Abstract: The present invention relates to methods for resolving convoluted peaks in a chromatogram into one or more constituent peaks using peak resolution values. The peaks methods of the invention determine empirical peak resolution values of “well-defined” or “isolated” peaks in the data, then extrapolate these empirical resolution values to peaks in neighboring regions to predict the number of constituent peaks at a given peak position. Predicted peak resolution values are compared to observed peak resolution values of low-resolution or convoluted peaks to determine the number of constituent peaks in the convoluted peaks. These methods enable extension of the region of data that can used for identifying nucleotide sequences, and increase base-calling accuracy in the low-resolution region (end region) of data.

Abstract: The present invention relates to methods for resolving convoluted peaks in a chromatogram into one or more constituent peaks using peak resolution values. The peaks methods of the invention determine empirical peak resolution values of “well-defined” or “isolated” peaks in the data, then extrapolate these empirical resolution values to peaks in neighboring regions to predict the number of constituent peaks at a given peak position. Predicted peak resolution values are compared to observed peak resolution values of low-resolution or convoluted peaks to determine the number of constituent peaks in the convoluted peaks. These methods enable extension of the region of data that can used for identifying nucleotide sequences, and increase base-calling accuracy in the low-resolution region (end region) of data.

Abstract: The present invention relates to methods and apparatus for detecting peaks in a sample nucleic acid data trace derived from a sample polynucleotide by (a) receiving a sequence signature of a reference polynucleotide, wherein the sequence signature comprises a profile of peak height at one or more peak position of a nucleic acid sequence data trace of one or more of reference polynucleotides; (b) receiving a sample nucleic acid sequence data trace of a sample polynucleotide corresponding to the reference polynucleotide, wherein the sample nucleic acid sequence data trace comprises a value of peak height at one or more peak position corresponding to the peak positions of the sequence signature; and (c) detecting peaks in the sample nucleic acid data trace having a peak height that correlates with the profile of peak height of the sequence signature at a corresponding peak position.

Abstract: The present invention is directed to a method for alignment of nucleic acid data traces. The method involves selecting reference alignment points from among internal peaks representing highly conserved bases, preferably consisting of heterogeneous multiplets. The alignment points may also optionally include the primer peak and/or the full-length peak. Reference position numbers are assigned to these alignment points reflecting the known relative position of the alignment point, a sequence position number is assigned to peaks in the data traces so as to maximize assigning the sequence position number and the reference position number to the same base. Optionally, the method may include the step of determining the average peak spacing interval between alignment points and assigning a sequence position number to peaks occurring at the intervals. The data traces are then aligned based on the assigned sequence position numbers.

Abstract: A simulated electrophoretic trace is prepared by first obtaining an input file containing an input base sequence comprising a string of letters (A, C, G and/or T) in an order corresponding to the input base sequence, and then modifying the input file using one or more functions to take into account perturbations associated with (1) changes in peak intensity as a function of base number; (2) peak shape as a function of base number; (3) peak skew; (4) spacing between peaks; (5) background; (6) noise; (7) spectral cross-talk; (8) instrumental effects and/or (9) gel electrophoresis effects to produce a modified file representing a simulated electrophoretic trace. The method may be performed using a specially adapted apparatus.

Abstract: A simulated electrophoretic trace is prepared by first obtaining an input file containing an input base sequence comprising a string of letters (A, C, G and/or T) in an order corresponding to the input base sequence, and then modifying the input file using one or more functions to take into account perturbations associated with (1) changes in peak intensity as a function of base number; (2) peak shape as a function of base number; (3) peak skew; (4) spacing between peaks; (5) background; (6) noise; (7) spectral cross-talk; (8) instrumental effects and/or (9) gel electrophoresis effects to produce a modified file representing a simulated electrophoretic trace. The method may be performed using a specially adapted apparatus.

Abstract: The sequence of a target DNA molecule is determined by preparing four chain termination reaction mixtures, one for each base type. The first set of fragments, indicative of the positions of a first type of base, are labeled with a first fluorescent label. The second set of fragments, indicative of the positions of a second type of base, are labeled with a second fluorescent label different from the first fluorescent label, The third set of fragments, indicative of the positions of a third type of base, are labeled with a third fluorescent label, different from the first and second fluorescent labels or with at least two labels, including at least one fluorescent label selected from among the first, second and third fluorescent labels.

Abstract: The sequence of a target DNA molecule is determined by preparing four chain termination reaction mixtures, one for each base type. The first set of fragments, indicative of the positions of a first type of base, are labeled with a first fluorescent label. The second set of fragments, indicative of the positions of a second type of base, are labeled with a second fluorescent label different from the first fluorescent label. The third set of fragments, indicative of the positions of a third type of base, are labeled with a third fluorescent label, different from the first and second fluorescent labels or with at least two labels, including at least one fluorescent label selected from among the first, second and third fluorescent labels.

Abstract: For evaluation of a target DNA sequence, a sample mixture is prepared containing one or more sets of sequencing polynucleotide fragments, each set containing fragments having lengths indicative of the positions of at least one base within the target DNA sequence. These sequencing fragment sets are each labeled with a different type of label (for example fluorescent labels). The sample mixture also includes a set of calibrant polynucleotide fragments having a plurality of known fragment lengths. The calibrant polynucleotide fragments are labeled with a spectroscopically-distinguishable calibrant label. The sample mixture is then electrophoretically separated to separate the polynucleotide fragments as a function of fragment length. Real-time detection is used to detect the label(s) on the set(s) of sequencing fragments and the calibrant label as they migrate in a common lane of the separation medium to produce a sequencing data trace and a calibrant data trace.

Abstract: A microelectrophoresis chip comprises a substrate in which there are formed one or more channels, one channel for each sample to be evaluated. The channels extend for the length of the chip, a distance of generally around 1 cm, and are about 1 to 10 &mgr;m wide and 1 to 10 &mgr;m in depth. The channels are filed with a homogeneous separation matrix which acts as an obstacle to the electrophoretic migration of the charged molecules. Microelectrodes disposed in the channels are used to induce an electric filed within the homogeneous separation medium. When a voltage is applied across two or more of the microelectrodes, the charged molecules are induced to move and separate according to the electric field density, the type of solvent film, and the charge, shape and size of the charged molecule.

Abstract: A microelectrophoresis chip comprises a substrate in which there are formed one or more channels, one channel for each sample to be evaluated. The channels extend for the length of the chip, a distance of generally around 1 cm, and are about 1 to 10 &mgr;m wide and 1 to 10 &mgr;m in depth. The channels are filled with a homogeneous separation matrix which acts as an obstacle to the electrophoretic migration of the charged molecules. Microelectrodes disposed in the channels are used to induce an electric filed within the homogeneous separation medium. When a voltage is applied across two or more of the microelectrodes, the charged molecules are induced to move and separate according to the electric field density, the type of solvent film, and the charge, shape and size of the charged molecule.

Abstract: A microelectrophoresis chip comprises a substrate in which there are formed one or more channels, one channel for each sample to be evaluated. The channels extend for the length of the chip, a distance of generally around 1 cm, and are about 1 to 10 &mgr;m wide and 1 to 10 &mgr;m in depth. The channels are filled with a homogeneous separation matrix which acts as an obstacle to the electrophoretic migration of the charged molecules. Microelectrodes disposed in the channels are used to induce an electric field within the homogeneous separation medium. When a voltage is applied across two or more of the microelectrodes, the charged molecules are induced to move and separate according to the electric field density, the type of solvent film, and the charge, shape and size of the charged molecule.

Abstract: Separation matrices useful in the formation of solid-state mm- to cm-scale devices for the rapid, high-resolution separation of single-stranded DNA ladder bands generated by the Sanger dideoxy- or Maxam/Gilbert chemical DNA sequencing procedures are formed from a solid support (1) having a plurality of posts (4) disposed on a first major surface thereof to form an obstacle course of posts (4) and pores (5). The posts are arranged in a regular X, Y array and are separated one from another by a distance of 100 nm or less, preferably 10 to 30 nm, and are optionally separated into lanes 2. The separation matrix can be manufactured by first forming a mold, preferably a reusable mold using lithography techniques. The mold is the reverse of the desired pattern of posts and pores of the obstacle course, and is used for casting the obstacle course. The cast obstacle course is then fused to a solid support and separated from the mold.

Abstract: Gel holders for electrophoresis gels are made using clad fibers, particularly glass fibers as spacers between substrates. A plurality of fibers with a high-melting interior core and a low-melting external cladding are placed between a first planar substrate and a second planar substrate. The fibers are heated to a temperature sufficient to at least soften the exterior cladding of the fibers without softening the interior core of the fibers, and then cooled while they are in contact with the first and second substrates to solidify the exterior cladding. This adheres the fibers to the first and second substrates, and forms a gel chamber between said first and second substrates. The gel chamber has a thickness defined by interior core of the fibers. The fibers may be heated before or after the second substrate is placed over the top of the fibers. The gel holders thus formed may be filled immediately with a gel forming solution such as a polyacrylamide, or they may be stored indefinitely and used as needed.

Abstract: A high dynamic range apparatus for separation and detection of polynucleotide fragments has a housing adapted to receive an electrophoresis gel holder containing an electrophoresis gel loaded with fluorophore-labeled samples; one or more laser diodes for providing radiation of a frequency suitable for excitation of the fluorophore which irradiates a an array of excitation/detection sites on the electrophoresis gel; an array of detectors aligned with the excitation/detection sites for collecting fluorescent emissions; and one or more components for increasing the dynamic range of the instrument by at least an order of magnitude.

Abstract: An apparatus and method for thermally cycling a reaction mixture in a reaction vessel to expose the mixture to the varying temperatures necessary to, for example, achieve PCR amplification or the preparation of sequencing fragments using a cycle sequencing operation makes use of flow-through reaction vessels, such as capillary tubes, for the preparation and thermal cycling of reaction mixtures. In order to prevent loss of the reaction mixture from the vessels during heating, the thermal cycling apparatus of the invention provides means for sealing the proximal and distal end of each reaction vessel. The proximal ends can be sealed by coupling to a pump which permits movement of the samples within the reaction vessels.

Abstract: Gel holders for electrophoresis gels are made using clad fibers, particularly glass fibers as spacers between substrates. A plurality of fibers with an high-melting interior core and a low-melting external cladding are placed between a first planar substrate and a second planar substrate. The fibers are heated to a temperature sufficient to at least soften the exterior cladding of the fibers without softening the interior core of the fibers, and then cooled while they are in contact with the first and second substrates to resolidify the exterior cladding. This adheres the fibers to the first and second substrates, and forms a gel chamber between said first and second substrates. The gel chamber has a thickness defined by interior core of the fibers. The fibers may be heated before or after the second substrate is placed over the top of the fibers. The gel holders thus formed may be filled immediately with a gel forming solution such as a polyacrylamide, or they may be stored indefinitely and used as needed.

Abstract: Gel holders for electrophoresis gels are made using fibers, particularly glass fibers, which are affixed to the substrates forming the gel holder using an adhesive. These gel holders can be made by placing a plurality of adhesive-coated fibers between a first planar substrate and a second planar substrate; and applying pressure to the outside of the substrates to adhere the fibers to the first and second substrates. This forms a gel chamber between the first and second substrates which has a thickness defined by diameter of the fibers. Alternatively, uncoated fibers may be laid down in pairs, with a line of adhesive disposed between each fiber of the pair. When the adhesive is cured, it binds the fibers in position as spacers. At the same time, the fibers isolate the adhesive from the gel compartment. In this way, interference of components of the adhesive with the polymerization of the a gel in the gel chamber can be avoided.