Abstract: A class of asymmetric monobenzoxanthene compounds useful as fluorescent dyes are disclosed having the structure ##STR1## wherein Y.sub.1, and Y.sub.2 are individually hydroxyl, amino, imminium, or oxygen, R.sub.1 -R.sub.8 are hydrogen, fluorine, chlorine, alkyl alkene, alkyne, sulfonate, amino, amido, nitrile, alkoxy, linking group, and combinations thereof, and R.sub.9 is acetylene, alkane, alkene, cyano, substituted phenyl and combinations thereof The invention ftrther includes novel intermediate compounds useful for the synthesis of asymmetric benzoxanthene compounds having the general structure ##STR2## where substituents R.sub.3 -R.sub.7 correspond to like-referenced substituents in the structure of described above, and Y.sub.2 is hydroxyl or amine. In another aspect, the invention includes methods for synthesizing the above dye compounds and intermediates.

Abstract: An electrophoresis system including means for reducing the distortion of a sample zone eluting from a capillary electrophoresis separation capillary is disclosed. The system includes one or more separation capillaries, each separation capillary having an inlet end and an outlet end; a first electrode in electrical communication with the inlet ends of the separation capillaries; a second electrode in electrical communication with the outlet ends of the separation capillaries; and one or more focusing electrodes in electrical communication with the outlet ends of the separation capillaries. In operation, the voltage of each of the electrodes is adjusted such that (i) the sample zone is transported from the inlet end to the outlet end of the separation capillaries and (ii) the distortion of the sample zone eluting from the separation capillaries is reduced.

Abstract: Apparatus and method for performing a nucleic acid amplification reaction and preferably a polymerase chain reaction (PCR) in a reaction mixture in at least one capillary tube. Several different embodiments are disclosed. One embodiment cycles a sample through a capillary tube loop passing through two thermostatted fluid baths. Another embodiment has the capillary tube routed alternatingly between two heat exchangers to that the sample makes only one pass through the tube. Other embodiments maintain the heat exchangers stationary and translate the samples between them. Still further embodiments maintain the samples stationary and either automatically translate or rotate the heat exchangers past the samples contained within the capillary tubes to perform the thermal cycles necessary for the amplification reaction.

Abstract: Propargylethoxyamino nucleosides are disclosed having the structure ##STR1## wherein R.sub.1 and R.sub.2 are --H, lower alkyl, or label; B is a 7-deazapurine, purine, or pyrimidine nucleoside base; W.sub.1 is --H or --OH; W.sub.2 is --OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3'-position; and W.sub.3 is --PO.sub.4, --P.sub.2 O.sub.7, --P.sub.3 O.sub.10, phosphate analog, or --OH. Additionaly, a primer extension method is provided employing the above propargylethoxyamino nucleosides.

Abstract: Method and composition for detecting one or more selected polynucleotide regions in a target polynucleotide. In the method, a mixture of sequence-specific probes are reacted with the target polynucleotide under hybridization conditions, and the hybridized probes are treated to selectively modify those probes which are bound to the target polynucleotide in a base-specific manner. The resulting labeled probes include a polymer chain which imparts to each different-sequence probe, a distinctive ratio of charge/translational frictional drag, and a detectable label. The labeled probes are fractionated by electrophoresis in a non-sieving matrix, and the presence of one or more selected sequences in the target polynucleotide are detected according to the observed electrophoretic migration rates of the labeled probes in a non-sieving medium.

Abstract: Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R.sub.21 Z.sub.1 C(O)R.sub.22 R.sub.28 where R.sub.21 is a C.sub.1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z.sub.1 is either NH, sulfur or oxygen, R.sub.22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R.sub.28 includes a functional group which attaches the linker to the acceptor dye.

Abstract: Methods and apparatus are disclosed for improved mapping and sequencing of carbohydrate polymers. In one aspect, the improvement includes a means for coupling two capillary electrophoresis (CE) tubes in such a way so as to (i) efficiently transfer a selected sample component from a first CE capillary to a second CE capillary or, (ii) introduce a supplementary reagent into the separation path between the two capillaries, e.g., an internal standard, binding agent, enzyme, and the like. In an additional aspect, the invention includes improved apparatus and methods for electrophoresis of labeled carbohydrates in which a sample carbohydrate labeled with a first label is separated by CE and its migration behavior is compared with an internal standard labeled with one or more second labels which are distinguishable from the first label.

Abstract: Apparatus and method for performing a nucleic acid amplification reaction and preferably a polymerase chain reaction (PCR) in a reaction mixture in at least one capillary tube. Several different embodiments are disclosed. One embodiment cycles a sample through a capillary tube loop passing through two thermostatted fluid baths. Another embodiment has the capillary tube routed alternatingly between two heat exchangers to that the sample makes only one pass through the tube. Other embodiments maintain the heat exchangers stationary and translate the samples between them. Still further embodiments maintain the samples stationary and either automatically translate or rotate the heat exchangers past the samples contained within the capillary tubes to perform the thermal cycles necessary for the amplification reaction.

Abstract: Method and composition for detecting one or more selected polynucleotide regions in a target polynucleotide. In one embodiment of the invention, a plurality of different-sequence probe pairs are added to a target polynucleotide, where each probe pair includes two polynucleotide probe elements which are complementary in sequence to adjacent portions of a selected one of the target sequences in the target polynucleotide. In each probe pair, one of the probe elements contains a non-polynucleotide polymer chain which imparts a distinctive mobility to the associated probe pair, when the elements in the pair are ligated. The other element in the pair contains a detectable reporter label. After the probe pairs have been allowed to hybridize with the target polynucleotide, the hybridized polynucleotides are treated under conditions effective to ligate the end subunits of target-bound probe elements when their end subunits are base-paired with adjacent target bases.

Abstract: Substituted propargylethoxyamido nucleosides are disclosed having the structure ##STR1## wherein X is selected from the group consisting of amino alkanoic acid, alkylamino benzoic acid, .alpha.-amino acid, and 4-amino-2-butynoic acid. R.sub.1 and R.sub.2 taken separately are selected from the group consisting of --H, lower alkyl, protecting group, and label; R.sub.3 is selected from the group consisting of --H and lower alkyl. B is a 7-deazapurine, purine, or pyrimidine nucleoside base. When B is purine or 7-deazapurine, the sugar moiety is attached at the N.sup.9 -position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N.sup.1 -position of the pyrimidine.

Abstract: A time of flight spectrometer having a dynode detector in which a set of dynode plates at an output end of the dynode is each connected to an associated capacitor that functions as a charge reservoir for said dynode, thereby substantially avoiding saturating this dynode. A grid is place adjacent to and parallel to a front surface of a target to produce an acceleration region that accelerates ions substantially perpendicularly away from said front surface, thereby reducing time of flight deviations caused by nonperpendicular emission of ions from the target. A biased guide wire aligned perpendicular to the front surface of the target produces an electric field that images ions from the target onto a detector.

Abstract: Apparatus and method for determining an unknown starting molar concentration of target nucleic acid molecules at the beginning of a polymerase chain reaction in a sample reaction mixture containing suitable buffers, two complementary kinds of oligonucleotide primers, a molar excess of four kinds of nucleoside triphosphates, a DNA polymerase, and the unknown starting molar concentration of target nucleic acid molecules, wherein the two kinds of primers are provided in a known concentration (C.sub.

Abstract: A connecting apparatus for conveyance of cryogenic fluid has a pair of connecting members formed of thermal insulating material. Each has a flange with an axial tubular protrusion and a rim extending over the protrusion so as to form an annular cavity therebetween. Each flange and its protrusion has a central bore. When the two members are fitted together, the combined bore conveys of cryogenic fluid therethrough. The protrusions have overlapping lips with a snug fit to substantially retain the cryogenic fluid in the combined bore. A detachable retainer holds the members in union with an o-ring seal between the rims. The respective cavities form an enclosed cavity that provides thermal insulation between the ring seal and the tubular protrusions for the cryogenic fluid in the common bore.

Abstract: A method is provided for determining a genotype by comparing the nucleotide sequence of members of a gene system which flank the polymorphous sections of a locus or loci of interest. In a general embodiment of the method, the nucleotide sequences chosen for comparison (i) contain one or two sequences that are completely conserved between the members of the gene family, where two or more members are selected from different sources, and (ii) the completely conserved sequences flank strongly conserved sections of genetic material. The completely conserved sequences are typically used to amplify, from different sources, the strongly conserved sections of genetic material. The resulting amplified nucleic acid sequences from the different sources are then compared to establish genotypes.

Abstract: An electrophoresis separation medium is disclosed comprising a matrix of a copolymer composed of a linear hydrophilic polymer segment having a selected substantially uniform segment length and hydrophobic polymer segments carried on each end. Also disclosed is an electrophoresis method employing the separation medium which permits high resolution separation of polynucleotides, especially in DNA sequencing operations.

Abstract: Cloning systems useful for the isolation of recombinant nucleic acid are disclosed in which the recombination of cloning-system nucleic acid and foreign nucleic acid is linked to the expression of a moiety on the surface of a host organism, the moiety being a first member of a binding pair. When recombination occurs between the nucleic acid and the foreign nucleic acid, the moiety is expressed on the surface of the host organism. The isolation of recombinant nucleic acid is then performed by attaching a second member of the binding pair to a solid support and contacting the host organism with the support. When the first member of the binding pair is expressed on the surface of the host organism, the host organism binds to the second member of the binding pair attached to the solid support, thereby selectively isolating those organisms. Other aspects of the invention include devices for the isolation of individual cells that differentially express binding moieties on their surface.

Abstract: An improved signal processing method in a signal processor for performing the deconvolution of a signal resulting from an analytical separation process is disclosed. In a first aspect, a signal representing a plurality of partially separated sample zones is measured, a point-spread-function of the signal is determined, and the Fourier transform of the signal and the point-spread-function is taken. Next, a noise component n of the signal is determined and the value of a result signal A(f) is calculated using the following filter ##EQU1## where D(f) is the Fourier transform of the signal, P(f) is the Fourier transform of the point-spread-function, and P*(f) is the complex conjugate of P(i). Finally, the inverse Fourier transform of the result A(f) is taken and reported as A(t). Preferably, the point-spread function is a Gaussian function having a standard deviation .sigma., where .sigma. is determined using either an .alpha..beta. tracker or the function .sigma.=(a+bt.sup.2).sup.

Abstract: The compounds of the invention are exemplified by the class of diglycolate synthesis supports particularly useful as support reagents for the direct synthesis of 3'-labeled polynucleotides. Generally, the compounds of the invention have the structure ##STR1## where T is an acid-cleavable hydroxyl protecting group, e.g., 4,4'-dimethoxytrityl; L.sub.1 is a linker for connecting a 3'-terminal nitrogen to carbon; L.sub.2 and L.sub.3 are linkers for connecting oxygen and carbon; W is a solid support, e.g., CPG or polystyrene; L.sub.4 is a linker for connecting the solid support to nitrogen; R.sub.1 and R.sub.2 are nitrogen substituents, e.g., hydrogen, lower alkyl, nitrogen protecting group, or label; and R.sub.3 through R.sub.7 are carbon substituents, e.g., hydrogen or lower alkyl. In a first particularly preferred embodiment, the synthesis supports of the invention are exemplified by compounds having the structure ##STR2## where DMT is 4,4'-dimethoxytrityl and W is polystyrene.

Abstract: The invention relates to passive internal references for use in quantitating the formation of amplification products in a nucleic amplification reaction. The internal amplification reference molecules of the invention comprise a first and second fluorophore joined together through a backbone connector. The first and second fluorophores are joined on the backbone in a configuration that permits the energy transfer from the first fluorophore to the second fluorophore. The backbone connector is selected so as not to bind to the target nucleic acid sequence under nucleic acid amplification conditions. Preferably, the backbone connector is a polynucleotide. Another aspect of the invention is to provide passive internal reference molecule containing reagent compositions for use in nucleic acid amplification reactions. The compositions comprise the internal amplification reference molecule of the invention and a nucleic acid amplification reaction buffer.

Abstract: An oligonucleotide probe is provided which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. The oligonucleotide probe is constructed such that the probe exists in at least one single-stranded conformation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucleotide where the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule. By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibit different fluorescence signal intensities when the probe is hybridized and unhybridized.