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 product 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 product is of smaller size. By evaluating the size of the ligation product, both the existence of a mutation and its approximate position can be identified.

Abstract: Evaluation of a sample for the presence and qualitative nature of a microorganism can be performed in a single vessel by combining a natural abundance DNA sample with a sequencing mixture containing a primer pair, a thermally stable polymerase such as ThermoSequenase.TM. which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than about 0.4 times the rate of incorporation of deoxynucleotides, nucleotide triphosphate feedstocks, and a chain terminating nucleotide triphosphate. The mixture is processed through multiple thermal cycles for annealing, extension and denaturation to produce a product mixture which is analyzed by electrophoresis.

Abstract: An electrophoresis microgel is formed in a gel holder. The gel holder comprises a top substrate, a bottom substrate and a spacer disposed between the top substrate and the bottom substrate. The spacer establishes a separation of from 25 to 250 microns between the top substrate and the bottom substrate. A gel compartment is formed by partially sealing the top substrate to the bottom substrate, while leaving an opening for the introduction of unpolymerized gel. The gel compartment is then filled with an unpolymerized gel, which is polymerized in the gel compartment. Electrodes may be printed on the substrates, may be contacts to an exposed edge of gel, or may be applied through windows cut into one of the substrates. One type of gel holder makes use of graded beads having a diameter of 25 to 250 microns slurried in an adhesive such as an acrylate adhesive as the spacer. The slurry is printed onto the surface of one or both substrates to form a spacer of the desired shape, and then hardened using heat or light.

Abstract: Normalization of experimental fragment patterns for nucleic acid polymers having putatively known sequences starts with obtaining at least one raw fragment pattern for the experimental sample. The raw fragment pattern represents the positions of a selected nucleic acid base within the polymer as a function of migration time or distance. This raw fragment pattern is conditioned using conventional baseline correction and noise reduction technique to yield a clean fragment pattern. The clean fragment pattern is then evaluated to determine one or more "normalization coefficients.

Abstract: The allelic type of a polymorphic genetic locus in a sample is identified by first combining the sample with a sequencing reaction mixture containing a polymerase, nucleotide feedstocks, one type of chain terminating nucleotide and a sequencing primer to form a plurality of oligonucleotide fragments of differing lengths, and then evaluating the length of the oligonucleotide fragments. As in a standard sequencing procedure, the lengths of the fragments indicate the positions of the type of base corresponding to the chain terminating nucleotide in the extended primer. Instead of performing and evaluating four concurrent reactions, one for each type of chain terminating nucleotide, however, the sample is concurrently combined with at most three, and preferably only one, sequencing reaction mixtures containing different types of chain terminating nucleotides. The information obtained from this test is evaluated prior to performing any additional tests on the sample.

Abstract: Sequencing of a selected region of a target nucleic acid polymer in a genomic DNA sample can be performed in a single vessel by combining the sample with a sequencing mixture containing a primer pair, a thermally stable polymerase such as Thermo Sequenase.TM. which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than about 0.4 times the rate of incorporation of deoxynucleotides, nucleotide feedstocks, and a chain terminating nucleotide. The mixture is processed through multiple thermal cycles for annealing, extension and denaturation to produce a product mixture which is analyzed by electrophoresis.

Abstract: A method for quantitative and qualitative analysis of a nucleic acid analyte in a sample suspected to contain the nucleic acid analyte first combines the sample with a control nucleic acid, and two primer pairs, a first primer pair effective to amplify a conserved region of the nucleic acid analyte if present in the sample to produce a conserved fragment having a first length and to amplify the control nucleic acid to produce a control fragment having a second length different from the first length, and a second primer pair effective to amplify a second region of the nucleic acid analyte to produce a sequencing fragment. One member of the first primer pair is labeled with a detectable label, and one member of the second primer pair may be labeled with a label such as biotin effective to permit capture of the primer.

Abstract: Amplification and sequencing of a selected region of a target nucleic acid polymer are be performed in a single vessel. The sample is added to an amplification mixture containing a thermally stable polymerase and nucleoside feedstocks. Chain terminating dideoxynucleosides are added either at the beginning of the amplification reaction or during the course of the amplification. A thermally stable polymerase which incorporates dideoxynucleotides into an extending oligonucleotide at a rate which is no less than about 0.4 times the rate of incorporation of deoxynucleosides can be used in the amplification mixture or added with the chain terminating nucleoside.

Abstract: An improved electrophoresis and fluorescence detection method for nucleotide sequences comprises a fluorescence sensing region along the path of nucleotide detection, coupled with amplification and integration in an integrator of output signals in the form of activity peaks. The output signal, which is converted to a voltage signal, is summed with a programmable offset generated by an inexpensive eight-bit D/A converter. The offset signal is selected to establish a lower starting point for the dynamic range of analog-digital conversion, and is selected to null some or all of the background fluorescence level. The integrator is switchable under program control. The integrator is switched on for long and short integration intervals. The short intervals permit sensing over a dynamic range accommodating very high levels of fluorescence; very high peaks may be measured and features of the peaks distinguished.

Abstract: Patient samples are identified by adding to the sample, preferably at the time it is taken, a plurality of identification oligonucleotides. The identification oligonucleotides are co-processed and sequenced at the same time as the sample. The resulting sequence analysis thus provides both the sequence of the region of interest in the sample DNA, and the sequence of the identification oligonucleotides which are used to confirm the identity of the patient. In an embodiment of the invention, a plurality of specially constructed identification oligonucleotides is used. Each identification oligonucleotide is constructed based upon a starting oligonucleotide and comprises(a) a primer site which is not homologous with DNA from the organism from which the sample is taken and which may be the same or different from the primer site of the other identification oligonucleotides; and(b) an identification region having the general formula-(M-N).sub.x - or -(M-N-N).sub.

Abstract: A virtual DNA sequencer combines a plurality of individual DNA sequencers. Samples of DNA or other nucleic acid from subjects are prepared and allocated in real time to particular lanes or sets of lanes in electrophoresis plates of the individual sequencers, with records kept of the allocations. The data resulting from the electrophoresis runs is collected and collated according to the identities of the subjects. The individual sequencers are networked, and each individual sequencer is preferably equipped with a data buffer large enough to accommodate all or substantially all of a data run, thus protecting the virtual sequencer from loss of valuable data in the event that the network is disrupted for some portion of the time of the data run. In this way, a plurality of sequencers is virtually the same as a single sequencer with a very large number of tracks each of which can run for a much longer sequencing run than an individual sequencer.

Abstract: An improved electrophoresis and fluorescence detection apparatus has an electromagnetic radiation sensor juxtaposed with a sensing region. The output signal from the electromagnetic radiation sensor is a current signal, and the current signal is converted to a voltage signal. The voltage signal is summed with a programmable offset generated by an inexpensive eight-bit D/A converter. The offset signal is selected to establish a lower starting point for the dynamic range of the A/D conversion, and is selected to null some or all of the background electromagnetic radiation level. The summed signal is amplified and integrated in an integrator. The integrator is switchable under program control. The integrator is switched on for long and short intervals. The short intervals permit sensing over a dynamic range accommodating very high levels of fluorescence; very high peaks may be measured and features of the peaks distinguished.

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

Abstract: An electrophoresis microgel is formed in a gel holder. The gel holder comprises a top substrate, a bottom substrate and a spacer disposed between the top substrate and the bottom substrate. The spacer establishes a separation of from 25 to 250 microns between the top substrate and the bottom substrate. A gel compartment is formed by partially sealing the top substrate to the bottom substrate, while leaving an opening for the introduction of unpolymerized gel. The gel compartment is then filled with an unpolymerized gel, which is polymerized in the gel compartment. Electrodes may be printed on the substrates, may be contacts to an exposed edge of gel, or may be applied through windows cut into one of the substrates. One type of gel holder makes use of graded beads having a diameter of 25 to 250 microns slurried in an adhesive such as an acrylate adhesive as the spacer. The slurry is printed onto the surface of one or both substrates to form a spacer of the desired shape, and then hardened using heat or light.

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.

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 quantitatively amplifying exons of the sample RB1 gene using primers complementary to intron regions flanking each exon; and then 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 of 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: A hierarchy of at least two assay techniques is utilized in testing for disease-associated mutations. The first assay in the hierarchy is selected to provide a highly specific test for the existence of the disease-associated mutation, although the accuracy of the test need not be high. The final assay in the hierarchy is selected to provide a highly accurate and highly specific test for the existence of the disease associated mutation. Intermediate tests of progressively greater accuracy may also be included in the hierarchy. Once the hierarchy has been selected for a given mutation-associated disease, a patient sample is analyzed the patient sample using the first, lowest accuracy assay in the hierarchy. If the result of the first assay is negative for the presence of a disease-associated mutation, then the next assay in the hierarchy is performed.

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 through 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.