Abstract: A class of 4,7-dichlororhodamine compounds useful as fluorescent dyes are disclosed having the structure ##STR1## wherein R.sub.1 -R.sub.6 are hydrogen, fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, sulfonate, sulfone, amino, amido, nitrile, lower alkoxy, linking group, or combinations thereof, or, when taken together, R.sub.1 and R.sub.6 is benzo, or, when taken together, R.sub.4 and R.sub.5 is benzo; Y.sub.1 -Y.sub.4 are hydrogen or lower alkyl, or, when taken together, Y.sub.1 and R.sub.2, Y.sub.2 and R.sub.1 Y.sub.3 and R.sub.3, or Y.sub.4 and R.sub.4 is propano, ethano, or substituted forms thereof, and X.sub.1 -X.sub.3 taken separately are selected from the group consisting of hydrogen, chlorine, fluorine, lower alkyl, carboxylate, sulfonic acid, --CH.sub.2 OH, and linking group. In another aspect, the invention includes reagents labeled with the 4,7-dichlororhodamine dye compounds, including deoxynucleotides, dideoxynucleotides, and polynucleotides.

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 further 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: A chain-termination type DNA sequencing method is disclosed wherein deoxynucleotide 2'-deoxythymidine-5'-triphosphate is replaced by 2'-deoxyuridine-5'-triphosphate, or analogs thereof Kits for performing the method are also provided.

Abstract: A one-piece sample tube for use in a PCR thermal cycler having a first substantially cylindrically shaped upper wall section having an external conically beveled shoulder moulded around the entire circumference of the section, the thickness of the wall section above the shoulder being about 0.022 inches and the thickness of the wall section below the shoulder being about 0.015 inches. The sample tube further includes a second lower wall section having a frustrum of a cone shape with an included angle of about 17 degrees, the lower wall section having a thickness of 0.009 inches the second section holding about 100 microliters of liquid. The tube is compliant and made from polypropylene and is autoclavable.

Abstract: An apparatus is provided which includes a sample holder for holding a reaction chamber which includes an optical interface, a fiber optic cable for delivering an excitation beam to a sample housed within the reaction chamber and for receiving light emitted by the sample, and a lens co-axially disposed with the fiber optic cable and positioned outside the reaction chamber for focusing the excitation beam through the optical interface and within a volume of the sample and for collecting and transmitting to the fiber optic cable light emitted within the volume of the sample.

Abstract: A class of aromatic-substituted xanthene compounds useful as fluorescent dyes is disclosed, the compounds having the general structure where Y.sub.1 and Y.sub.2 taken separately are selected from the group consisting of hydroxyl, oxygen, imminium, linking group and amine, or Y.sub.1 taken together with R.sub.2 is cyclic imine, or Y.sub.2 taken together with R.sub.3 is cyclic amine; R.sub.2, R.sub.3, R.sub.5, and R.sub.7 taken separately are selected from the group consisting of hydrogen, fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, sulfonate, sulfone, amino, imminium, amido, nitrile, lower alkoxy, phenyl, and linking group; R.sub.1 taken separately is selected from the group consisting of phenyl, substituted phenyl, polycyclic aromatic, substituted polycyclic aromatic, linking group and electron-rich heterocycle, or when taken together with R.sub.7 is selected from the group consisting of electron-rich heterocycle and indene; R.sub.

Abstract: The present invention relates to improved methods of generating polynucleotide sequencing reaction products from cycle sequencing and relates to various instruments and reagents for use in the subject methods. The use of "step-down" thermal profiles in conjunction with cycle sequencing is described. Step-down thermal profiles are formed by combining several thermal cycle sets such that the annealing temperature of each thermal cycle set is less than the annealing temperature of the preceding thermal cycle set. One embodiment of the invention is a method of generating a plurality of polynucleotide sequencing reaction products in parallel by subjecting a plurality of sequencing solution preparations to a step-down thermal profile, i.e., exposure to repeated thermal cycle sets, each thermal cycle set having an annealing temperature that is lower than the annealing temperature of the annealing phases of the preceding thermal cycle set.

Abstract: A method of purifying a hydrophobically substituted polynucleotide by reverse phase HPLC is described. The hydrophobic substituent may be removed from the polynucleotide under non-acidic conditions; the purification method is thus especially useful for acid sensitive polynucleotide analogs.

Abstract: A method is provided for determining aneuploidy of selected chromosomes. An important feature of the invention is the quantitative amplification of STR markers with repeat units of at least 3 nucleotides. The amplified STR DNA is separated by size and the respective quantities of the amplified components are determined and related to chromosome copy number. The highly polymorphic nature of the STR markers permits a more sensitive and reliable quantitative analysis of the amplified DNA.

Abstract: The present invention relates to the electrophoretic separation of bio-organic molecules using a slab gel electrophoresis apparatus having a pair of spaced, confronting plates defining a multi-lane separation zone adapted to hold a separation medium and an upper loading zone. In one aspect of the invention, at least one of the plates is shaped to provide an expanded loading zone. The plate-to-plate distance within the expanded loading zone is greater than the plate-to-plate width within the separation zone. In another aspect of the invention, a comb is provided with each tooth having a gradual taper beginning at a support member and extending along most of the tooth's longitudinal axis, and a sharp taper beginning immediately beyond the gradual taper and extending to the end of the tooth.

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-sieiving medium.

Abstract: An improved fluorescence monitoring apparatus for measuring fluorescent emission from a sample in response to sample irradiation by an emission beam is disclosed. The apparatus employs a sample tube having surface roughness characteristics which substantially reduce background fluorescence emission due to contamination of the tube holder in the apparatus. Also disclosed is a method of reducing such background, by texturing of a sample tube to produce desired roughness characteristics.

Abstract: The invention relates to new methods of RNA isolation that exploit the surprising discovery that RNA may be differentially precipitated from DNA. The subject methods result in the formation of an RNA-containing precipitate that has an RNA content at least two fold enriched with respect to DNA, as compared with the RNA to DNA ratio of the solution from which the RNA-containing precipitate is derived. The invention also includes various methods of DNA isolation that employ the selective precipitation of RNA. The degree of RNA enrichment achieved is often much greater than two fold; enrichment by a factor of ten or greater is frequently obtained. By precipitating RNA from a solution, the RNA may be collected by simple procedures such as centrifugation or filtration, thereby avoiding the need to bind the RNA to solid phase. The collected RNA precipitate may then be solubilized.

Abstract: A method is provided for genotyping a target sequence at at least two allelic sites by a 5' nuclease amplification reaction. In one embodiment, the method includes performing a nucleic acid amplification on a target sequence having at least two different allelic sites using a nucleic acid polymerase having 5'.fwdarw.

Abstract: The invention provides a method for diagnosis of genetic abnormalities or other genetic conditions which can be readily automated. The method is used to determine the presence or absence of a target sequence in a sample of denatured nucleic acid and entails hybridizing the sample with a probe complementary to a diagnostic portion of the target sequence (the diagnostic probe), and with a probe complementary to a nucleotide sequence contiguous with the diagnostic portion (the contiguous probe), under conditions wherein the diagnostic probe remains bound substantially only to the sample nucleic acid containing the target sequence. The diagnostic probe and contiguous probe are then covalently attached to yield a target probe which is complementary to the target sequence, and the probes which are not attached are removed.

Abstract: A primary chromatographic system is operated with a standard sample at several temperatures to generate primary retention times which are fitted to a function to determine thermodynamic constants to relate the times to temperature. A target chromatographic system is operated with the standard sample to generate secondary retention times. The function is used with these times to determine and an effective column parameter for the target system. The function then is used with this parameter and the primary times to determine a pressure program. Further operation of the target system with a application sample and the pressure program effects standardized retention times. A particular searching technique is utilized to apply the function. A temperature calibration technique with a selected sample measures column temperature for the target system, and a validation is done on the target system.

Abstract: Reporter-quencher probe assays of nucleic acid amplification, such as PCR, are rendered more meaningful by the addition of internal control reagents. An internal control polynucleotide is amplified with internal control primers and the product is measured by correlation with increased fluorescence by polymerase mediated-exonuclease cleavage or hybridization of the internal control probe. Probes specific for target and internal control polynucleotides are labelled with spectrally resolvable reporters, allowing for concurrent detection and measurement of target and control amplification. A kit of all PCR reagents can be dispensed into reaction chambers in a high-throughput system for rapid and accurate nucleic acid amplification assay, with real-time or end-point measurements. Fluorescent signals correlated to target and internal control levels are spectrally resolvable and measured concurrently.

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: In a first aspect, the method of the invention includes the steps of annealing a primer to a target nucleic acid; performing a first primer extension reaction using a first primer extension reagent; separating the target-primer hybrid and unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent; measuring a signal produced by the label; treating the label so as to render the label undetectable; and repeating the above steps until the signal is substantially less than a signal detected in a previous cycle.

Abstract: Apparatus are disclosed that thermally cycles samples between at least two temperatures. These apparatus operate by impinging fluid jets onto the outer walls of a sample containing region. Because the impinging fluid jets provide a high heat transfer coefficient between the jet and the sample containing region, the sample containing regions are uniformly cycled between the two temperatures. The heat exchange rate between the jets and the sample regions are substantially uniform.