Abstract: Electrophoresis gels are formed using a gel-forming insert having a beveled edge which results in loading sites having a beveled bottom surface. The gel forming insert can have a continuous beveled edge across the entire width of the gel, in which case a special loading insert is used which matches the bevel of the gel. Alternatively, the gel forming insert may be formed with a plurality of fingers with beveled ends, each finger defining a well in the gel. In one form of the gel-forming insert, the fingers are formed from a soft, flexible polymer such as silicone applied on a rigid support.

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: Electrophoresis gels are formed using a gel-forming insert having a beveled edge which results in loading sites having a beveled bottom surface. The gel forming insert can have a continuous beveled edge across the entire width of the gel, in which case a special loading insert is used which matches the bevel of the gel. Alternatively, the gel forming insert may be formed with a plurality of fingers with beveled ends, each finger defining a well in the gel. In one form of the gel-forming insert, the fingers are formed from a soft, flexible polymer such as silicone applied on a rigid support.

Abstract: A method is provided for simultaneously determining the positions of a selected nucleotide base in a target region of both strands of a denatured duplex nucleic acid polymer. The nucleic acid polymer is combined with a reactant mixture comprising first and second oligonucleotide primers, said primers binding to the sense and antisense strands, respectively, of the nucleic acid polymer at a location flanking the target region; a thermostable DNA polymerase; a chain-terminating nucleotide triphosphate complementary to the selected nucleotide base; and other reagents for synthesis of chain extension products to form a reaction mixture. This mixture is processed through a plurality of thermal cycles, each including at least a chain extension phase and a denaturation phase to produce chain extension products. These chain extension products are evaluated to determine the positions of the selected bases.

Abstract: Rapid and cost effective diagnosis of p53 mutations of a sample of patients is achieved by employing a selected plurality of diagnostic tools, in a hierarchy of increasing accuracy and cost per tool, in which each tool detects essentially no false positives. Diagnostic tests that may be included among the plurality of tests selected include, in order of increasing accuracy and cost:(a) immunoassays,(b) analysis of DNA from a patient sample by quantitative amplification of p53 exons using amplification primers complementary to intron regions flanking each exon and examination of the length or quantity of each amplified fragment for nucleotide insertions or deletions relative to the normal p53 gene.

Abstract: Reliable and cost effective testing for mutations in the RB1 gene can be accomplished by (a) quantitatively amplifying exons of the sample RB1 gene using primers complementary to intron regions flanking each exon; and (b) determining the lengths and/or quantities of the amplification products for each exon and comparing that length or quantity to the length or quantity of amplification products obtained when a wild-type RB1 gene is amplified using the same primers. Differences in length between an amplified sample exon and the corresponding amplified wild-type exon reflect the occurrence on an insertion or deletion mutation in the sample RB1 gene. Differences in quantity reflect the complete absence of an exon, or heterozygosity for a mutant exon. Next, the nucleic acid sequence of each exon found to contain an insertion or deletion mutation is determined, or of all exons in the event no insertion or deletion mutations are identified.

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: The sequence of human papillomavirus present in a sample is determined by amplifying a portion of the L1 open reading frame of human papillomavirus genome to form L1 amplicons containing plus and minus amplified strands using MY09 and MY11 amplification primers, and then determining the positions of at least the A bases in the antisense amplicon using a consensus sequencing primer which is shifted six bases with respect to the MY09 primer (MY09-6). This primer has the sequence ARRGGAWACT GATCWARDTC (Seq. ID No.

Abstract: Electrophoretic separation of an analyte species in a sample is achieved with increased resolution by loading the sample onto a loading site of an electrophoresis gel, said loading site having a gel/buffer interface and then applying a focusing electric field to a first pair of electrodes to cause the analyte species to migrate to a narrow region disposed at or near the loading site to produce a focused sample. Then a separation electric field is applied to cause the analyte species in the focused sample to migrate through the electrophoresis gel and to be separated into bands. This method is preferably performed in an electrophoresis apparatus that is particularly adapted to practicing the method by virtue of the a pair of focusing electrodes which are positioned to cause migration of sample to a narrow region near the buffer/gel interface within the sample loading site of the gel.

Abstract: A ligase-based assay which relies only upon knowledge of the wild-type sequence of a gene or gene fragment is used to detect all types of mutations, i.e., point mutations, insertions and deletions. The assay makes use of a set of oligonucleotide probes, which may be packaged in kit form, which hybridize in series along the length of the gene. The ligation of the probes together form a ligation product, the size of which is evaluated. When the gene or gene fragment being analyzed corresponds to the normal sequence and thus perfectly matches the probes, all of the probes in the set are ligated together, and the ligation produce has a certain resulting size. When a mutation appears in the gene, the hybridization of the probe overlapping the mutation is impaired, with the result that some or all of the ligation produce is of smaller size. By evaluating the size of the ligation product, both the existence of a mutation and its approx. position can be identified.

Abstract: The instant invention relates to methods and products for the diagnosis of mutations in the von Hippel-Lindau tumor suppressor gene. More specifically, the products are DNA oligonucleotides for use in amplifying and sequencing genomic DNA and kits including these oligonucleotides. The method of the invention relates to a hierarchical system for cost-effective diagnosis of von Hippel-Lindau tumor suppressor disease-associated mutations.

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: A streamlined, hierarchical method for obtaining information about the allelic type of a sample of genetic material derived from an HIV-infected sample relies on the observation that 93-95% of the known mutational variants of the reverse transcriptase and protease genes of HIV-1 can be determined by evaluating the positions of the A and T nucleotides within the sample. Thus, a substantial fraction of all mutational variations can be unequivocally identified by performing two initial sequencing reactions on the sample in which only ddA and ddT are used as chain terminators. For the small fraction of samples which are not identifiable based on the positions of these two bases, a second test is performed in which the sequence is determined in the 3'-direction for all four bases. This test will identify substantially all of the remaining samples.

Abstract: An apparatus for the rapid preparation of electrophoresis gels comprises: (a) a housing; (b) a support fixture removably disposed within the housing and adapted to receive a gel holder having an internal gel compartment, the support fixture being optionally adapted to permit filling of the gel holder within the housing; (c) an optional injection system, which is connectible to a reservoir for holding a polymerizable solution; (d) an optional solution injection connector adapted to couple the injection system to a gel holder placed within the filling fixture, (e) an optional controller for the injection system, which causes the injection system to inject polymerizable solution from the reservoir into the gel compartment; and (f) a radiation source disposed within the housing in a location effective to irradiate polymierizable solution within the gel compartment of a gel holder in the support fixture.

Abstract: Improved detection methods and apparatus which may be used individually or in combinations enhance the ability of the electrophoresis apparatus to detect fluorophore-labeled materials in short periods of time.

Abstract: The sequence of a target nucleic acid polymer can be determined by (a) performing a first chain-extension sequencing reaction on the target nucleic acid polymer in a reaction mixture containing first and second chain-terminators to produce a first product mixture containing commonly-labeled polynucleotide fragments complementary to a first strand of the target nucleic acid polymer, each fragment in the mixture being terminated with the first or second chain-terminator; (b) performing a second chain extension sequencing reaction on the target nucleic acid polymer in a reaction mixture containing the first and a third chain-terminator to produce a second product mixture containing commonly-labeled polynucleotide fragments complementary to the first strand of the target nucleic acid polymer, each fragment in the mixture being terminated with the first or the third chain-terminator, said first, second and third chain-terminators each being different; and (c) evaluating the lengths of the polynucleotide fragments

Abstract: Data traces from four channels of an automated electrophoresis detection apparatus are aligned by identifying peaks in each of the four data traces; normalizing the heights of the peaks in each of the data traces to a common value to generate four normalized data traces; combining the four normalized data traces in an initial alignment; and determining coefficients of shift and stretch for selected data points within each normalized data trace. The coefficients are determined by optimizing a cost function which reflects the extent of overlap of peaks in the combined normalized data traces to which the coefficients have been applied. The cost function is optimized when the extent of overlap is at a minimum. The coefficients are then used to generate a warp function for each normalized data trace. These warp function are applied to their respective data traces to produce four warped data traces which are aligned to form an aligned data set.

Abstract: Amplification primer and sequencing primer sites have been identified which permit sequence-based typing of each of the classical HLA genes in highly robust and consistent reactions without allelic dropout. To determine the DNA sequence, and thus the type of at least one exon of an HLA-A, HLA-B and HLA-C gene present in a sample, the sample is combined with an amplification reaction mixture containing the amplification primers and amplified to form an amplification product including exon 2 and exon 3 of the gene together in a single fragment. The amplification product is then combined with a sequencing reaction mixture containing one or more oligonucleotide sequencing primers which hybridize to a conserved regions the amplification product. The oligonucleotide sequencing primers between them are effective to produce sequencing fragments from all known alleles of exon 2 or exon 3 of the gene under suitable conditions to produce sequencing fragments.

Abstract: Changes in polarized light incident on a detection zone within a separation matrix are used to detect optically active molecules within the separation matrix. The separation and detection of optically active molecules within the detection zone is done by loading a sample containing optically active molecules onto a separation matrix; applying a motive force to cause the sample to migrate though the separation matrix and to separate into a plurality of subgroups of optically active molecules; directing an incident beam of polarized radiation to the detection zone; processing the collected exiting beam with an optical component which discriminates between radiation having the same polarization as the incident beam and radiation having a different polarization from the incident beam; and measuring the intensity of the processed exiting beam.

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.