least 3, and more preferably at least 5. The invention also provides a composition comprising a plurality of sets of compounds, each set of compounds having the formula Tms-L-MO I wherein , Tms is an organic group that can be detected by means of mass spectrometry, which comprises carbon, at least one of hydrogen and fluorine, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine. In the formula, L is an organic group that allows a portion containing Tms to be separated from the remainder of the compound, wherein the portion containing Tms comprises a functional group that supports an individual ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid. In the formula, MO I is a nucleic acid fragment in which L is conjugated with MOI in a location other than the 3 'end of MOI. In the composition, the compounds within a set have the same elution time but non-identical Tms groups. In addition, the invention provides equipment for forming genotypes.

The equipment comprises a plurality of pairs of amplification primers, wherein at least one of the primers has the formula: Tms-L-MOI wherein, Tms is an organic group that can be detected by means of mass spectrometry, which it comprises carbon, at least one of hydrogen and fluorine, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine. In the formula, L is an organic group that allows a portion containing Tms to be separated from the remainder of the compound, wherein the portion containing Tms comprises a functional group that supports an individual ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid. In the formula, MOI is a fragment of nucleic acid in which L is conjugated with MOI at a location other than the 3 'end of MOI; each pair of primers is associated with a different location. In the equipment, the plurality of preference is at least 3, and more preferably at least 5. As mentioned above, the present invention provides compositions and methods for determining the sequence of neclic acid molecules. Briefly, such methods generally comprise the steps of (a) generating labeled nucleic acid fragments that are complementary to a selected nucleic acid molecule (e.g., labeled fragments) from a first term in a second term of an acid molecule nucleic), wherein a tag is correlated with a particular or selected nucleotide, and can be detected by any of a variety of methods, (b) separating tagged fragments by sequence length, (c) separating a tag from a fragment labeled, and (d) detecting the labels and thus determining the sequence of the nucleic acid molecule. Each of the aspects will be discussed in more detail below.

B. DIAGNOSTIC METHODS 1. Introduction As mentioned above, the present invention also provides a wide variety of methods in which the labels and / or linkers described above can be used in place of traditional labels (eg, radioactive or enzymatic), in order to improve the specificity, sensitivity or the number of samples that can be analyzed simultaneously, within a given method. Representative examples of such methods that can be improved include, for example, RNA amplification (see Lizardi et al., Bio / Technology 6: 1197-1202, 1988; Kramer et al., Nature 339: 401-402, 1989; others, Clinical Chem. 35 (9): 1826-1831; U.S. Patent No. 4,786,600), and DNA amplification using CSF or polymerase chain reaction ("PCR") (see, U.S. Patent No. 4,683,195 , 4,683,202 and 4,800,159). In one aspect of the present invention, methods are provided for determining the identity of a nucleic acid molecule or fragment (or for detecting the presence of a selected nucleic acid molecule or fragment), comprising the steps of (a) generating molecules of labeled nucleic acid from one or more selected target nucleic acid molecules, wherein a label is correlated to a particular nucleic acid molecule and can be detected by means of non-fluorescent spectrometry or potentiometry, (b) separating labeled molecules by size, (c) separating the labels of the labeled molecules and (d) detecting the labels by means of non-fluorescent spectrometry or potentiometry, and thus determining the identity of the nucleic acid molecules. In a related aspect of the invention, methods are provided for detecting a selected nucleic acid molecule, comprising the steps of (a) combining the labeled nucleic acid tests with target nucleic acid molecules under conditions and for a sufficient time to allow Hybridization of a labeled nucleic acid test for a selected target nucleic acid sequence, where a labeled nucleic acid test can be detected by means of non-fluorescent spectrometry or potentiometry, (b) altering the size of hybridized labeled tests, non-hybridized tests or target molecules, or test hybrids: objective, (c) separating the tests marked by size, (d) separating the marks from the marked tests and (e) detecting the marks by means of non-fluorescent spectrometry or potentiometry, and thus detecting the selected nucleic acid molecule. These, and other related techniques, are described in more detail later. 2. PCR PCR can amplify a desired DNA sequence from any source (virus, bacteria, plant or human) hundreds of millions of times in a matter of hours. PCR is especially valuable because the reaction is highly specific, easily automated and capable of amplifying minute amounts of sample. For these reasons, PCR has had a great impact on clinical medicine, the diagnoses of genetic disease, forensic science and the biology of evolution. Briefly, PCR is a procedure based on a specialized polymerase, which can synthesize a complementary strand for a given strand of DNA in a mixture containing 4 base pairs of DNA and 2 DNA fragments (primers, each approximately one length 20 bases) flanking the target sequence. The mixture is heated to separate the double-stranded DNA strands containing the target sequence and then cooled to allow (1) the primers to find and bind to their complementary sequences on the separate strands and (2) the polymerase to extend the primers in complementary strands. The repeated heating and cooling cycles multiply the target DNA exponentially, since each new double strand separates to become two tempered for further synthesis. In about 1 hour, 20 cycles of PCR can be amplified a million times the target. In one embodiment of the invention, methods are provided to determine the identity of a nucleic acid molecule, or to detect the selected nucleic acid molecule in, for example, a biological sample, using the PCR technique. Briefly, such methods comprise the steps of generating a series of fragments or nucleic acid molecules labeled during PCR t to separate the resulting fragments by size. The size separation step can be achieved using any of the techniques described herein, including for example electrophoresis (e.g., polyacrylamide gel electrophoresis) or preferably HPLC. Then the marks can be separated from the separated fragments and detected by means of the respective detection technology. Examples of such technologies have been described herein, and include for example mass spectrometry, infrared spectrometry, potentiostatic amperometry or ultraviolet ray spectrometry. 3. RNA Traces and Differential Deployment When tempering is RNA, the first step in the trace is inverted transcription. Liang and Pardee (Science 257: 967, 1992) were the first to describe an RNA fingerprint protocol, using a primer for oligo-based transcription (dT), but with an 'anchor' of two bases at the 5 'end ( for example, oligo 5 '- (dT11) CA-3 \ The start occurs mainly at the 5' end of poly (rA) tail and mainly in sequences that end 5'-UpG-poly (rA) -3 ', with a focusing selectively on one of 12 polyadenylated RNAs After transcription and denaturation, arbitrary initiation is performed on the first resulting strand of cDNA PCR can now be used to generate a footprint of products that match primers better and that derived from the 3 'end of polyadenylated heterogeneous mRNA and RNA.This protocol has been termed "differential display." Alternatively, an arbitrary primer can be used in the first inverted transcription step, selecting those regions internal to the RNA having 6- 8 pairs that match the 3 'end of the primer. This is followed by arbitrary initiation of the first strand resulting from cDNA with the same or a different primer and then subjected to PCR. This particular protocol is shown anywhere in the RNA, including reading frames (Welsh et al., Nuc.Acids, Res. 20: 4965, 1992). In addition, it can be used in RNAs that are not polyadenylated, such as bacterial RNAs. This variant of RNA fingerprinting by means of arbitrarily initiated PCR has been called RAP-PCR. If RNA traces are made with arbitrarily initiated PCR in samples derived from cells, tissues and other biological material that has undergone different experimental treatments or has different histories of development, between the samples can be detected differences in the expression of the gene. For each reaction, it is assumed that the same number of effective PCR duplication events occur and any differences in the initial concentrations of cDNA products are preserved as a ratio of intensities in the final footprint. There are no meaningless relationships between the intensities of the bands within an individual lane or a gel, which are a function of coincidence and abundance. However, the ratio between lanes is maintained for each sample RNA, allowing differentially expressed RNAs to be detected. The ratio of starting materials between samples is maintained even when the number of cycles is sufficient to allow the PCR reaction to saturate. This is because the number of duplications needed to achieve saturation is almost completely controlled by the non-variant products that create the most of the footprint. In this respect, fingerprinting with PCR is different from conventional PCR of a single product in which the ratio of starting materials between samples is not preserved unless the products are sampled in the exponential phase of amplification. In one embodiment of the invention methods are provided for determining the identity of a nucleic acid molecule in, for example, a biological sample, using the RNA fingerprint technique. Briefly, such methods usually comprise the steps of generating a series of labeled nucleic acid fragments. The fragments are generated by means of PCR or similar amplification schemes and then subsequently separated by size. This size separation step can be, for example, any of the techniques described herein, including for example gel electrophoresis (e.g., polyacrylamide gel electrophoresis) or preferably H PLC. Then the marks are separated from the separated fragments, and then the marks are detected by means of the respective detection technology. Representative examples of suitable technologies include mass spectrometry, infrared spectrometry, potentiostatic metrometry or ultraviolet ray spectrometry. The relative amounts of any given fragment of nucleic acid are not important, but the size of the band is informative when referred as a control sample. 4. Individual Chain Conformation Polymorphism of Fluorescence-based PCR (PCR-SSCP) A number of methods are available in addition to the RFLP approach to analyze base substitution polymorphisms. Orita and others have discovered a way to analyze these polymorphisms on the basis of conformational differences in denatured A DN. Briefly, the digestion of restriction enzymes or PCR is used to produce small fragments of DNA that are then denatured and resolved by electrophoresis in undenatured polyacrylamide gels. Differences in conformation in the individual strand DNA fragments resulting from base substitutions are detected by electrophoretic mobility changes. The making of internal chain pairs creates individual chain conformations that are highly specific sequence and distinctive electrophoretic mobillity. However, the detection rates in different studies using conventional sscp vary from 35% to almost 100% with the highest detection rates generally requiring several different conditions. In principle, the method can also be used to analyze polymorphisms based on inserts or short separations. This method is one of the most powerful tools for identifying DNA mutation and separation sites (SSCP-PCR, Dean et al., Cell 61: 863, 1990). In one embodiment of the invention methods are provided for determining the identity of a nucleic acid molecule, or for detecting a selection nucleic acid molecule, for example, in a biological sample, using the PCR-SSP technique. Briefly, such methods generally comprise the steps of general a series of labeled nucleic acid fragments. Then the fragments generated by means of PCR are separated by size. Preferably, the size separation step is non-denatured and the nucleic acid fragments are denatured prior to the separation methodology. The separation step by size can be achieved, for example by gel electrophoresis (e.g., polyacrylamide gel electrophoresis) or preferably HPLC. The marks of separating fragments are then separated, and then the marks are detected by means of the respective detection technology (for example, mass spectrometry, infrared spectrometry, potentiostatic amperometry or ultraviolet ray spectrometry).

. Traces of Dideoxy (ddF) Another method has been described (ddF, Sakar et al., Genomics 13: 441, 1992) that detected 100% of individual base changes in the human factor IX gene when tested in a retrospective and prospective manner . In total, 84 of 84 different sequence changes were detected when analyzed in genomic DNA from patients with hemophilia B. Briefly, in the applications of brands to form genotypes or other purposes, one method that can be used is the dideoxy trace. The principle of the method is as follows: a target nucleic acid to be sequenced is placed in a reaction having a dideoxy terminator complementary to the base which is known to mutate in the target nucleic acid. For example, if the mutation results in an A- >change; G, the reaction would be carried out in a dideoxy terminator reaction C. PCR primers are used to locate and amplify the target sequence of interest, if the hypothetical target sequence contains the change A->. G, the size of the sequence population is changed due to the incorporation of a dideoxy terminator in the amplified sequences. In this particular application of marks, a fragment would be generated that would have a predictable size in the case of a mutation. The tags would bind to the 5 'end of the PCR primers and provide a' map 'for sampling type and dideoxy terminator type. A PCR amplification reaction would take place, the resulting fragments would be separated by size for example HPLC or PAGE. At the end of the separation procedure, the DNA fragments are collected in a temporal reference frame, the marks are separated and the presence or absence of mutation is determined by means of the length of the chain due to the premature chain terminator by means of the incorporation of a dideoxy terminator dice. It is important to note that ddf results in the gain or loss of a dideoxy termination segment and / or a change in the mobility of at least one of the termination segments or products. Therefore, in this method, a search is made for the change of a fragment mobility in a high environment of other fragments of another molecular weight. An advantage is the knowledge of the fragment length associated with a given mutation. In one embodiment of the invention methods are provided for determining the identity of a nucleic acid molecule, or for detecting a selection nucleic acid molecule, for example, in a biological sample, using the ddF technique. Briefly, such methods usually comprise the steps of generating a series of labeled nucleic acid fragments, followed by separation based on size. Preferably, the size separation step is not denatured and the nucleic acid fragments are denatured before the separation methodology. The step of separation by size is achieved, for example by gel electrophoresis (e.g., polyacrylamide gel electrophoresis) or preferably HPLC. The marks are then separated from the separated fragments, and then the marks are detected by means of the respective detection technology (for example, mass spectrometry, infrared spectrometry, potentiostatic amperometry or ultraviolet ray spectrometry). 6. Restriction maps and RFLPs Restriction endonucleases recognize short DNA sequences and separate DNA molecules at specific sites. Some restriction enzymes (rare separators) separate DNA very infrequently, generating a small number of very large fragments (several thousand to one million bp). Most enzymes separate DNA more frequently, thus generating a large number of small fragments (less than one hundred to more than one thousand bp). On average, restriction enzymes with 4-base recognition sites will produce base length of 256 pieces, 6-base recognition sites will produce base length of 4,000 pieces, and 8-base recognition sites will produce length of 64 bits, 000 pieces. Since hundreds of different restriction enzymes have been characterized, the A DN can be separated into many small fragments. A great variety of techniques for the analysis of DNA polymorphisms has been developed. The most widely used method, the restriction fragment length polymorphism approach (R FPL), combines restriction enzyme digestion, gel electrophoresis, membrane staining and hybridization of a cloned DNA test. Polymorphisms are detected as variations in the lengths of the fragments marked in the dyeings. The RFPL approach can be used to analyze base substitutions when the sequence change falls within a restriction enzyme site or to analyze minisatellites / VNTRs by choosing restriction enzymes that separate the outer part of the repeating units. Agarose gels usually do not have the resolution necessary to distinguish minisatile / VNTR alleles that differ by a single repeat unit, but many of the minisatellites / VNTRs are so variable that highly informative markers can still be obtained. In one embodiment of the invention methods are provided for determining the identity of a nucleic acid molecule, or for detecting a selection nucleic acid molecule, for example, in a biological sample, using the technique of restriction mapping or RFLPs. Briefly, such methods usually comprise the steps of generating a series of labeled nucleic acid fragments in which the generated fragments are digested with restriction enzymes. The labeled fragments are generated by conducting a hybridization step of the labeled tests with the digested target nucleic acid. The hybridization step can take place before or after digestion with restriction nuclease. The step can be achieved, for example, by gel electrophoresis (e.g., polyacrylamide gel electrophoresis) or preferably HPLC. Then the marks of the separated fragments are separated, and then the marks are detected by means of the respective detection technology (for example, mass spectrometry, infrared spectrometry, potentiostatic amperometry or ultraviolet ray spectrometry). 7. DNA fingerprint The fingerprint of DNA involves the deployment of a set of DNA fragments from a specific DNA sample. A variety of DNA fingerprinting techniques are now available (Jeffries et al., Nature 314: 67, 1985, Welsh and McClelland, Nuc.Acids, Res. 19; 861, 1991), most of which use PCR to generate fragments The choice of which footprint technique to use depends on the application, for example, type of DNA, making DNA marker maps and the organisms under investigation, for example, prokaryotes, plants, animals, humans. In recent years, a large number of fingerprint methods have been developed that meet these requirements, including random amplified polyomorphic DNA (RAPD). Traces of DNA amplification (DAF) and arbitrarily primed PCR (AP-PCR). These methods are based on the amplification of random genomic DNA fragments by the arbitrary selection of PCR primers. The DNA fragment patterns can be generated in the sequence of the PCR primers and the nature of the tempered DNA. PCR is performed at low tempering temperatures to allow the primers to be quenched at multiple locations in the DNA. The DNA fragments are general when the primer binding sites are within a distance allowing amplification. At first, an individual primer is sufficient to generate band patterns. A new technique for DNA fingerprints has been described, called AFLP (Vos et al, Nuc Acids Res. 23: 4407, 1995). The AFLP technique is based on the detection of genomic restriction fragments by means of PCR amplification, and can be used for DNA of any origin or complexity. Briefly, fingerprints are produced without prior knowledge of sequence using a limited set of generic primers. The number of fragments detected in a single reaction can be "harmonious" by means of the selection of primer sets. The AFLP technique is strong and reliable because the reaction conditions are used to anneal the primer; The reliability of the RFLP technique is combined with the energy of the PCR technique. In one embodiment of the invention methods are provided for determining the identity of a nucleic acid molecule, or for detecting a selection nucleic acid molecule, for example, in a biological sample, using the DNA fingerprint technique. Briefly, such methods usually comprise the steps of generating a series of labeled nucleic acid fragments, followed by the separation of the fragments by size. The separation step by size can be achieved, for example by gel electrophoresis (e.g., polyacrylamide gel electrophoresis) or preferably HPLC. Then the marks are separated from the separated fragments, and then the marks are detected by means of the respective detection technology (for example, mass spectrometry, infrared spectrometry, potentiostatic amperometry or ultraviolet ray spectrometry). 8. Application of Separable Marks for Formation of Genotypes and Detection of Polymorphism a. Introduction Although a few known human DNA polymorphisms are based on insertions, separations or other rearrangements of non-repeated sequences, the vast majority is based on either individual base substitutions or variations in the number of double repeats. Base substitutions are very abundant in the human genome, occurring on average once every 200-500 bp. Variations of length in blocks of double repeats is also common in the genome, with at least tens of thousands of scattered polymorphic sites (called loci). Repetition lengths for double repeat polymorphisms range from 1 bp in sequences (dA) n (dT) n to at least 170 bp in alpha-satellite DNA. The double-repeat polymorphisms can be divided into two main groups consisting of mini-satellites / variable number of double repeats (VNTRs), with typical repetition lengths of ten of base pairs and with from ten to thousands of repeating units, and microsatellites, with repetition lengths of up to 6 bp and with maximum total lengths of approximately 70 bp. Most of the microsatellite polymorphisms identified so far are based on dinucleotide repeat sequences (dC-dA) n or (dG-dT) n. The analysis of microsatellite polymorphisms involves the amplification by means of polymerase chain reaction (PCR) of a small DNA fragment containing a block of repeats followed by electrophoresis of DNA amplified in denatured polyacrylamide gel. The PCR primers are complementary to the unique sequences flanking the blocks of repeats. Polyacrylamide gels, rather than agarose gels, are traditionally used for microsatellites since alleles usually differ in size only by individual repetition. Thus, in one aspect of the present invention methods for determining the genotype of a selected organism are provided, comprising the steps of (a) generating labeled nucleic acid molecules from a selected target molecule, wherein a brand is correlative to a particular fragment and can be detected by means of non-fluorescent spectrometry or potentiometry, (b) separating the labeled molecules by sequence length, (c) separating the label from the labeled molecule and (d) detecting the label by means of non-fluorescent spectrometry or potentiometry and thus determine the genotype of the organism. In another aspect of the invention, methods are provided for determining the genotype of a selected organism, comprising the steps of (a) combining a labeled nucleic acid molecule with a selected target molecule under conditions and for a time sufficient to allow hybridization of the molecule marked with the target molecule, wherein a mark is correlated with a particular fragment and can be detected by means of non-fluorescent spectrometry or penciometry, and thus determine the genotype of the organism. b. Application of Separable Marks to Detect the Genotype A PCR approach to identify the restriction fragment length polymorphism (RFPL) combines gel electrophoresis and tag detection associated with specific PCR primers. In general, a PCR primer will have a specific label. Therefore, the tag will represent a set of PCR primers and, therefore, a predetermined DNA fragment length. Polymorphisms are detected as variations of the fragments marked on a gel or eluted from a gel. Polyacrylamide gel electrophoresis will usually have the resolution needed to distinguish minisatélite / VNTR alleles that differ by a single repeat unit. The analysis of microsatellite polymorphisms involves the amplification by means of the reaction Polymerase chain (PCR) of a small DNA fragment containing a block and repeats followed by electrophoresis of the amplified DNA in denatured polyacrylamide gel or followed by separation of DNA fragments by HPLC. The amplified DNA will be labeled using primers having separable labels at the 5 'end of the primer. The primers are incorporated into the newly synthesized chains by chain extension. The PCR primers are complementary to the unique sequences flanking the repeat blocks. The mini-satellite / VNTR polymorphisms can also be amplified, as well as the microsatellites described above. Descriptions of many types of DNA sequence polymorphisms have provided the fundamental basis for understanding the structure of the human genome (Botstein et al., Am. J. Human Genetics 32: 0314, 1980; Donis-Keller, Cell 51: 319, 1987; Weissenbach et al., Nature 359: 794). The construction of extensive framework link maps has been facilitated through the use of these DNA polymorphisms and has provided a practical means for locating disease genes by binding. The microsatellite dinucleotide markers are proving to be very powerful tools in the identification of human genes that have been shown to contain mutations and in some cases cause disease. Genomic dinucleotide repeats are highly polymorphic (Weber, 1990, Genomic Analysis, Vol. 1, pp. 159-181, Cold Spring Laboratory Press, Cold Spring Harbor, NY, Weber and Wong, 1993, Hum. Mol. Genetics, 2 , p1123) and can have up to 24 alleles. The microsatellite dinucleotide repeats can be amplified using primers complementary to the unique regions surrounding the dinucleotide repeat by PCR. Following the amplification, several loci are amplified and combined (multiplexed) before the size separation step. The process of applying amplified microsatellite fragments to a separation step by size and then identifying the size and thus the allele is known as genotype determination. Chromosome specific markers that allow a high level of multiplexing have been reported to perform complete genome tests for linkage analysis (Davies et al., 1994, Nature, 371, p130). The marks can be used for greater effect in the determination of the genotype with microsatellites. Briefly, the PCR primers are constructed to carry labels and are used in a carefully chosen PC reaction to amplify repeats of di-, tri- or tetra-nucleotides. The amplification products are then separated according to size by means of methods such as HPLC or PAGE. Then the DNA fragments are collected temporarily, the marks separated from repetitive DNA fragments and the longitus deduced from the comparison with internal standards in the size separation step. The allele identification is made with reference to the size of the amplified products. With the separable mark approach to determine the genotype, it is possible to combine multiple samples in an individual separation step. There are two general ways in which this can be done. The first general method to fully examine is the detection of an individual polymorphism in a large group of individuals. In this senario an individual set or network of PCR primers is used and each amplification is done with a DNA sample type by reaction. The number of samples that can be combined in the separation step is proportional to the number of separable marks that can be generated by detection technology (for example, 400-600 for mass spectrometer marks). Therefore, it is possible to simultaneously identify 1 to several polymorphisms in a large group of individuals. The second approach is to use multiple sets of PCR primers that can identify numerous polymorphisms in a single DNA sample (for example, to determine the genotype of an individual). In this approach the PCR primers are combined in an individual amplification reaction that generates PCR products of different length. Each pair of primer or network set is encoded by means of a specific separable tag, which implies that each PCR fragment will be encoded with a specific tag. The reaction is carried out in a single separation step (see below). The number of samples that can be combined in the separation step is proportional to the number of separable marks that can be generated by detection technology (for example, 400-600 for mass spectrometer marks). c. Enzymatic Mutation Detection and Applications Markings In this particular application or method, inequalities in heteroduplexes are detected by enzymatic separation of unequal base pairs in a nucleic acid duplex. The DNA sequences to be tested for the presence of a mutation are amplified by PCR using a specific set of primers, the amplified products are denatured and mixed with denatured reference fragments and hybridized, which results in the formation of heteroduplexes. Then the heteroduplexes are treated with enzymes that recognize and separate the duplex if an inequality is found. Such enzymes are S1 nucleases, Mung beam nuclease, "resolvases", T4 endonuclease IV, etc. Essentially any enzyme that recognizes in vitro inequalities and separates the resulting inequality can be used. Treatment with the appropriate enzyme, DNA duplexes are separated by size, for example, by HPLC or PAGE. The DNA fragments are temporarily collected. The marks are separated and detected. The presence of a mutation is detected by the change in the mobility of fragments relative to a wild-type reference fragment. d. Marking Applications for the Oligonucleotide Linkage Assay (OLA) The oligonucleotide linkage assay as originally described by Landergren et al. (Landergren et al., Science 241: 487, 1988) is a useful technique for identifying sequences (known ) in very large and complex genomes. The principle of the Ola reaction is based on the ability to covalently link two diagnostic oligonucleotides as they hybridize adjacent to one another on a given DNA target. If the sequences in the test joints do not have perfectly based pairs, the tests will not be bound by ligase. The ability of a thermostable ligase to discriminate individual base pair differences when placed at the 3 'end of the "upstream" test provides the opportunity for individual base pair resolution (Barony, PNAS USA 88: 189, 1991) . In the application of marks, the marks would join the test that is linked to the amplified product. After finishing the OLR, the fragments are separated based on size, the marks are separated and detected by means of mass spectrometry. and. Sequence Specific Amplification PCR primers with a 3 'end complementary to either a mutant or normal oligonucleotide sequence can be used to selectively amplify one or the other allele (Newton et al., Nuc Acids Res., 17, p2503; others, 1989, J. Lab. Clin. Med., 114, p105; Sommer et al., 1989, Mayo Clin. Proc., 64, 1361; Wu et al., PNAS USA, 86, p2757). In general, PCR products are visualized after amplification by PAGE, but the principle of sequence specific amplification can be applied to solid phase formats.

F. Potential Application of Marks to some Amplification-based Assays Genotype determination: A potential application of tags is the identification or identification of virus genotype by hybridization with labeled tests. For example, F + RNA coliphages may be useful candidates as indicators for contamination with enteric viruses. The determination of genotype by means of nucleic acid hybridization methods is a reliable, fast, simple and inexpensive alternative to determine serotypes (Kafatos et al., Nucleic Acids Res., 7: 1541, 1979). Amplification techniques and nucleic acid hybridization techniques have been successfully used to classify a variety of microorganisms including E. coli (Feng, Mol Cell Probes 7: 151, 1993), rotavirus (Sethabutr et al., J. Med. Virol., 37: 192, 1992) m hepatitis C virus (Stuyver et al., J. Gen. Virol. 74: 1093, 1993) and herpes simplex virus (Matsumoto et al., J. Virol. Methods 40: 119, 1992). The prediction applications of mutation analysis in cancers: have described genetic alterations in a variety of experimental mammalian and human neoplasms and represent the morphological basis for the sequence of morphological alterations observed in carcinogenesis (Vogelstein et al., NEJM 319: 525, 1988 ). In recent years with the advent of molecular biology techniques, allelic losses in certain chromosomes or the mutation of tumor suppressor genes as well as mutations in several onco genes (eg, c-myc, c-jun, and the ras family) they have been the most studied entities. Previous work (Finkelstein et al., Arch. Surg. 128: 526, 1993) has identified a correlation between specific types of point mutations in the K-ras oncogene and the stage in diagnosis in colorectal carcinoma. The results suggest that the analysis can provide important information about the aggressiveness of the tumor, including the pattern and spread of metastasis. More recently, the prognostic value of the TP53 and K-ras nutation analysis in stage III of colon carcinoma has been demonstrated (Pricolo et al., Am. J. Surg. 171: 41, 1996). Therefore, it is evident that the determination of the genotype of tumors and precancerous cells, and the detection of specific mutation will be increasingly important in the treatment of cancers in humans.

C. Separation of Nucleic Acid Fragments A sample that requires analysis is usually a mixture of many components in a complex matrix. For samples containing unknown compounds, the components must be separated from each other so that each individual component can be identified by other analytical methods. The separation properties of the components in a mixture are constant under constant conditions, and therefore once determined they can be used to identify and quantify each of the components. Such procedures are typical in chromatographic and electrophoretic analytical separations. 1. High Performance Liquid Chromatography (HPLC) High performance liquid chromatography (HPLC) is a chromatographic separation technique to separate compounds that dissolve in the solution. The HPLC instruments consist of a mobile phase reserve, a pump, an injector, a separation column and a detector. The compounds are separated by injecting an aliquot of the sample mixture into the column. The different components in the mixture pass through the column at different speeds due to the differences in their division behavior between the mobile liquid phase and the stationary phase. Recently, IP-RO-HPLC in non-porous PS / DVB particles with chemically bonded alkyl chains have shown to be fast alternatives for capillary electrophoresis in the analysis of both double and simple nucleic acids that provide similar degrees of resolution (Huber et al., Anal Biochem., 212: 351, 1993, Huber et al., 1993, Nuc.Aids Res. 21: 1061, Huber et al., Biotechniques 16: 898, 1993). In contrast to ion exchange chromatography, which does not always retain double-stranded DNA as a function of chain length (since the base pairs at interact with the positively charged stationary phase, more strongly than the base pairs GC), IP-RP-HPLC make separation strictly dependent on size possible. A method using 100 mM of triethylammonium acetate as reagents to make ion pairs has been developed. Phosphodiester oligonucleotides could be successfully separated into alkylated non-porous particles of 2.3 uM poly (styrene-divinylbenzene) by means of high performance liquid chromatography (Oefner et al., Anal. Biochem. 223: 39, 1994). The described technique allowed the separation of PCR products that differ only from 4 to 8 base pairs in length on a size scale of 50 to 200 nucleotides. 2. Electrophoresis Electrophoresis is a separation technique that is based on the mobility of ions (or DNA as is the case described here) in an electric field. The negatively charged DNA migrates to a positive electrode and the positively charged ions migrate towards a negative electrode. For safety reasons one electrode usually makes ground and the other deviates positively or negatively. The loaded species have different migration speeds depending on their load, size and total configuration and therefore can be separated. An electrode apparatus consists of a high voltage power supply, electrodes, regulator and a support for the regulator such as a polyacrylamide gel, or a capillary tube. Open capillary tubes are used for many types of samples and gel supports are usually used for biological samples such as mixtures of proteins or DNA fragments. 3. Capillary Electrophoresis (CE) Capillary electrophoresis (CE) in its various manifestations (free solution, isotachophoresis, isoelectric focusing, polyacrylamide gel, micellar electrokinetics "chromatography") is being developed as a method for rapid high resolution separations of small sample volumes of complex mixtures, in combination with the sensitivity and inherent selectivity of MS, CE-MS is a powerful potential technique for bioanalysis. In the novel application described in this, the interface of these methods will lead to superior DNA sequencing methods that overshadow the speed of current sequencing methods by several orders of magnitude. The correspondence between CE and the electrospray ionisation flow rate (ESI) and the fact that both are provided by means (mainly used for) of ionic species in solution provides the basis for an extremely attractive combination. The combination of capillary zone electrophoresis (EZC) and capillary isotachophoresis with ESI-based spectrometers has been described (Olivares et al., Anal. Chem. 59: 1230, 1987; Smith and others, Anal. Chem. 60: 436, 1988; Loo and others, Anal. Chem. 179: 404, 1989; Edmonds et al., J. Chroma. 474: 21; 1989; Loo et al., J. Microcolumn Sep. 1: 223, 1989; Lee and others, J. Chromatog. 458: 313, 1988; Smith et al., J. Chromatog. 480: 211, 1989; Grese et al., J. Am. Chem. Soc. 111: 2835, 1989). Small peptides are easily subjected to EZC analysis with good sensitivity (femtomole). The most powerful separation method for DNA fragments is polyacrylamide gel electrophoresis (PAGE), generally in a thick gel format. However, the main limitation of the current technology is the relatively long time necessary to perform gel electrophoresis of DNA fragments produced in the sequence formation reactions. An increasing magnitude (10 times) can be achieved with the use of capillary electrophoresis, which uses ultra-thin gels. In the free solution in a first approximation all the DNA migrates with the same mobility as the addition of a base results in the compensation of mass and charge. In polyacrylamide gels, DNA fragments pass and migrate as a function of length and this approach has now been applied to CE. A remarkable number of plates per meter with interlaced polyacrylamide (plates 10 + 7 per meter, Cohen and others, Proc. Nati, Acad. Sci., E.U.A. 85: 9660, 1988) has now been achieved. Such CE columns described can be used to form DNA sequences. The EC method is in principle 25 times faster than coarse gel electrophoresis in a standard sequencer. For example, you can read about 300 bases per hour. The separation rate is limited in coarse gel electrophoresis by the magnitude of the electric field that can be applied to the gel without excessive heat production. Therefore, the higher CE speed is achieved by the use of higher field strengths (300 v / cm in CE versus 10 V / cm in thick gel electrophoresis). The capillary format reduces the amperage and in this way the energy and the resulting heat generation. Smith et al. (Smith et al., Nuc Acids, Res. 18: 4417, 1990) have suggested using multiple capillaries in parallel to increase passage. Similarly, Mathies and Huang (Mathies and Huang, Nature 359: 167, 1992) have introduced capillary electrophoresis in which separations are performed in a parallel array of capillaries and high step sequence formation is demonstrated (Huang and others, Anal, Chem. 64: 967, 1992, Huang et al., Anal. Chem. 64: 2149, 1992). The main disadvantage of capillary electrophoresis is the limited amount of sample that can be loaded into the capillary. By concentrating a large amount of sample at the beginning of the capillary, before separation, the loading capacity is increased, and the levels of detection can be decreased by several orders of magnitude. The most popular preconcentration method in CE is sample stacking. Sample stacking has recently been revised (Chien and Burgi, Anal, Chem. 64: 489A, 1992) - Sample stacking depends on the matrix difference, (pH, ionic strength) between the sample regulator and the regulator capillary, so that the electric field through the sample area is more than in the capillary region. In sample stacking, a large volume of sample is introduced into a low concentration regulator for preconcentration in the heat of the capillary column. The capillary is filled with a pH regulator of the same composition, but at a higher concentration. When the sample ions reach the pH regulator and the lower electric field, they are stacked in a concentrated zone. Sample stacking has increased detection by 1-3 orders of magnitude. Another method of preconcentration is to apply isotachophoresis (ITP) before separation by EC of analyte-free zone. ITP is a technique of. electrophoresis that allows microliter volumes of sample to be loaded into the capillary, in contrast to the low nL injection volumes commonly associated with CE. The technique depends on inserting the sample between two pH regulators (conduction and drag electrolytes) of higher or lower mobility, respectively, than that of the analyte. The technique is inherently a concentration technique, where the analytes are concentrated in pure zones that migrate with the same speed. The technique is currently less popular than the stacking methods described above due to the need for various conduction and entrainment electrolyte choices and the ability to separate only cationic or anionic species during the separation process. The heart of the DNA sequence formation process is the markedly selective electrophoretic separation of DNA or oligonucleotide fragments. It is remarkable because each fragment resolves and differs only by means of a nucleotide. Separations of up to 1000 fragments (1000 bp) have been obtained. An additional advantage of forming sequences with separable marks is the following. There is no need to use a thick gel format when the DNA fragments are separated by means of polyacrylamide gel electrophoresis when separable labels are employed. Since the numerous samples are combined (4 to 2000) there is no need to make samples in parallel as is the case with the current dye-primer and dye-terminator methods (eg, AB1373 sequencer). Since there is no reason to make parallel rows, there is no reason to use a thick gel. Therefore, a tube gel format can be employed for the electrophoretic separation method. Grossman (Grosman et al., Genet, Anal. Tech. Appl. 9: 9, 1992) has shown that a considerable advantage is obtained when using a tube gel format instead of a thick gel format. This is due to Joule's greater ability to dissipate heat in a tube format compared to a thick gel which results in faster completion times (by 50%), and much higher resolution of DNA fragments from high molecular weight (greater than 1000 nt). Long readings are critical in genomic sequence formation. Therefore, the use of separable tags in sequence formation has the additional advantage of allowing the user to employ the most efficient and sensitive DNA separation method, which also has the highest resolution. 4. Microfabricated Devices Capillary electrophoresis (CE) is a powerful method for forming DNA sequences, forensic analysis, PCR product analysis and restriction fragment size. CE is much faster than traditional thick PAGE since a much higher potential field can be applied with capillary gels. However, CE has the disadvantage of allowing only one sample to be processed per gel. The method combines the fastest separation times of CE with the ability to analyze multiple samples in parallel. The underlying concept behind the use of microfabricated devices is the ability to increase the information density in electrophoresis by miniaturizing the swath dimension to approximately 100 micrometers. The electronics industry commonly uses microfabrication to make circuits with characteristics of less than one size. The current density of the capillary arrangements is limited to the outer diameter of the capillary tube. Microfabrication of channels produces a higher density of arrangements. Microfabrication also allows physical assembly that is not possible with glass fibers and joins the channels directly with other devices on a chip. Some devices have been built in microchips for separation technologies. A gas chromatograph (Terry et al., IEEE Trans. Electron Device, ED-26: 1880, 1979) and a liquid chromatograph (Manz et al., Sens. Actuators B1: 249, 1990) have been manufactured on silica chips, but these devices have not been widely used. Several groups have reported fluorescent separation dyes and amido acids in prefabricated devices (Manz et al., J Chromatography 593: 253, 1992; Effenhauser et al., Anal Chem. 65: 2637, 1993). Recently, Wooley and Mathies (Woiley and Mathies, Proc. Nati. Acad. Sci. 91: 1 1348, 1994) have shown that photolithography and chemical etching can be used to make large numbers of separation channels on substrates. of glass. The channels are filled with hydroxyethyl cellulose (HEC) separation matrices. It was shown that DNA restriction fragments can be separated in just ten minutes.

D. Marker Separation As described above, different linker designs will confer separation capacity ('instability') under different specific physical and chemical conditions. Examples of conditions that serve to separate various linker designs include acid, base, oxidation, reduction, fluorine, thiol exchange, photolysis and enzymatic conditions.

Examples of separable linkers that satisfy the general criteria for the linkers listed above will be well known to those skilled in the art and include those found in the available catalog of Pierce (Rockford, IL). Examples include: - ethylene glycobis (succinimidylsuccinate) (EGS), an amine reactive entanglement reagent, which is separable by hydroxylamine (1 M at 37 ° C for 3-6 hours); - disuccinimidyl tartarate (DST) and sulfo-DST, which are reactive amine interlacing reagents, separable by 0. 015 M sodium periodate; bi [2- (succinimidyloxycarbonyloxy) ethyl] sulfone (BSOCOES) and sulfo-BSOCOES, which are reactive amine crosslinking reagents, separable by base (pH 11.6); 1,4-di- [3 '- (2'-pyridyldithio (propionamido) butane (DPDPB), a pyridyldithiol interlayer which can be separated by thiol reduction or exchange; N- [4- (p-azidosalicylamido) -butyl] -3 '- (2'-pyrididithio) propionamide (APDP), a pyridyldithiol interleaver that can be separated by thiol reduction or exchange; - bi- [beta-4- (azidosalicylamido) ethyl] -disulfide, a photoreactive crosslinker which can be separated by thiol reduction or exchange; - N-succinimidyl- (4-azidophenyl) -1,3'-dithiopropionate (SADP), a photoreactive crosslinker which can be separated by thiol reduction or exchange; sulfosuccinimidyl-2- (7-azido-4-methylcumarin-3-acetamide) etiI-1,3'-dithiopropionate (SAED), a photoreactive interlayer that can be separated by thiol reduction or exchange; sulfosuccinimidyl-2- (m-azido-o-nitrobenzamido) -ethyl-1,3'-dithiopropionate (SAND), a photoreactive crosslinker which can be separated by thiol reduction or exchange. Other examples of linkers that can be separated are the separation conditions that can be used to release labels are the following. A silyl linker group can be separated by acidic conditions. A substituted 3-, 4-, 5- or 6- 2-nitrobenzyloxy substituted or substituted 2-, 3-, 5- or 6- 4-nitrobenzyloxy group can be separated by means of a photon source (photolysis). A substituted 3-, 4-, 5- or 6- 2-alkoxyphenoxy or substituted 2-, 3-, 5- or 6- 4-alkoxyphenoxy group can be separated by means of Ce (NH4) 2 (N03) 6 ( oxidation). A 3-pentenyl, 2-butenyl, or 1-butenyl linking group can be separated by means of 03, OS, 04/04, or KMn04 (oxidation). A substituted 2- [3-, 4- or 5-furyl] oxy group can be separated by means of 02, Br2, MeOH or acid. Conditions for the separation of other stable linking groups include: t-alkyloxy linking groups that can be separated by acid; methyl (dialkyl) methoxy or 4-2-substituted-l, 3-dioxlan-2-yl linking groups which can be separated by means of H30 +; 2-silylethoxy linker groups which can be separated by means of fluorine or acid; linker groups 2 - (X) -ethoxy (where X = keto, amide ester, cyano, sulfide, sulfoxide, sulfone) which can be separated under alkaline conditions; 2-, 3-, 4-, 5- or 6-benzyloxy substituted which can be separated by acid or under reductive conditions; 2-butenyloxy linker groups which can be separated by (Ph3P) 3RhCl (H), substituted 3-, 4-, 5- or 6- 2-bromophenoxy linking groups which can be separated by Li, Mg or BuLi; Methylthiomethoxy linker groups which can be separated by means of Zn or Mg; 2-hydroxyethyloxy linker groups which can be removed by oxidation (for example, with Pb (OAc) 4). Preferred linkers are those that are separated by acid or photolysis. Several unstable acid linkers have been discovered for solid-phase peptide syntheses that are useful for attaching labels with MOIs. Some of these linkers are described in a recent review by Lloyd-Wílliams et al. (Tetrahedron 49: 1 1065-1,1333, 1993). A useful type of linker is based on p-alkoxybenzyl alcohols, of which two, 4-hydroxymethylphenoxy acid and 4- (4-hydroxymethyl-3-methoxyphenoxy) butyric acid, are commercially available from Advanced ChemTech (Louisville, KY) . Both linkers can be attached to a label by means of an ester linkage with benzyl alcohol, and with an MO I containing amine via an amide linkage with the carboxylic acid. The labels bound by these molecules are released from MOI with varying concentrations of trifluoroacetic acid. The separation of these linkers results in the release of carboxylic acid in the label. The acid separation of the linked labels by related linkers, such as 2,4-dimethoxy-4 '- (carboxymethyloxy) -benzylhydrylamine (available from Advanced Chem Tech in FMOC-protected form), results in the release of an amide. carboxylic in the released brand. The photoinstable binders useful for this application have been mostly developed for solid phase peptide synthesis (see Lloyd-Williams magazine). These linkers are commonly based on 2-nitrobenzyl esters or 2-nitrobenzylamides. Two examples of photoinstable linkers that have been recently reported in the literature are 4- (4- (1-Fmoc-am ino) ethyl) -2-methoxy-5-nitrophenoxy) butanoic acid (Holmes and Jones, J. Org. 60: 2318-2319, 1995). Both linkers can be linked by carboxylic acid to an amine in MO I. The binding of the label to the linker is formed by forming an amide between a carboxylic acid in the label and the amine in the linker. The separation of photoinstable linkers is usually carried out with ultraviolet rays of 350 nm wavelength at intensities and times known to those skilled in the art. Examples of commercial sources of photochemical separation instruments are Aura Industries, Inc. (Staten Island, NY) and Agrenetics (Wilmington, MA). The separation of the linkers results in the release of a primary amide in the label. Examples of photoseparation linkers include nitrophenyl glycine esters, exo-endo-2-benxonorboneyl chlorides and methane sulphonates, and 3-amino-3 (2-nitrophenyl) propionic acid. Examples of enzymatic separation include sterases that will separate ester bonds, nucleases that will separate phosphodiester bonds, proteases that separate peptide bonds, etc.

E. Mark Detection Detection methods are commonly supported in absorption and emission in some type of spectrum field. When atoms and molecules absorb light, the input energy excites a quantized structure at a higher energy level. The type of excitation depends on the wavelength of the light. The electrons are promoted to higher orbits by means of ultraviolet or visible rays, the molecular vibrations are excited by means of infrared rayor, and the rotations are excited by means of microwaves. An absorption spectrum is the absorption of light as a function of wavelength. The spectrum of an atom or molecule depends on its energy level structure. The absorption spectra are useful for the identification of compounds. Specific absorption spectroscopic methods include atomic absorption spectroscopy (AA), infrared spectroscopy (R l) and ultraviolet ray spectroscopy (vs-uv). Atoms and molecules that are excited at higher energy levels can decay to lower levels by emitting radiation. This emission of light is called fluorescence if the transition is between states of the same rotation, and phosphorescence if the transition occurs between different rotation states. The emission intensity of an analyte is linearly proportional to the concentration (at low concentrations), and is useful for quantifying the emitting species. Specific emission spectroscopy methods include atomic emission spectroscopy (EEA), atomic fluorescence spectroscopy (EFA), laser induced fluorescence (FIL) and x-ray fluorescence (FRX). When electromagnetic radiation passes through matter, most of the radiation continues in its original direction but a small fraction is diverted in other directions. Light that deviates at the same wavelength as the incoming light is called Rayleigh deviation. The light that deviates in transparent solids due to vibrations (phonos) is called Brillouin deviation. The Brillouin deviation commonly moves by 0.1 to 1 wave number from the incident light. The light that is deflected due to vibrations in the molecules or optical fononos in opaque solids is called Raman deviation. The deviated Raman light moves as much as 4000 wave buffers from the incident light. Specific deviation spectroscopy methods include Raman spectroscopy. The spectroscopy (Rl) is the measurement of the wavelength and the intensity of the absorption of average infrared rays by means of a sample. The average infrared rays (2.5 - 50μM, 4000 - 200cm-1) is sufficiently energetic to excite molecular vibrations at higher energy levels. The wavelength of the Rl ablosion bands are characteristic of specific types of chemical bonds and the Rl spectroscopy is generally more useful for the identification of organic and organometallic molecules. Near infrared absorption spectroscopy (IRC) is the measurement of the wavelength and the intensity of infrared absorption by a sample. Near infrared light extends the 800 nm - 2.5 μM (12, 500 - 4000 cm '1) scale and is energetic enough to excite overtones and combinations of molecular vibrations at higher energy levels. IRC spectroscopy is commonly used for quantitative measurement of organic functional groups, especially O-H, N-H and C = 0. The components and design of IRC instrumentation are similar to the vs-uv absorption spectrometers. The light source is usually a tungsten lamp and the detector is commonly a PbS solid-state detector. The sample supports can be glass or quartz and typically the solvents are CCI and CS2. The convenient instrumentation of I RC spectroscopy makes it suitable for online monitoring and process control. Ultraviolet and visible abosorption spectroscopy (vs-uv) is the measurement of the wavelength and absorption intensity of near ultraviolet light and visible by a sample. Absorption in UV vacuum occurs at 100-200 nm; (105 - 50, 000 cm "1) UV quartz at 200-350 nm (50, 000-28, 570 cm" 1) and visible at 350-800 nm; (28, 570-12, 500 cm "1) and is described by the Beer-Lambert-Bouguet law, ultraviolet and visible light are sufficiently energetic to promote external electrons at higher energy levels. vs-uv can be applied to molecules and inorganic ions or complexes in the solution.Vis-uv spectra are limited by clear characteristics of the spectra.The light source is usually a hydrogen lamp or deuterium for measurements of uv and a tungsten lamp for visible measurement The wavelengths of these continuous light sources are selected with a wavelength separator such as a prism or grating monochromator.The spectra are obtained by scrutinizing the wavelength separator and quantitative measurements can be made from a spectrum or at a simple wavelength .. Mass spectrometers use the difference in mass to charge ratio (m / z) of atoms or molecules ionized cells and also for chemical determination and structural information about molecules. Molecules have distinctive fragmentation patterns that provide structural information to identify compounds. The general operations of a mass spectrometer are as follows. Gas phase ions are created, the ions are separated in space and time based on their mass to charge ratio, and the number of ions of each mass to charge ratio is measured. The energy of the ion separation of a spectrometer is described by means of resolution, which is defined as R = m / delta m, where m is the mass of ion and delta m is the difference in mass between two resolviral peaks in a mass spectrum. For example, a mass spectrometer with a resolution of 1000 can solve an ion with an m / z of 100.1. In general, a mass spectrometer (MS) consists of an ion source, a mass-selective analyzer, and an ion detector. Magnetic, quadripole, and time-of-flight sector designs also require optical extraction and acceleration ions to transfer ions from the source region to the mass analyzer. The details of various designs of mass analyzers (for EM magnetic sector, EM quadrupole or in time of flight) are described below. The individual focus analyzers for EM magnetic sector use a particle beam path of 180, 90 and 60 degrees. The various forces that influence the separated ions of particle with different mass relationships with charge. With the double-focus analyzers, an electrostatic analyzer is added to this type of instrument to separate particles with difference in kinetic energies. A quadrupole earth filter for EM quadrupole consists of metal rods arranged in parallel. Applied voltages affect the path of ions traveling down the flight path centered between the four rods. For given DC and AC voltages, only ions of some mass ratio with charge pass through the quadrupole filter and all other ions are sent to their original path. A mass spectrum is obtained by monitoring the ions that pass through the quadrupole filter as the voltages in the rods vary. A time-of-flight spectrometer uses the differences in transit time through a "region of inertia" to separate ions of different masses. It operates in a pulse mode so that the ions must be produced in pulses and / or extracted in pulses. A pulsed electric field accelerates all ions in a region of field-free inertia with a kinetic energy of q V, where q is the ion change and V is the applied voltage. Since the kinetic energy is 0.5 m V2, the lighter ions have a higher velocity than the heavier ions and reach the detector faster in the end of the region of inertia. The output of an ion detector is displayed on an oscilloscope as a function of time to produce the mass spectrum. The ion formation process is the starting point for mass spectrometric analysis. Chemical ionization is a method that uses a reactive ion to react with analyte molecules (brands) to form ions either through a proton or by hydride transfer. Reactive ions are produced by introducing a large excess of methane (relative to the brand) into an electron impact source (EI). Electron shocks produce CH + and CH3 + that react more with methane to form CHS + and C2HS +. Another method to ionize brands is by means of plasma and luminosity discharge. The plasma is partially hot ionized gas that excites and ionizes the atoms effectively. A brightness discharge is a low pressure plasma maintained between two electrodes. Electron impact ionization employs an electron beam, commonly generated from a tungsten filament, to ionize gas phase molecules or atoms. A beam electron pushes an electron out of the analyte atoms or molecules to create ions. Electrospray ionization uses a very fine needle and a series of skimmers. A sample solution is sprayed into the source chamber to form droplets. The drops carry charge when they leave the capillary and as the solvent vaporizes the drops disappear leaving highly charged analyte molecules. The rapid bombardment of atoms (RBA) uses a high energy beam of neutral atoms, commonly Xe or Ar, which hits a solid sample causing desorption and ionization. This is used for large biological molecules that are difficult to obtain in the gas phase. RBA causes small fragmentation and usually gives a large molecular ion peak, making it useful for molecular weight determination. The atomic beam is produced by accelerating ions from an ion source through a load cell. The ions collect an electron in collisions with neutral atoms to form a beam of high energy atoms. Laser ionization (EMIL) is a method in which a laser pulse removes material from the surface of a sample and creates a microplasm that ionizes some of the constituents of the sample. Ionization by assisted matrix laser desorption (IDLMA) is an EMIL method of vaporization and ionization of large biological molecules such as proteins or DNA fragments. The biological molecules are dispersed in a solid matrix such as nicotinic acid. A UV laser pulse ejects the matrix that carries some of the large molecules into the gas phase in an ionized form so that they can be extracted in a mass spectrometer. The ionization of plasma desorption (DP) uses the decrease of 252 Cf that produces two fission fragments that travel in opposite directions. A fragment hits the sample by hitting 1-10 analyte ions. The other fragment hits a detector and activates the start of data acquisition. This ionization method is especially useful for large biological molecules. Resonance ionization (EM I R) is a method in which one or more laser beams are harmonized with the resonance for transitions of a gas phase molecule or atom. To promote it in a gradual way about its ionization potential to create an ion. Secondary ionization (EMSI) uses an ion beam; such as 3He +, 160+ or 40Ar +; it focuses on the surface of a sample and sparkles material in the gas phase. The spark source is a method that ionizes solid samples by pressing an electric current through two electrodes. A tag can be loaded before, during or after the separation of the molecule to which it is attached. Ionization methods based on ion "de-settlement", the direct formation or emission of ions from solid or liquid surfaces have allowed to increase the application to non-volatile and thermally unstable compounds. These methods eliminate the need for neutral molecule volatization prior to ionization and generally minimize the thermal degradation of the molecular species. These methods include field desorption (Becky, Principles of Field lonization and Field Desorption Mass Espectometry, Pergamon, Oxford, 1977), plasma desorption (Sundqvist and Macfarlane, Mass Spectrom, Rev. 4: 421, 1985), laser desorption (Karas and H illenkamp, Anal, Chem. 60: 2299, 1988; Karas et al., Angew. Chem. 101: 805, 1989). fast particle bombardment (e.g., rapid particle bombardment, BRP, and secondary ion mass spectrometry, EM IS, Barber et al., Anal. Chem. 54: 645A, 1982) and ionisation with thermal spraying (TA) (Vestal, Mass Spectrom Rev. 2: 447, 1983). Thermospray is widely applied for the on-line combination with liquid chromatography. The BRP continuous flow methods (Caprioli et al., Anal. Chem. 58: 2949, 1986) have also shown significant potential. A more complete list of combinations of ionization / mass spectrometry is mass spectrometry of ion trap, mass spectrometry of ionization by electroaspersion, mass spectrometry of ion spray, liquid ionization mass spectrometry, mass spectrometry of ionization by atmospheric pressure, ionization spectrometry with electron, ionization mass spectrometry by bombardment of metastable atom, ionization mass spectrometry by fast bombardment of atom, mass spectrometry IDLMA, mass spectrometry by time of flight for photoionization, spectrometry of laser drip mass, mass spectrometry IDLMA-TDV, APCI mass spectrometry. nanospray mass spectrometry, resonance ionization mass spectrometry, secondary ionization mass spectrometry, mass spectrometry by thermal spraying. Docile ionization methods to non-volatile biological compounds have variations of applicability that overlap. The ionization efficiencies are highly dependent on the matrix composition and the type of compound. The currently available results indicate that the upper molecular mass for TA is approximately 8000 daltons (Jones and Krolik, Rapid Comm, Mass Spectrom, 1:67, 1987). Since TA is mainly practiced with cadmipolar mass spectrometers, the sensitivity usually suffers disproportionately at higher mass with charge ratios (m / z). Time-of-flight mass spectrometers (TDV) are commercially available and have the advantage that the m / z variation is limited only by the efficiency of the detector. Recently, two additional ionization methods have been presented. These two methods are now referred to as laser-assisted matrix desorption (ILMA, Karas and Hillenkamp, Anal., Chem. 60: 2299, 1988, Karas et al., Angew, Chem. 101: 805, 1989) and electroerosing ionization (IEA). ). Both methodologies have very high ionization efficiency (for example, [molecular ions producedj / fmollecules consumed] very high). The sensitivity, which defines the ultimate potential of the technique, depends on the size of the sample, the amount of ions, the flow rate, the detection efficiency and the actual ionization efficiency. EM-Electrospray is based on an idea proposed first in the decade of the '60s (Dole and others, J. Chem. Phys. 49: 2240. 1 968) Electrospray ionization (IEA) is a means to produce molecules for analysis by mass spectroscopy. Briefly, electrospray ionization produces highly charged drops by nebulizing liquids in a strong electrostatic field. Highly charged drops, usually formed in a dry bath gas at atmospheric pressure, shrink by evaporation of neutral solvent until the repulsion of the charge exceeds the cohesive forces, leading to a "Coulomb explosion." The exact mechanism of ionization is controversial and several groups have hypothesized (Blades et al., Anal. Chem. 63: 2109-14, 1991; Kerbarle and others, Anal. Chem. 65: A972-86, 1993; Fenn, J. Am. Soc. Mass Spectrom. 4: 524-35, 1993). Without taking into account the ultimate ion formation process, IEA produces charged molecules from a solution under mild conditions. The ability to obtain data from useful spectra in small quantities of an organic molecule is supported by the efficient production of ions. The ionization efficiency by IEA refers to the degree of positive charge associated with the molecule. Experimentally improving ionization has usually involved using acidic conditions. Another method for improving ionization has been the use of quaternary amines when possible (see Aebersold et al., Protein Science 1: 494-503, 1992; Smith et al., Anal. Chem. 60: 436-41, 1988). Electrospray ionization is described in more detail in the following manner. The production of ion by electroaspersion requires two steps: dispersion of highly charged drops at near atmospheric pressure, followed by conditions to induce evaporation. A solution of analyte molecules is passed through a needle that remains at high electrical potential. At the end of the needle, the solution dispersed in a vapor of highly charged small droplets containing the analyte molecules. The small droplets evaporate quickly and, through a process of field desorption or residual evaporation, protonated protein molecules are released in the gas phase. An electrospray is usually produced by applying a high electric field to a small flow of liquid (usually 1 -10 uL / min) from the capillary tube. A potential difference of 3-6 kV is commonly applied between the capillary electrode and the opposite located 0.2-2 cm away (where ions, charged groups, and even charged droplets, depending on the degree of desolvation, can be sampled by medium of EM through a small hole). The electric field results in the accumulation of charge on the liquid surface in the capillary terminal; thus, the liquid flow rate, resistivity, and surface tension are important factors in the production of drops. The high electric field results in the disruption of the liquid surface and the formation of highly charged liquid droplets. Drops positively or negatively charged may occur depending on capillary deviation. The negative ion mode needs the presence of an electron scavenger such as oxygen to inhibit the electric discharge. A wide variety of liquids can be electrostatically sprayed in a vacuum or with the aid of a nebulizer. The use of only electrical fields for nebulization leads to some practical restrictions in the liquid conductivity and dielectric constant scale. The conductivity of the solution of less than 10-5 ohms is needed at room temperature for a stable electrospray at liquid flow rates that correspond to an aqueous electrolyte solution of < 10-4 M. In the most useful found mode for IEA-EM, an appropriate liquid flow rate results in the dispersion of the liquid as fine vapor. A short distance from the capillary the diameter of the drop is usually quite uniform and in the order of 1μm. It is of particular importance that the total electrospray ion current increases only slightly for higher liquid flow rates. There is evidence that heating is useful for handling electroaspersion. For example, the light heating allows the aqueous solutions to be easily electrified, presumably dubbed at the desired viscosity and surface tension. Both the thermally assisted and the gas atomizing assisted atomisation allow higher liquid flow rates to be used, but decrease the degree of drop loading. The formation of molecular ions requires conditions to effect the evaporation of the initial drop population. This can be achieved at higher pressures by means of a flow of dry gas at moderate temperatures (<60 ° C), heating during transportation through the interface, and (particularly in the case of methods to trap the ion) by energetic shocks at a relatively low pressure. Although the detailed processes underlying the IEA remain uncertain, the small droplets produced through IEA seem to allow almost any species to have a net charge in the solution to be transferred to the gas phase after the evaporation of residual solvent. The mass spectrometric detection then requires that the ions have a variation of m / z that can be followed (<400 daltons for quadripolar instruments) after desolvation, and that they are produced and transmitted with sufficient efficiency. The wide range of solutions already found to receive the EM-IEA, and the lack of substantial dependence on ionization efficiency with molecular weight, suggests a highly non-discriminative and widely applicable ionization process. The "source" of electrospray ion works near atmospheric pressure. The "source" of electrospray is typically a metal or capillary glass that incorporates a method for electrically diverting the liquid solution with respect to a counter electrode. Solutions, typically water-methanol mixtures containing the analyte and often other additives such as acetic acid, flow to the capillary terminal. An ESI source has been described (Smith et al., Anal, Chem. 62: 885, 1990) which can essentially accommodate any solvent system. Typical flow rates for ESI are 1-10 μL / min. The main requirement of an ESI-MS interface is to test and transport ions from the high pressure region in the MS as efficiently as possible. The efficiency of ESI can be very high, by providing the basis for extremely sensitive measurements, which is useful for the invention described herein. Current instrument performance can provide a total ion current at the detector of approximately 2 x 10"12 A or approximately 107 counts / sec for individually charged species At the base of instrumental operation, concentrations as low as 10" 10 M or approximately 10"1 S / mol of an individually charged species will give detectable ions current (approximately 10 counts / s) if the analyte is completely ionized, For example, low atomole detection limits have been obtained for Quaternary ammonium ions using an ESI interface with capillary zone electrophoresis (Smith et al., Anal Chem 59: 1230, 1988) For a compound with a molecular weight of 1000, the average number of charges is 1, the number approximate load states is 1, the maximum width (m / z) is 1 and the maximum intensity (ions / s) is 1 x 1 01 2. Notably, little sample is actually consumed when obtaining an ESI mass spectrum (Smith and others , Anal, Chem. 60: 1 948, 1988). Substantial gains can also be obtained by the use of disposition detectors with sector instruments, allowing simultaneous detection of portions of the spectrum. Since only about 10.5 of the ions formed by ESI are currently detected, attention to the factors limiting instrumental functioning can provide a basis for improved sensitivity.It will be apparent to those skilled in the art that the present invention contemplates and accommodates improvements in ionization and detection.An interface is preferably placed between the separation instrumentation (for example, gel) and the detector (for example, mass spectrometer) .The interface preferably has the following properties: (1) the ability to collect the DNA fragments at discrete time intervals, (2) concentrate the DNA fragments, (3) remove the DNA fragments from the electrophoresis regulators, (4) cut the DNA fragment mark, ( 5) separating the brand of the DNA fragment, (6) arranging the DNA fragment, (7) placing the mark in a volatile solution, (8) volatize and ionize the brand, and (9) place or transport the brand to an electrospray device that introduces the brand into the mass spectrometer. The interface also has the ability to "collect" DNA fragments as they exit the bottom of a gel. The gel can be composed of a muddy gel, a tubular gel, a capillary gel, etc. The DNA fragments can be collected by several methods. The first method uses an electric field where DNA fragments are collected on or near an electrode. A second method is where the DNA fragments are collected by flowing a current or liquid that passes the bottom of a gel. Aspects of both methods can be combined in which DNA collected in a flowing stream that can then be concentrated by the use of an electric field. The end result is that the DNA fragments were removed from the medium under which the separation method was performed. That is, DNA fragments can "crawl" from one type of solution to another by the use of an electric field. Once the DNA fragments are in the appropriate solution (compatible with electroaspersion and mass spectrometry) the tag can be cut from the DNA fragment. The DNA fragment (or remnants thereof) can be separated after labeling by the application of an electric field (preferably, the label is of opposite charge to that of the DNA label). The mark is then introduced into the electrospray device by the use of an electric field or a flowing liquid. Fluorescent labels can be identified and quantified more directly by their wavelengths and absorption and fluorescence emission emissions. Although a conventional spectrofluorometer is extremely flexible, by providing continuous excitation scales and emission wavelengths (lE ?, i, ls2), more specialized instruments such as flow cytometers and laser scanning microscopes require probes that can be excited at a single fixed wavelength. In contemporary instruments, this is usually the 488 nm line of the argon laser. The fluorescence intensity per probe molecule is proportional to the product of e and QY. The scale of these parameters between fluorophores of current practical importance is approximately 10,000 to 100,000 cm'1 M "1 for e and 0.1 to 1.0 for QY When the absorption is directed towards saturation by high intensity illumination, the irreversible destruction of the Excited fluorophore (photobleaching) becomes the factor that limits fluorescence detection The practical impact of photobleaching depends on the fluorescent detection technique in question It will be apparent to a person skilled in the art that a device (an interface) can be interposed between the separation and detection steps to allow the continuous operation of size separation and brand detection (in real time) This unites the separation and instrumentation methodology with the detection and instrumentation methodology that make up a device. is interposed between a separation and detection technique by mass spectrometry or amperometry potentiostatic The function of the interface is mainly the release of the brand from the analyte (for example, mass spectrometry). There are several implements representative of the interface. The design of the interface depends on the choice of linkers that can be cut. In the case of linkers that can be photo-cut, a source of energy or photons is required. In the case of an unstable acid linker, an unstable base linker, or a disulfide linker, reagent addition is required within the interface. In the case of unstable heat linkers, a source of energy heat is required. Enzyme addition is required for an enzyme-sensitive linker such as a specific protease and a peptide linker, a nuclease and an RNA or DNA linker, a glycosylase, H RP or phosphatase and a linker that is unstable after cutting (for example, similar to chemiluminescent substrates). Other characteristics of the interface include minimum bandwidth, DNA separation of marks before injection in a spectrometer. Separation techniques include those based on electrophoretic methods and techniques, affinity techniques, size retention (dialysis), filtration, and simlar.

EXAMPLES EXAMPLE 1 PREPARATION OF UNSTABLE ACID LINERS TO BE USED IN MW IDENTIFIER SEQUENCE THAT CAN BE CUT Synthesis of pentafluorophenyl esters of mass spectroscopy labels that can be cut chemically, to release labels with carboxyl amide terminals Figure 1 shows the reaction scheme. Step A. Resin TentaGel S AC (compound II, available from ACT, 1 eq.) Is suspended with DMF in the collection container of the peptide synthesizer ACT357 (ACT). Compound I (3 eq.), HATU (3 eq.) And DIEA (7.5 eq.) In DMF are added and the collection vessel is stirred for 1 hour. The solvent is removed and the resin is washed with NMP (2X), MeOH (2X), and DMF (2X). The coupling of I to the resin and the washing steps are repeated, to give compound III. Step B. The resin (compound III) is mixed with 25% piperidine in DMF and stirred for 5 minutes. The resin is filtered, then mixed with 25% piperidine in DMF and stirred for minutes. The solvent is removed, the resin is washed with NMP (2X), MeOH (2X) and DMF (2X), and used directly in step C. Step C. The unprotected resin from step B is suspended in DMF and an FMOC-protected amino acid, which contains amine functionality in its side chain, is added. (compound IV, for example, alpha-N-FMOC-3- (3-pyridyl) -alanine, available from Synthetech, Albany, OR, 3 eq.), HATU (3 eq.), and DIEA (7.5 eq.) in DMF. The vessel is stirred for 1 hour. The solvent is removed and the resin is washed with NMP (2X), MeOH (2X), and DMF (2X). The IV coupling to the resin and washing steps are repeated to give compound V. Step D. The resin (compound V) is tested with piperidine as described in step B to remove the FMOC group. The deprotected resin is then divided equally by ACT357 of the collection vessel into 16 reaction vessels. Step E. The 16 aliquots of unprotected resin from step D are suspended in DMF. To each reaction vessel is added the appropriate carboxylic acid V-16 (R?.? SC02H; 3 eq.), HATU (3 eq.), And DIEA (7.5 eq.) In DMF. The containers are stirred for 1 hour. The solvent is removed and the resin aliquots are washed with NMP (2X), MeOH (2X) and DMF (2X). The coupling of Vl? -16 to the aliquots of resin and the washing steps are repeated, to give compounds VI .iß.

Step F. The resin aliquots (compounds Vll? 16) are washed with CH 2 Cl 2 (3X). To each reaction vessel is added 1% TFA in CH 2 Cl 2 and the vessels are stirred for 30 minutes. The solvent is filtered from the reaction vessels in individual tubes. The resin aliquots are washed with CH2Cl2 (2X) and MeOH (2X) and the filtrates are combined in the individual tubes. The individual tubes are evaporated under vacuum, providing compounds Vll .ie-Step G. Each of the free carboxylic acids Vll .ie is dissolved in DMF. Pyridine (1.05 eq.) Is added to each solution, followed by pentafluorophenyl trifluoroacetate (1.1 eq.). The mixtures are stirred for 45 minutes at room temperature. The solutions are diluted with EtOAc, washed with 1 M aqueous citric acid (3X) and 5% aqueous NaHCO3 (3X), dried over Na2SO, filtered, and evaporated in vacuo, by providing compounds IX1-16.

B. Synthesis of pentafluorophenyl esters of mass spectroscopy labels that can be cut chemically, to be released with carboxyl acid terminals Figure 2 shows the reaction scheme. Step A. 4- (Hydroxymethyl) phenoxybutyric acid (Compound I; 1 eq.) Is combined with DIEA (2.1 eq.) And allyl bromide (2.1 eq.) In CHCl3 and heated at reflux for 2 hours. The mixture is diluted with EtOAc, washed with 1 N HCl (2X), pH 9.5 carbonate buffer (2X) and brine (1X), dried over Na2SO4, and evaporated in vacuo to give the allyl ester of compound I. Step B. The allyl ester of compound I of step A (1.75 eq.) Is combined in CH2Cl2 with an FMOC-protected amino acid containing amine functionality in its side chain (compound II, eg, alpha-N-FMOC- 3- (3-pyridyl) -alanine, available from Synthetech, Albany, OR; 1 eq.), N-methylmorpholine (2.5 eq.) And HATU (1.1 eq.), And stirred at room temperature for 4 hours. The mixture is diluted with CH2Cl2, washed with 1 M aqueous citric acid (2X), water (1X), and 5% aqueous NaHCO3 (2X), dried over Na2SO4, and evaporated in vacuo. Compound III is isolated by flash chromatography (CH2Cl2-> EtOAc). Step C. Compound III is dissolved in CH2Cl2, Pd (PPh3) (0.07 eq.) And N-methylaniline (2 eq.) Are added, and the mixture is stirred at room temperature for 4 hours. The mixture is diluted with CH2Cl2, washed with 1 M aqueous citric acid (2x) and water (1X), dried over Na2SO4, and evaporated in vacuo. Compound IV is isolated by flash chromatography (CH2Cl2-> EtOAc + HOAc). Step D. Resin TentaGel S AC (Compound V; 1 eq.) Is suspended with DMF in the collection container of the peptide synthesizer ACT357 (ACT), Louisville, KY). Compound IV (3 eq.), HATU (3 eq.) And DIEA (7.5 eq.) In DMF are added and the collection vessel is stirred for 1 hour. The solvent is removed and the resin is washed with NMP (2X), MeOH (2X), and DMF (2X). The coupling of IV to the resin and the washing steps are repeated, to give compound VI. Step E. The resin (compound VI) is mixed with 25% piperidine in DMF and stirred for 5 minutes. The resin is filtered, then mixed with 25% piperidine in DMF and stirred for 10 minutes. The solvent is removed, the resin is washed with NMP (2X), MeOH (2X) and DMF (2X). The deprotected resin is then evenly divided by ACT357 of the collection vessel into 16 reaction vessels. Step F. The 16 aliquots of unprotected resin from step E are suspended in DMF. To each reaction vessel is added the appropriate carboxylic acid Vll1-16 (R? -? ßCO2H; 3 eq.), HATU (3 eq.), And DIEA (7.5 eq.) In DMF. The containers are stirred for 1 hour. The solvent is removed and the resin aliquots are washed with NMP (2X), MeOH (2X) and DMF (2X). The coupling of Vll1-16 to the aliquots of resin and the washing steps are repeated, to give Vll.-Iß compounds. Step G. The resin aliquots (Vlll? .6 compounds) are washed with CH2Cl2 (3X). To each reaction vessel is added 1% TFA in CH 2 Cl 2 and the vessels are stirred for 30 minutes. The solvent is filtered from the reaction vessels in individual tubes. The resin aliquots are washed with CH2Cl2 (2X) and MeOH (2X) and the filtrates are combined in the individual tubes. The individual tubes are evaporated in vacuo, by providing compounds IX1.1β- Step H. Each of the free carboxylic acids IX? -? 6 is dissolved in DMF. Pyridine (1.05 eq.) Is added to each solution, followed by pentafluorophenyl trifluoroacetate (1.1 eq.). The mixtures are stirred for 45 minutes at room temperature. The solutions are diluted with EtOAc, washed with 1 M aqueous citric acid (3X) and 5% aqueous NaHCO3 (3X), dried over Na2SO4, filtered, and evaporated in vacuo, by providing the compounds X? -? 6 EXAMPLE 2 DEMONSTRATION OF FOTOLYTIC CUTTING OF T-L-X A compound of T-L-X as prepared in Example 11 was irradiated with UV light for 7 minutes at room temperature. A Rayonett fluorescence UV lamp (Southern New England Ultraviolet Co., Middletown, CT) with an emission peak at 350 nm as a UV light source is used. The chamber is placed 15 cm away from the Petri dishes with samples. SDS gel electrophoresis shows that > 85% of the conjugate is cut under these conditions.

EXAMPLE 3 PREPARATION OF TWO FLUORESCENT MARKERS AND FLUOROFORO CUTTING DEMONSTRATION Synthesis and Purification of Oligonucleotides Oligonucleotides (ODNs) are prepared in automatic DNA synthesizers by using the normal foforamidite chemistry provided by the vendor, or the chemistry of H-phofonate (Glenn Research Sterling, VA). The appropriately blocked dA, dG, dC and T phosphoramidites are commercially available in these forms, and synthetic nucleosides can be converted to the appropriate form. Oligonucleotides are prepared by using the normal phosphoramidite provided by the vendor, or the chemistry of H-phosphonate. The oligonucleotides are purified by adaptations of normal methods. Oligonucleotides with d-trityl groups are chromatographed on H PLC using a 12, 300 # Rainin micrometer (Emeryville, CA), Dynamax C-8 4.2x250 mm inverted phase column using a gradient of 15% to 55% MeCN in 0.1 N Et3NH + OAc ", pH 7.0, over 20 minutes When detritylation is performed, the oligonucleotides are further purified by gel exclusion chromatography.The analytical checks for the quality of the oligonucleotides are conducted with a column of PRP ( Altech, Deerfield, IL) at alkaline pH and by PAGE The preparation of oligonucleotides derived from 2,4,6-trichlorotriazine: 10 to 1000 μg of linked 5'-amino terminal oligonucleotide react with an excess of recrystallized cyanuric chloride in 10% of n-methyl-pyrrolidone in alkaline buffer (pH 8.3 to 8.5 preferably) at 19CC at 25 ° C for 30 to 120 minutes The final reaction conditions consist of 0.15 M sodium borate at pH 8.3, 2 mg / ml of recrystallized cyanuric chloride and 500 μg / ml of respective oligonucleotide. The unreacted cyanuric chloride is removed by size extrusion chromatography on a G-50 Sephadex column (Pharmacia, Piscataway, NJ). The activated purified oligonucleotide is then reacted with a molar excess of 100 cystamine doublings in 0.15 M sodium borate at pH 8.3 for 1 hour at room temperature. Unreacted cystamine is removed by size exclusion chromatography on a G50 Sephadex column. The derived ODNs then react with amine-reactive fluorochromes. The preparation of derivatized ODN is divided into 3 portions and each portion reacts with (a) a molar excess of 20 red Texas sulfonyl chloride doublings (Molecular Probes, Eugene, OR), with (b) a molar excess of 20 doublings of sulfonyl chloride of lysamin (Molecular Probes, Eugene, OR), (c) a molar excess of 20 doublings of fluorescein isothiocyanate. The final reaction conditions consist of 0.15 M sodium borate at pH 8.3 for 1 hour at room temperature. The unreacted fluorochromes are removed by size exclusion chromatography on a G-50 Sephadex column. To cut the oligonucleotide fluorochrome, the ODNs are adjusted to 1 x 10"5 molar and then dilutions are made (12 dilutions of 3 bends) in TE (TE is 0.01 M Tris, pH 7.0, 5 mM EDTA). 100 μl of ODNs is added 25 μl of 0.01 M dithiothreitol (DTT) .There is no DTT added to an identical series of controls.The mixture is incubated for 15 minutes at room temperature.The fluorescence is measured on a black microtiter plate. solution is removed from the incubation tubes (150 microliters) and placed on a black microtiter plate (Dynatek Laboratories, Chantilly, VA) The plates are then read directly using a Fluoroskan II fluorometer (Fiow Laboratories, McLean, VA) when using an excitation wavelength of 495 nm and verification emission at 520 nm for fluorescein, when using an excitation wavelength of 591 nm and verification emission at 612 nm for Texas red, and when using a wavelength of excitation of 570 nm and em verification at 590 nm for lysine.

Modes of fluorochrome RFU not cut RFU cut RFU free 1. 0 x 105M 6.4 1200 1345 3.3 x 106M 2.4 451 456 1.1 x 106M 0.9 135 130 3.7 x 107M 0.3 44 48 1.2 x 107M 0.12 15.3 16.0 4.1 x 107M 0.14 4.9 5.1 1. 4 x 108M 0.13 2.5 2.8 4.5 x 109M 0.12 0.8 0.9 The data indicates that there is approximately an increase of 200 doubling in relative fluorescence when the fluorochrome is crushed from the ODN.

EXAMPLE 4 PREPARATION OF M13 SEQUENCE CEUTERS MARKED AND DEMONSTRATION OF BRAND CUTTING Preparation of 2,4,6-trichlorotriazine-derived oligonucleotides: 1000 μg of 5'-amino-terminal linked oligonucleotide (5'-hexylamine-TGTAAAACGACGGCCAGT-3") (SEQ ID No. 1) are reacted with an excess of recrystallized cyanuric chloride in 10% n-methyl-pyrrolidone in alkaline buffer (pH 8.3 to 8.5 preferably) at 19 ° C at 25 ° C for 30 to 120 minutes The final reaction conditions consist of 0.15 M sodium borate at pH 8.3, 2 mg / ml recrystallized cyanuric chloride and 500 μg / ml of respective oligonucleotide The unreacted cyanuric chloride is removed by size extrusion chromatography on a G-50 Sephadex column The activated purified oligonucleotide is then reacted with a molar excess of 100 mg / ml. doubling of cystamine in 0.15 M sodium borate at pH 8.3 for 1 hour at room temperature Unreacted cystamine is removed by size exclusion chromatography on a G50 Sephadex column. Onan with amine-reactive fluorochromes. The derivatized ODN preparation is divided into 12 portions and each portion reacts (25 molar excess) with (1) 4-methoxybenzoic acid, (2) 4-fluorobenzoic acid, (3) toluic acid, (4) benzoic acid, (5) ) indole-3-acetic acid, (6 = 2,6-difluorobenzoic acid, (7) nicotinic acid N-oxide, (8) 2-nitrobenzoic acid, (9) 5-acetylsalicylic acid, (10) 4-ethoxybenzoic acid (11) cinnamic acid, (12) 3-aminonicotinic acid The reaction is for 2 hours at 37 ° C in 0.2 M NaBorato pH 8.3 The derivative ODNs are purified by size exclusion chromatography on G-50 Sephadex. cut the oligonucleotide label, the ODNs are adjusted to 1 x 10"s molar and then dilutions are made (12 dilutions of 3 bends) in TE (TE is 0.01 M Tris, pH 7.0, 5 mM EDTA) with 50% EtOH (V / V) To volumes of 100 μl of ODNs is added 25 μl of 0.01 M dithiothreitol (DTT) .There is no DTT added to an identical series of controls.The mixture is incubated for 30 minutes at room temperature. The environment is then added to 0.1 M NaCl and 2 volumes of EtOH to precipitate the ODNs. The ODNs are removed from the solution by centrifugation at 14.00 x G at 4 ° C for 15 minutes. The supernatants are reserved, dried to totality. The pellet is then dissolved in 25 ul of MeOH. The pellet is then tested by mass spectrometry for the presence of marks. The mass spectrometer used in this work is an external ion source Fourier transformation mass spectrometer (FTMS). Samples prepared for MALDI analysis are deposited in a direct probe and inserted into the ion source. When a sample is irradiated with a laser pulse, ions are withdrawn from the source and passed in a long quadrupole ion guide that focuses and transports them to an FTMS analyzer cell located inside the hole of a superconducting magnet. The spectrum produced the following information. Peaks that vary in intensity from 25 to 100 units of intensity relative to the following molecular weights: (1) 212.1 amu indicating 4-methoxybenzoic acid derivative, (2) 200.1 indicating 4-fluorobenzoic acid derivative, (3) 196.1 amu indicating derivative of toluic acid, (4) 182.1 amu indicating benzoic acid derivative, (5) 235.2 amu indicating indole-3-acetic acid derivative, (6) 218.1 amu indicating 2,6-difluorobenzoic acid derivative, (7) 199.1 amu indicating derivative of nicotinic acid N-oxide, (8) 227.1 amu indicating 2-nitrobenzamide, (9) 179.18 amu indicating 5-acetylsalicylic acid derivative, (10) 226.1 amu indicating 4-ethoxybenzoic acid derivative, (11) 209.1 amu indicating cinnamic acid derivative, (12) 198.1 amu indicating 3-aminonicotinic acid derivative. The results indicate that the MW identifiers are cut from the primers and can be detected by mass spectrometry.

EXAMPLE 5 PR UAE R ESE A NN I N T I O N S P U S T O R S FOR THE R 1 -36LYS (e-I N I P) -AN P-TFP Figure 3 illustrates the parallel synthesis of a series of TLX compounds (X = Lh), where Lh is an activated ester (specifically, tetrafluorophenyl ester), L2 is an ortho-nitrobenzylamine group with L3 being a methylene group linking Lh and L2, T has a modular structure in which the lysine carboxylic acid group has been bonded to the nitrogen atom of the L2 group benzylamine to form an amide bond, and a variable weight component R? -36, (wherein these R groups correspond to T2 as defined herein, and can be introduced via any of the specific carboxylic acids listed herein) is linked through the α-amino group of the lysine, while a spectrum enhancer group of mass (introduced via N-methylisonipecotic acid) is bound through the e-amino group of lysine. Referring to Figure 3: Step A. NovaSyn HMP resin (available from Novabiochem; 1 eq.) Is suspended with DMF in the collection container of the ACT357 peptide synthesizer. Compound I (available ANP of ACT, 3 eq.), HATU (3 eq.) And NMM (7.5 eq.) In DMF are added and the collection vessel is stirred for 1 hour. The solvent is removed and the resin washed with N MP (2X), MeOH (2X), and DMF (2X). The IV coupling to the resin and washing steps are repeated, to give compound II.

Step B. The resin (compound II) is mixed with 25% piperidine in DMF and stirred for 5 minutes. The resin is filtered, then mixed with 25% piperidine in DMF and stirred for minutes. The solvent is removed, the resin is washed with NMP (2X), MeOH (2X) and DMF (2X), and used directly in step C. Step C. The deprotected resin from step B is suspended in DMF and added an FMOC-protected amino acid, containing an amine functionality protected in its side chain (Fmoc-Lysine (Aloc) -OH, available from PerSecptive Biosystems, eq.), HATU (3 eq.), and NMM (7.5 eq.) in DMF. The vessel is stirred for 1 hour. The solvent is removed and the resin is washed with NMP (2X), MeOH (2X) and DMF (2X). The coupling of Fmoc-Lys (Aloc) -OH to the resin and washing steps are repeated, to give compound IV. Step D. The resin (compound IV) is washed with CH2Cl3 (2X), and then suspended in a solution of (PPh3) 4Pd (0) (0.3 eq.) And PhSiH3 (10 eq.) In CH2Cl2. The mixture is stirred for 1 hour. The solvent is removed and the resin is washed with CH2Cl2 (2X). The palladium step is repeated. The solvent is removed and the resin is washed with CH2Cl2 (2X), N, N-diisopropylethylammonium diethyldithiocarbamate in DMF (2x), DMF (2X) to give compound V. Step E. The unprotected resin from step D is coupled with N-methylisonipecotic acid as described in step C to give compound VI. Step F. The Fmoc VI protected resin is divided equally by the ACT357 of the collection vessel in 36 reaction vessels to give the compounds Vl? -36.

Step G. The resin (compounds I-36) is treated with piperidine as described in step B to remove the FMOC group. Step H. The 36 aliquots of unprotected resin from step G are suspended in DMF. To each reaction vessel is added the appropriate carboxylic acid (R1-16C02H; 3 eq.), HATU (3 eq.), And DIEA (7.5 eq.) In DMF. The containers are stirred for 1 hour. The solvent is removed and the resin aliquots are washed with NMP (2X), MeOH (2X) and DMF (2X). The coupling of R? -36 to the aliquots of resin and the washing steps are repeated, to give compounds Vlll? -36. Step I. The resin aliquots (Villas compounds) are washed with CH2Cl2 (3X). To each reaction vessel is added 1% TFA in CH 2 Cl 2 and the vessels are stirred for 30 minutes. The solvent is filtered from the reaction vessels in individual tubes. The resin aliquots are washed with CH2Cl2 (2X) and MeOH (2X) and the filtrates are combined in the individual tubes. The individual tubes evaporate under vacuum, by providing compounds IX1.36. Step J. Each of the free carboxylic acids IX1.36 is dissolved in DMF. Pyridine (1.05 eq.) Is added to each solution, followed by pentafluorophenyl trifluoroacetate (1.1 eq.). The mixtures are stirred for 45 minutes at room temperature. The solutions are diluted with EtOAc, washed with 1 M aqueous citric acid (3X) and 5% aqueous NaHCO3 (3X), dried over Na2SO4, filtered, and evaporated in vacuo, by providing compounds X1-36.

EXAMPLE 6 PREPARATION OF A SERIES OF COMPOUNDS OF FORMULA R1-36LYS (e-INIP) -ANP-TFP Figure 4 illustrates the parallel synthesis of a series of TLX compounds (X = Lh), where Lh is an activated ester (specifically, tetrafluorophenyl ester), L2 is an ortho-nitrobenzylamine group with L3 being a methylenic linking group Lh and L2, T has a modular structure in which the lysine carboxylic acid group has been bonded to the nitrogen atom of the L2 group benzylamine to form an amide bond, and a variable weight component R? -36, (wherein these R groups correspond to T2 as defined herein, and can be introduced via any of the specific carboxylic acids listed herein) is linked through the α-amino group of the lysine, while a spectrum enhancer group of mass (introduced via N-methylisonipecotic acid) is bound through the e-amino group of lysine. Referring to Figure 4: Step A. NovaSyn HMP resin is coupled with compound I (NBA prepared according to the procedure of Brown et al., Molecular Diversity, 1, 4 (1995)) in accordance with the procedure described in step A of example 5, to give compound II. Step B. The resin (compound II) is treated as described in steps B-J of example 5 to give compounds X? -36.

EXAMPLE 7 PREPARATION OF A SERIES OF COMPOUNDS OF THE FORMULA INIP-LYS (e-R1.36) -ANP-TFP Figure 5 illustrates the parallel synthesis of a series of TLX compounds (X = Lh), where Lh is an activated ester (specifically, tetrafluorophenyl ester), L2 is an ortho-nitrobenzylamine group with L3 being a methylene group that binds Lh and L2, T has a modular structure in which the lysine carboxylic acid group has been bonded to the nitrogen atom of the L2 group benzylamine to form an amide bond, and a variable weight component R? -36, (wherein these R groups correspond to T2 as defined herein, and can be introduced via any of the specific carboxylic acids listed herein) is linked through the α-amino group of the lysine, while a spectrum enhancer group of mass (introduced via N-methylisonipecotic acid) is bound through the e-amino group of lysine. Referring to Figure 5: Steps A-C. The same as in Example 5. Step D. The resin (compound IV) is treated with piperidine as described in step B of example 5 to remove the FMOC group.

Step E. The unprotected α-amine in the resin in step D is coupled with N-methylisonipecotic acid as described in step C of Example 5 to give compound V. Step F. Same as in Example 5.

Step G. The resin (compounds VI1-36) is treated with palladium as described in step D of example 5 to remove the Aloe group. Steps H-J. The compounds X1-3e are prepared in the same manner as in example 5.

EXAMPLE 8 PREPARATION OF A SERIES OF COMPOUNDS OF FORMULA R1-36-GLU (? -DIAEA) -ANP-TFP Figure 6 illustrates the parallel synthesis of a series of compounds of T-L-X (X = Lh), where Lh is an activated ester (specifically, tetrafluorophenyl ester), L2 is an ortho-nitrobenzylamine group with L3 being a methylene group linking Lh and L2, T has a modular structure wherein the lysine carboxylic acid group has been bonded to the nitrogen atom of the L2 group benzylamine to form an amide bond, and a component of variable weight R? -3β, (wherein these R groups correspond to T2 as defined herein, and can be introduced via any of the specific carboxylic acids listed herein) is attached through the α-amino group of the lysine, while that a mass spectrum sensitivity enhancer group (introduced via N-methylisonipecotic acid) binds through the α-carboxylic acid of glutamic acid. Referring to Figure 6: Steps A-B. Same as in example 5.

Step C. The deprotected resin (compound III) is coupled to Fmoc-Glu- (OAI) -OH by using the coupling method described in step C of example 5 to give compound IV. Step D. The resin (compound IV) is treated with piperidine as described in step B of example 5 to remove the FMOC group.

Step E. The deprotected α-amine in the resin in step D is coupled with N-methylisonipecotic acid as described in step C of example 5 to give compound V. Step E. The unprotected resin from step D is suspended in DMF and a FMOC-protected amino acid, which contains amine functionality in its side chain (compound IV, eg, alpha-N-FMOC-3- (3-pyridyl) -alanine, available from Synthetech, Albany, OR, is added. 3 eq.), HATU (3 eq.), And DIEA (7.5 eq.) In DMF. The vessel is stirred for 1 hour. The solvent is removed and the resin is washed with NMP (2X), MeOH (2X), and DMF (2X). The coupling of IV to the resin and the washing steps are repeated, to give compound VI. Steps F-J. Same as in example 5.

EXAMPLE 9 PREPARATION OF A SERIES OF COMPOUNDS OF FORMULA INIP-LYS (e-R? .36) -ANP-LYS (e-NH2) -NH2 Figure 7 illustrates the parallel synthesis of a series of TLX compounds (X = Lh), where Lh is an activated ester (specifically, tetrafluorophenyl ester), L2 is an ortho-nitrobenzylamine group with L3 being a methylene group linking Lh and L2, T has a modular structure in which the carboxylic acid group of lysine has been bonded to the nitrogen atom of the L2 group benzylamine to form an amide bond, and a variable weight component R? -36, (wherein R groups correspond to T2 as defined herein, and can be introduced via any of the specific carboxylic acids listed herein) is linked through the α-amino group of the lysine, while a spectrum enhancer group of mass (introduced via N-methylisonipecotic acid) is bound through the e-amino group of lysine. Referring to Figure 7: Step A. Mix Fmoc-Lys (Boc) -SRAM resin (available from ACT, compound I) with 25% piperidine in DMF and stir for 5 minutes. The resin is filtered, then mixed with 25% piperidine in DMF and stirred for 10 minutes. The solvent is removed, the resin is washed with NMP (2X), MeOH (2X) and DMF (2X), and used directly in step B. Step B. The resin (compound II), ANP (available from ACT; 3 eq.), HATU (3 eq.) And NMM (7.5 eq.) In DMF are added and the collection vessel is stirred for 1 hour. The solvent is removed and the resin is washed with NMP (2X), MeOH (2X), and DMF (2X). The coupling of I to the resin and the washing steps are repeated, to give compound III. Steps C-J. The resin (compound III) is treated as in steps B-l in example 5 to give the compounds X?, 36.

EXAMPLE 10 PREPARATION OF A SERIES OF COMPOUNDS OF FORMULA INIP-LYS (e-R1 -36) -ANP-LYS (e-NH2) -NH2 Figure 8 illustrates the parallel synthesis of a series of compounds of T-L-X (X = Lh), where Lh is an activated ester (specifically, tetrafluorophenyl ester), L2 is an ortho-nitrobenzylamine group with L3 being a methylene group linking Lh and L2, T has a modular structure in which the carboxylic acid group of lysine has been bonded to the nitrogen atom of the L2 group benzylamine to form an amide bond, and a component of variable weight R? -36, (wherein these R groups correspond to T2 as defined herein, and can be introduced via any of the specific carboxylic acids listed herein) is attached through the a-amino group of the lysine, while that a mass spectrum sensitivity enhancer group (introduced via N-methylisonipecotic acid) binds via the e-amino group of lysine. Referring to Figure 8: Steps A-E. These steps are identical to steps A-E in Example 5. Step F. The resin (compound VI) is treated with piperidine as described in step B in example 5 to remove the FMOC group. Step G. The deprotected resin (compound VIII) is coupled to Fmoc-Lys (Tfa) -OH by using the coupling method described in step C of example 5 to give compound VII I.

Steps H-K. The resin (compound VI I I) is treated as in steps F-J in example 5 to give compounds Xl? -36.

EXAMPLE 1 1 PREPARATION OF A SERIES OF COMPOUNDS OF FORMULA R1-3e-YS (e-INI) -ANP-5'-AH-ODN Figure 9 illustrates the parallel synthesis of a series of TLX compounds (X = MOI, where MOI is a nucleic acid fragment, ODN) derived from the esters of Example 5 (the same procedure could be used with other TLX compounds where X is an activated ester). The MOI is conjugated to T-L through the 5 'end of the MOI, via a phosphodiester-alkyleneamine group. Referring to Figure 9: Step A. Compounds XI 11 -36 are prepared according to a modified biotinylation procedure in Van Ness et al., Nucleic Acids Res., 19, 3345 (1991). To a solution of one of the 5'-aminohexyl oligonucleotides (compounds Xl? -36, 1 mg) in 200 μM sodium borate (pH 8.3, 250 mL) is added one of the tetrafluorophenyl esters (compounds X1 - 36 of Example A, molar excess of 100 doublings in 250 mL of NMP). The reaction is incubated overnight at room temperature. Unreacted and hydrolyzed tetrafluorophenyl esters are removed from compounds Xll? -3S by Sephadex G-50 chromatography.

EXAMPLE 12 PREPARATION OF A SERIES OF COMPOUNDS OF FORMULA R1-36-LYS (e-INIP) -ANP-LYS (e- (MCT-5'-AH-ODN)) - NH2 Figure 10 illustrates the parallel synthesis of a series of TLX compounds (X = MOI, where MOI is a nucleic acid fragment, ODN) derived from the esters of example 11 (the same procedure could be used with other TLX compounds in where X is an amine). The MOI is conjugated to T-L through the 5 'end of the MOI, via a phosphodiester-alkyleneamine group. Referring to Figure 10: Step A. Prepare the 5 '- [6- (4,6-dichloro-1, 3,5-triazin-2-ylamino) hexyl] oligonucleotides Xl -36 as described in Van Ness and others, Nucleic Acids Res., 19, 3345 (1991). Step B. To a solution of one of the 5 '- [6- (4,6-dichloro-1, 3,5-triazin-2-ylamino) hexyl] oligonucleotides (compounds Xll? -36) at a concentration of 1 mg / ml in 100 mM sodium borate (pH 8.3) was added a molar excess of 100 doublings of a primary amine selected from R1-36-LYS (e-INIP) -ANP-LYS (e- (MCT- 5'-AH-ODN)) - NH 2 (compounds X? -36 of example 11). The solution is mixed overnight at room temperature. The unreacted amine is removed by ultrafiltration through a 3000 MW cut-off membrane (Amicon, Beverly, MA) when using H20 as the wash solution (3X). The compounds XIII1-36 are isolated by reducing the volume to 100 mL.

EXAMPLE 13 DETECTION OF THE DETECTION OF M ULTIPLE BRANDS BY MASS SPECTROMETRY This example provides a description of the ability to simultaneously detect multiple compounds (labels) by mass spectrometry. In this particular example, 31 compounds are mixed with a matrix, deposited and dried on a solid support and then desorbed with a laser. The resulting ions are then introduced into a mass spectrometer. The following compounds (purchased from Aldrich, Milwaukee, Wl) are mixed together on a molar basis equal to a final concentration of 0.002 M (per compound) base: benzamide (121.14), nicotinamide (122.13), pyrazinamide (123.12), 3-amino-4-pyrazolecarboxylic acid (127.10), 2-thiophenecarboxamide (127.17), 4-aminobenzamide (135.15), tolumide (135.17), 6-methylnicotinamide (136.15), 3-aminonicotinamide (137.14), nicotinamide N-oxide (138.12), 3-hydropicolinamide (138.13), 4-fluorobenzamide (139.13), cinnamamide (147.18), 4-methoxybenzamide (151 .17), 2,6-difluorobenzamide (157.12), 4-amino-5-imidazole-carboxyamide (162.58), 3,4-pyridine-dicarboxyamide (165.16), 4-ethoxybenzamide (165.19), 2,3-pyrazindicarboxinamide (166.14), 2-nitrobenzamide (166.14), 3-fluoro-4-methoxybenzoic acid (170.4), indole-3-acetamide (174.2), 5-acetylsalicylamide (179.18), 3,5-dimethoxybenzamide (181.19), 1-naphthalene acetamide (185.23), 8-chloro-3,5-diamino-2-pyrazinecarboxyamide (187.59), 4 -trifluoromethylbenzamide (189.00), 5-amino-5-phenyl-4-pyrazole-carboxamide (202.22), 1-methyl-2-benzyl-malonamate (207.33), 4-amino-2, 3,5,6-tetrafluorobenzamide (208.11), 2,3-naphthalenedicarboxylic acid (212.22). The compounds are placed in DMSO at the concentration described above. One ul of the material is mixed with alpha-cyano-4-hydroxy cinnamic acid matrix (after a dilution of 1: 10,000) and deposited on a solid stainless steel support. The material is then desorbed by a laser when using the Protein TOF mass spectrometer (Bruker, Manning Park, MA) and the resulting ions are measured in the linear and operating modes of reflectrons. The following values of m / z are observed (figure 11): 121 .1 - »benzamide (121 .14) 122.1 - > nicotinamide (122.13) 123.1 ^ pyrazinamide (123.12 124.1 125.2 127.3 ^ 3-amino-4-pyrazolecarboxylic acid (127.10) 127.2- »2-thiophenecarboxamide (127.17) 135.1 -» 4-aminobenzamide (135.15) 135.1 - »tolumide (135.17) 136.2 - »6-Methylnicotinamide (136.15) 137.1 -» 3-aminonicotinamide (137.14) 138.2- »N-oxide of nicotinamide (138.12) 138.2 ^ 3-Hydropicolinamide (138.13) 139.2-» 4-Fluorobenzamide (139.13) 140.2 147.3 ^ Cinnamamide ( 147.18) 148.2 149.2 4-methoxybenzamide (151.17) 152.2 2,6-difluorobenzamide (157.12) 158.3 4-amino-5-imidazole-carboxyamide (162.58) 163. 3 165.2- 3,4-pyridine-dicarboxyamide (165.16) 165.2 ^ 4-ethoxybenzamide (165.19) 166.2- * 2, 3-pyrazine di carboxamide (166.14) 166.2 ^ 2-nitrobenzamide (166.14) 3-fluoro-4-methoxybenzoic acid (170.4) 171. 1 172.2 173.4 indole-3-acetamide (174.2) 178.3 179.3- > 5-acetylsalicylamide (179.18) 181.2- > 3,5-dimethoxybenzamide (181.19) 182.2- »1-naphthalene acetamide (185.23) 186.2 8-chloro-3,5-diamino-2-pyrazinecarboxamide (187.59) 188.2 189.2 ^ 4-trifluoromethyl-benzamide (189.00) 190.2 191.2 192.3 5- a mino-5-phenyl-4-pyrazole-carboxamide (202.22) 203.2 203.4 1-methyl-2-benzyl-malopamate (207.33) 4-amino-2,3,5,6-tetrafluorobenzamide (208.11) 212.2- acid 2 , 3-naphthalenedicarboxylic (212.22) 219.3 221.2 228.2 234.2 237.4 241.4 The data indicate that 22 of 31 compounds appeared in the spectrum with anticipated mass, 9 of 31 compounds appeared in the spectrum with an n + H mass (1 unit of atomic mass , amu) on the anticipated mass. The last phenomenon is probably due to the protonation of an amine within the compounds. Therefore, 31 of 31 compounds are detected by MALDI Mass Spectroscopy. More important, the example demonstrates that multiple marks can be detected simultaneously by a spectroscopic method. The alpha-cyano matrix alone (figure 11) gives peaks at 146.2, 164.1, 172. 1, 173.1, 189.1, 190.1, 191.1, 192.1, 212.1, 224. 1, 228.0, 234.3. Other masses identified in the spectrum are due to contaminants in the compounds acquired since no effort was made to further purify the compounds.

EXAMPLE 14 MARKERS OF M ICROSATÉLITE.AM PLI FICATION IS OF PCR The microsatellite markers are amplified to! use the following normal PCR conditions. In short, PCR reactions are performed in a total volume of 50 μl, containing 40 ng of genomic DNA, 50 pmol of each primer, 0.125 mM dNTPs and 1 unit of Taq polymerase. The 1 X amplification regulator contains 10 mM Tris base, pH 9, 50 mM KCl, 1.5 mM MgCl, 0.1% Triton X-100 and 0.01% gelatin. Reactions are performed using a "warm start" procedure: Taq polymerase is added only after a first denaturation step of 5 minutes at 96 ° C. Amplification of 35 cycles is carried out: denaturation (94 ° C for 40 seconds) and cancellation (55 ° C for 30 seconds). An extension process (72 ° C for 2 minutes) ends the process after the last cancellation. Since the amplification products to be obtained are short (90 to 350 long base pairs) and the time interval to raise the temperature from 55 ° C to 94 ° C (obtained with a ramp rate of 1 ° C / second) is long enough, DNA elongation termination can be achieved without a step at 72 ° C. From the foregoing, it will be appreciated that, although the specific embodiments of the invention have been described for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention.

Claims (5)

1. CLAIMS 1. - A method for determining the presence of a nucleic acid molecule, comprising: (a) generating the labeled nucleic acid molecules of one or more selected labeled nucleic acid molecules, wherein a tag is correlated with a nucleic acid fragment particular and can be detected by spectrometry or potentiometry; (b) separating said labeled molecules by size; (c) cutting said tag of said labeled molecule; and (d) detecting said mark by spectrometry or potentiometry, and determining the presence of said nucleic acid molecule.
2. 2. A method for detecting a selected nucleic acid molecule, comprising: (a) combining a labeled nucleic acid probe with target nucleic acid molecules under conditions and for a time sufficient to allow hybridization of said nucleic acid probe labeled to a complementary selected target nucleic acid sequence, wherein said labeled nucleic acid probe comprises a tag that correlates with a particular fragment and can be detected by spectrometry or potentiometry; (b) altering the size of said hybrid labeled probes, the size of non-hybrid probes or target molecules, or the size of the probe hybrids: target; (c) separating the probes marked by size; (d) cutting said tag from said marked probe; and (e) detecting said tag by spectrometry or potentiometry, and detecting said selected nucleic acid molecule. 3. The method according to claim 1 or 2, wherein the detection of the mark is by mass spectrometry, infrared spectrometry, ultraviolet spectrometry, or potentiostatic amperometry. 4. The method according to claim 1, wherein more than 4 labeled nucleic acid fragments are generated and wherein each tag is unique to a selected nucleic acid fragment. 5. The method according to claim 2, wherein more than 4 labeled nucleic probes are used and wherein each tag is unique to a selected nucleic acid probe. 6. The method according to claim 1 or 2, wherein said target nucleic acid molecule is generated by primer extension. 7. The method according to claim 2, wherein the size of said hybrid labeled probes, non-hybrid probes or target molecules, or probe: target bridges are altered by a method selected from the group consisting of extension of polymerase, ligation, exonuciease digestion and endonuclease digestion. 8. The method according to claim 1 or 2, wherein said labeled molecules are separated by a method selected from the group consisting of gel electrophoresis, capillary electrophoresis, micro-channel electrophoresis, H PLC, chromatography of Size exclusion and filtration. 9. The method according to claim 1 or 2, wherein said labeled molecules are cut by a method selected from the group consisting of oxidation, reduction, unstable acid methods, base unstable, enzymatic, electrochemical, thermal and photo-stable. 10. The method according to claim 1 or 3, wherein said mark is detected by time-of-flight mass spectrometry, quadrupole mass spectrometry, magnetic sector mass spectrometry and electric sector mass spectrometry. 1. The method according to claim 10, wherein said mark is detected by potentiostatic amperometry when using detectors selected from the group consisting of colometric detectors and amperometric detectors. 12. The method according to claim 1 or 2, wherein the steps of separating, cutting and detecting are performed continuously.
3. 3. The method according to claim 1 or 2, wherein the steps of separating, cutting and detecting are carried out continuously in a single device. 14. The method of conformity with claim 1 3, where the steps of separating, cutting and detecting are automatic. 15. The method according to claim 1 or 2, wherein said labeled molecules or probes are generated from oligonucleotide primers marked at 5 '. 16. - The method according to claim 1 or 2, wherein said labeled molecules or probes are generated from labeled dideoxynucleotide terminators. 17. The method according to any of claims 1 -2, 4-9 and 12-16, wherein said mark is detected by non-fluorescent spectrometry or potentiometry. 1 8. A method for generating a selected organism, comprising: (a) generating nucleic acid molecules labeled from a selected target molecule, wherein a label correlates with a particular fragment and can be detected by spectrometry or potentiometry; (b) separating said labeled molecules by size; (c) cutting said tag of said labeled molecule; and (d) detecting said mark by spectrometry or potentiometry, and determining the genotype of said organism. 9. A method for genotyping a selected organism, comprising: (a) combining a labeled nucleic acid molecule with a selected target molecule under conditions and for a time sufficient to allow hybridization of said labeled molecule to said target molecule , where a mark correlates with a particular fragment and can be detected by spectrometry or potentiometry; (b) separating said labeled molecules by size; (c) cutting said tag of said labeled molecule; and (d) detecting said mark by spectrometry or potentiometry, and determining the genotype of said organism. 20. The method according to claim 18 or 19, wherein said molecules are generated from an extraction of clones selected from the group consisting of genomic clones, cDNA clones and RNA clones. 21. The method according to claim 18 or 19, wherein said labeled molecules are generated by polymerase chain reaction. 22. The method according to claim 18 or 19, wherein the detection of the mark is by mass spectrometry, infrared spectrometry, ultraviolet spectrometry, or potentiostatic amperometry. 23. The method according to claim 18 or 19, wherein more than 4 labeled nucleic acid molecules are generated and wherein each tag is unique to a selected nucleic acid fragment. 24. - The method according to claim 18 or 19, wherein said target nucleic acid molecule is generated by primer extension. 25. The method according to claim 18 or 19, wherein said labeled molecules are generated by a method selected from the group consisting of gel electrophoresis, capillary electrophoresis, micro-channel electrophoresis, H PLC, chromatography of Size exclusion and filtration. 26. The method according to claim 18 or 9, wherein said labeled molecules are cut by a method selected from the group consisting of oxidation, reduction, acid unstable, base unstable, enzymatic, electrochemical, thermal and photoinnatable. 27. The method according to claim 18 or 19, wherein said mark is detected by mass spectrometry of time of flight, quadrupole mass spectrometry, mass spectrometry of magnetic sector and mass spectrometry of electric sector. 28. The method according to claim 18 or 18, wherein said mark is detected by potentiostatic amperometry when using detectors selected from the group consisting of colometric detectors and amperometric detectors. 29. The method according to claim 18 or 19, wherein the steps of separating, cutting and detecting are carried out continuously. 30. - The method according to claim 18 or 19, wherein the steps of separating, cutting and detecting are carried out continuously in a single device. 31. The method according to claim 18 or 19, wherein the steps of separating, cutting and detecting are automatic. 32. The method according to claim 18 or 19, wherein said labeled molecules are generated from 3 'non-labeled oligonucleotide primers. 33. The method according to claim 18 or 19, wherein said labeled molecules are generated from labeled dideoxynucleotide terminators. 34. The method according to claim 1, 2 or 18, wherein said labeled molecule is obtained from a biological sample. 35. The method according to any of claims 18-21, 23-26 and 29-34, wherein said mark is detected by non-fluorescent spectrometry or potentiometry. 36.- A composition comprising a plurality of compounds of the formula: Tm sL-MOI wherein, Tms is an organic group that can be detected by mass spectrometry, comprising carbon, at least one of hydrogen and fluorine, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; L is an organic group that allows a portion containing Tms to be cut from the rest of the compound, wherein the portion containing Tms comprises a functional group that supports a single state of ionized charge when the compound is subjected to mass spectrometry and selects from tertiary amine, quaternary amine and organic acid; MOI (molecule of interest) is a fragment of nucleic acid; and wherein at least two compounds have the same Tms but the MOI groups of those molecules have non-identical nucleotide lengths. 37.- A composition comprising a plurality of compounds of the formula: Tms-L-MOI wherein, Tms is an organic group that can be detected by mass spectrometry, comprising carbon, at least one of hydrogen and fluorine, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; L is an organic group that allows a portion containing Tms to be cut from the rest of the compound, wherein the portion containing Tms comprises a functional group that supports a single state of ionized charge when the compound is subjected to mass spectrometry and selects from tertiary amine, quaternary amine and organic acid; MOI is a fragment of nucleic acid; and wherein at least two compounds have the same Tms but those compounds have non-identical elution times as measured by H PLC when using a column of poly (styrene-divinylbenzene) particles-C18 2.3 μm non-porous, 50 x 4.6 mm i. d. , and a mobile phase formed by a combination of regulators A and B, where regulator A is 0.1 M trisbutylammonium acetate at pH 7 with 5% acetonitrile, and regulator B is 0.1 M triethylammonium acetate at pH 7 with 95 % acetonitrile, where regulators A and B combine to provide a linear gradient of 6.25 to 16.25% acetonitrile in 5 minutes, with a flow rate of 1 ml / min and a temperature of 40 ° C, where the compounds are detected by using uv detection at 254 nm. 38.- A composition comprising a plurality of compounds of the formula: Tms-L-MOI wherein, Tms is an organic group that can be detected by mass spectrometry, comprising carbon, at least one of hydrogen and fluorine, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; L is an organic group that allows a portion containing Tms to be cut from the rest of the compound, wherein the portion containing Tms comprises a functional group that supports a single state of ionized charge when the compound is subjected to mass spectrometry and selects from tertiary amine, quaternary amine and organic acid; MOI is a fragment of nucleic acid; and where no two compounds that have the same length of MOI nucieotide also have the same Tms. 39.- A composition according to any of claims 36-38, wherein the plurality is greater than 2. 40.- A composition according to any of claims 36-38, wherein the plurality is greater than 4. 41. A composition according to any of claims 36-38, wherein the nucleic acid fragment has a sequence complementary to a portion of a vector, wherein the fragment is capable of priming polynucleotide synthesis. 42.- A composition according to any of claims 36-38, wherein the Tms groups of members of the plurality differ by at least 2 amu. 43.- A composition according to any of claims 36-38, wherein the groups T s of members of the plurality differ by at least 4 amu. 44.- A composition comprising a plurality of series of compounds, each series of compounds having the formula: Tms-L-MOI wherein, Tms is an organic group that can be detected by mass spectrometry, comprising carbon, by at least one of hydrogen and fluorine, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; L is an organic group that allows a portion containing Tms to be cut from the rest of the compound, wherein the portion containing Tms comprises a functional group that supports a single state of ionized charge when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid; MO I is a fragment of nucleic acid; and members within a first set of compounds have identical Tm groups, however they have non-identical MOI groups with numbers differing from n-nucleotides in MO I and there are at least ten members within the first series, wherein acts, the Tms groups differ by at least 2 amu. 45. - A com position according to claim 44, wherein the plurality is at least 3. 46.- A composition according to claim 44, wherein the plurality is at least 5. 47. A composition comprising a plurality of series of compounds, each series of compounds having the formula: T sL-MOI wherein, T s is an organic group that can be detected by mass spectrometry, comprising carbon, by at least one of hydrogen and fluorine, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; L is an organic group that allows a portion containing Tms to be cut from the rest of the compound, wherein the portion containing Tms comprises a functional group that supports a single state of ionized charge when the compound is subjected to mass spectrometry and selects from tertiary amine, quaternary amine and organic acid; MOI is a fragment of nucleic acid; and wherein the compounds within a series have non-identical Tm groups but the same elution time by HPLC when using a column of poly (styrene-divinylbenzene) particles -C -C 8 2.3 μm non-porous, 50 × 4.6 mm id, and a mobile phase formed by a combination of regulators A and B, wherein regulator A is 0.1 M trisbutylammonium acetate at pH 7 with 5% acetonitrile, and regulator B is 0.1 M triethylammonium acetate at pH 7 with 95% of acetonitrile, where regulators A and B combine to provide a linear gradient of 6.25 to 16.25% acetonitrile in 5 minutes, with a flow rate of 1 ml / min and a temperature of 40 ° C, where the compounds they are detected when uv detection is used at 254 nm. 48.- A team for genotyping, comprising a plurality of amplification primer pairs, wherein at least one of the primers has the formula: Tms-L-MOI where, Tms is an organic group that can be detected by mass spectrometry, comprising carbon, at least one of hydrogen and fluorine, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; L is an organic group that allows a portion containing Tms to be cut from the rest of the compound, wherein the portion containing Tms comprises a functional group that supports a single state of ionized charge when the compound is subjected to mass spectrometry and it is selected from tertiary amine, quaternary amine and organic acid; MOI is a fragment of nucleic acid; and each pair of primer is associated with a different loci. 49 - A device according to claim 48, wherein the plurality is at least 3. 50.- A device according to claim 48, wherein the plurality is at least 5. SUMMARY Labels and linkers designed specifically for a wide variety of nucleic acid reactions are described, which are suitable for a wide variety of nucleic acid reactions where separation of nucleic acid molecules based on size is required.