Abstract: A class of asymmetric monobenzoxanthene compounds useful as fluorescent dyes are disclosed having the structure wherein Y1 and Y2 are individually hydroxyl, amino, imminium, or oxygen, R1–R8 are hydrogen, fluorine, chlorine, alkyl, alkene, alkyne, sulfonate, amino, amido, nitrile, alkoxy, linking group, and combinations thereof, and R9 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 where substituents R3–R7 correspond to like-referenced substituents in the structure of described above, and Y2 is hydroxyl or amine. In another aspect, the invention includes methods for synthesizing the above dye compounds and intermediates.

Abstract: The invention provides an asymmetric cyanine dye compound having the structure including substituted forms thereof, wherein, at least one of R1 and R2 is linking group, X is O, S, or Se, and n ranges from 0 to 2. The invention further provides reporter-quencher dye pairs comprising the asymmetric cyanine dyes, dye-labelled polynucleotides incorporating the asymmetric cyanine dyes, and hybridization detection methods utilizing the dye-labelled polynucleotides.

Abstract: Propargylethoxyamino nucleosides are disclosed having the structure wherein R1 and R2 are —H, lower alkyl, or label; B is a 7-deazapurine, purine, or pyrimidine nucleoside base; W1 is —H or —OH; W2 is —OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3?-position; and W3 is —PO4, —P2O7, —P3O10, phosphate analog, or —OH. Additionaly, a primer extension method is provided employing the above propargylethoxyamino nucleosides.

Abstract: Compositions and methods for the analysis of multiple nucleic acid target sequences are disclosed. The compositions comprise a probe comprising a target-specific portion for sequence-specific hybridization to a target nucleic acid sequence, and a tag; and a mobility-modifier comprising a tail and a tag complement for binding to the tag.

Abstract: A nucleoside/tide compound having the structure: NUC-L-S-LB/LG is described wherein NUC is a nucleoside/tide having a nucleobase portion B, L is a rigid linkage, S is a spacer; and LB/LG is a member of a linkage pair or a label. NUC is attached to L through B such that when B is a purine, L is attached to the 8-position of the purine, when B is 7-deazapurine, L is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, L is attached to the 5-position of the pyrimidine. In an important aspect of the invention, L has the structure: wherein each of n, o and p are integers ranging from 0 to 3, and the sum of n, o and p is at least 2, and each of W, X, Y and Z is selected from the group consisting of carbon and nitrogen. The invention further includes polynucleotide compounds comprising the nucleoside/tide, and primer extension methods utilizing the nucleoside/tide, particularly when used in combination with certain mutant polymerase enzymes.

Abstract: Dibenzorhodamine compounds having the structure: are disclosed, including nitrogen- and aryl-substituted forms thereof. In addition, two intermediates useful for synthesizing such compounds are disclosed, a first intermediate having the structure: including nitrogen- and aryl-substituted forms thereof, and a second intermediate having the structure including nitrogen- and aryl-substituted forms thereof, wherein substituents at positions C14 to C18 taken separately are selected from the group consisting of hydrogen, chlorine, fluorine, lower alkyl, carboxylic acid, sulfonic acid, —CH2OH, alkoxy, phenoxy, linking group, and substituted forms thereof. The invention further includes energy transfer dyes comprising the dibenzorhodamine compounds, nucleosides labeled with the dibenzorhodamine compounds, and nucleic acid analysis methods employing the dibenzorhodamine compounds.

Abstract: The invention provides an asymmetric cyanine dye compound having the structure including substituted forms thereof, wherein, at least one of R1 and R2 is linking group, X is O, S, or Se, and n ranges from 0 to 2. The invention further provides reporter-quencher dye pairs comprising the asymmetric cyanine dyes, dye-labelled polynucleotides incorporating the asymmetric cyanine dyes, and hybridization detection methods utilizing the dye-labelled polynucleotides.

Abstract: A method is disclosed for effecting the concentration of a polar analyte in an alternating electric field. In the method, a relative translation of the polar analyte and an alternating electric field along a translation path is effected. A portion of the polar analyte is then trapped and concentrated in a concentration zone formed by the intersection of the translation path and the alternating electric field. Also disclosed are various devices for carrying out the forgoing method.

Abstract: An oligonucleotide probe is disclosed, the probe including an oligonucleotide, a fluorescer molecule attached to a first end of the oligonucleotide and a quencher molecule attached to the opposite end of the oligonucleotide. The probe is rendered impervious to digestion by the 5′→3′ exonuclease activity of a polymerase and the 3′→5′ extension of by a polymerase. The invention also includes methods for performing combined PCR amplification and hybridization probing, one such method including the steps of contacting a target nucleic acid sequence with PCR reagents and an oligonucleotide probe as described above, and subjecting these reagents to thermal cycling. One preferred refinement of the above method further includes the addition of a strand displacer to facilitate amplification.

Abstract: An oligonucleotide probe is disclosed, the probe including an oligonucleotide, a fluorescer molecule attached to a first end of the oligonucleotide and a quencher molecule attached to the opposite end of the oligonucleotide. The probe is rendered impervious to digestion by the 5′→3′ exonuclease activity of a polymerase and the 5′→3′ extension of by a polymerase. The invention also includes methods for performing combined PCR amplification and hybridization probing, one such method including the steps of contacting a target nucleic acid sequence with PCR reagents and an oligonucleotide probe as described above, and subjecting these reagents to thermal cycling. One preferred refinement of the above method further includes the addition of a strand displacer to facilitate amplification.

Abstract: Compositions and methods for the analysis of multiple nucleic acid target sequences are disclosed. The compositions comprise a probe comprising a target-specific portion for sequence-specific hybridization to a target nucleic acid sequence, and a tag; and a mobility-modifier comprising a tail and a tag complement for binding to the tag.

Abstract: Methods for reducing peak broadening associated with the establishment of a run field in a capillary electrophoresis process, and apparatus useful for carrying out such methods, are described. Such methods include defining a maximum ramp rate to be used during an initial electric field ramp, defining a minimum period over which the run field is established, and/or maintaining a temperature of a separation medium to within certain ranges during the initial electric field ramp. In addition, methods for determining a ramp rate effective for reducing peak broadening are disclosed.

Abstract: The invention provides uncharged water-soluble silica-adsorbing polymers for suppressing electroendoosmotic flow and to reduce analyte-wall interactions in capillary electrophoresis. In one aspect of the invention, one or more of such polymers are employed as components of a separation medium for the separation of biomolecules, such as polynucleotides, polysaccharides, proteins, and the like, by capillary electrophoresis. Generally, such polymers are characterized by (i) water solubility over the temperature range between about 20° C. to about 50° C., (ii) concentration in a separation medium in the range between about 0.001% to about 10% (weight/volume), (iii) molecular weight in the range of about 5×103 to about 1×106 daltons, and (iv) absence of charged groups in an aqueous medium having pH in the range of about 6 to about 9.

Abstract: The invention provides uncharged water-soluble silica-adsorbing polymers for suppressing electroendoosmotic flow and to reduce analyte-wall interactions in capillary electrophoresis. In one aspect of the invention, one or more of such polymers are employed as components of a separation medium for the separation of biomolecules, such as polynucleotides, polysaccharides, proteins, and the like, by capillary electrophoresis. Generally, such polymers are characterized by (i) water solubility over the temperature range between about 20° C. to about 50° C., (ii) concentration in a separation medium in the range between about 0.001% to about 10% (weight/volume), (iii) molecular weight in the range of about 5×103 to about 1×106 daltons, and (iv) absence of charged groups in an aqueous medium having pH in the range of about 6 to about 9.

Abstract: Dibenzorhodamine compounds having the structure are disclosed, including nitrogen and aryl-substituted forms thereof. In addition, two intermediates useful for synthesizing such compounds are disclosed, a first intermediate having the structure including nitrogen- and aryl-substituted forms thereof, and a second intermediate having the structure including nitrogen- and aryl-substituted forms thereof, wherein substituents at positions C-14 to C18 taken separately are selected from the group consisting of hydrogen, chlorine, fluorine, lower alkyl, carboxylic acid, sulfonic acid, —CH2OH, alkoxy, phenoxy, linking group, and substituted forms thereof. The invention further includes energy transfer dyes comprising the dibenzorhodamine compounds, nucleosides labeled with the dibenzorhodamine compounds, and nucleic acid analysis methods employing the dibenzorhodamine compounds.

Abstract: Disclosed is a method for determining the number of repeat units in a repeat region of a target nucleic acid. 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: A class of asymmetric monobenzoxanthene compounds useful as fluorescent dyes are disclosed having the structure wherein Y1 and Y2 are individually hydroxyl, amino, imminium, or oxygen, R1-R8 are hydrogen, fluorine, chlorine, alkyl, alkene, alkyne, sulfonate, amino, amido, nitrile, alkoxy, linking group, and combinations thereof, and R9 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 where substituents R3-R7 correspond to like-referenced substituents in the structure of described above, and Y2 is hydroxyl or amine. In another aspect, the invention includes methods for synthesizing the above dye compounds and intermediates.

Abstract: Propargylethoxyamino nucleosides are disclosed having the structure wherein R1 and R2 are —H, lower alkyl, or label; B is a 7-deazapurine, purine, or pyrimidine nucleoside base; W1 is —H or —OH; W2 is —OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3′-position; and W3 is —PO4, —P2O7, —P3O10, phosphate analog, or —OH. Additionaly, a primer extension method is provided employing the above propargylethoxyamino nucleosides.

Abstract: A multi-channel capillary electrophoresis apparatus is disclosed. The apparatus includes a capillary array assembly comprising a plurality of capillaries, each capillary having a capillary outlet, and an outlet support for supporting the capillary outlets. The apparatus further includes a cuvette defining a receiving slot, a gap region, and a detection zone, where the receiving slot is adapted to removably receive the outlet support, and wherein when the outlet support is inserted into the receiving slot, the capillary outlets are positioned in the gap region in proximity to the detection zone, and a flow channel is formed by the outlet support and the receiving slot such that the flow channel is in fluid communication with the gap region. In addition, the apparatus includes a front plumbing block in fluid communication with the flow channel for supplying a fluid flow through the gap region sufficient to transport material downstream from the capillary outlets to the detection zone.

Abstract: Dibenzorhodamine compounds having the structure are disclosed, including nitogen- and aryl-substituted forms thereof. In addition, two intermediates useful for synthesizing such compounds are disclosed, a first intermediate having the structure including nitrogen- and aryl-substituted forns thereof, and a second intermediate having the structure including nitrogen- and aryl-substituted forms thereof, wherein substituents at positions C-14 to C18 taken separately are selected from the group consisting of hydrogen, chlorine, fluorine, lower alky, carboxylic acid, sulfonic acid, —CH2OH, alkoxy, phenoxy, linking group, and substituted forms thereof. The invention further includes energy transfer dyes comprising the dibenzorhodamine compounds, nucleosides labeled with the dibenzorhodamine compounds, and nucleic acid analysis methods employing the dibenzorhodamine compounds.