Visible Genetics Inc.

Other

Hsc Research and Development Limited

James M Dunn

John K Stevens

James Leushner

Jean-Michel Lacroix

May Hui

Marina T Larson

Eleftherios P Diamandis

Thomas D Yager

Brenda L Gallie

Robert Shipman

Denis Capatos

David E Matthews

Alexandre M Izmailov

Paul Waterhouse

John A Renfrew

Henryk Zaleski

Rodney D Gilchrist

William A Adams

James W Cassidy

Gregory Dee

Ronald J Green

Thomas J Digby

A 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 using a second nested set of sequencing primers ensures the maximum potential for sequencing a particular sample. To perform the method, reagents suitable for performing the tests are suitably packaged as a kit.

Method and kit for evaluation of HIV mutations

The invention relates to a device for managing battery packs by measuring and monitoring the operating capacity of individual battery modules in a battery pack. A programmable logic controller directs the selective closing of relays to allow individual battery modules to be load-tested using a variable discharge load unit, without compromising useful battery pack capacity. A battery module whose useful capacity falls below a predefined threshold may be connected to a battery charger for replenishment and then electrically realigned with the remaining modules in the pack for continued operation. Alternatively, an alarm may be triggered which alerts the user that the module is due for replacement. This sequence of events is performed on all cells in the pack at a predetermined interval.

Monitoring battery packs

Sequencing of a selected region of a target nucleic acid polymer in a natural abundance 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&trade; 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 reaction mixture also includes an unconventional nucleotide and an appropriate enzyme for degradation of nucleic acid polymers containing the unconventional nucleotide. The mixture is processed through multiple thermal cycles for annealing, extension and denaturation to produce a product mixture which is analyzed by electrophoresis.

Method for sequencing of nucleic acid polymers

Samples are tested for mutations in the BRCA1 gene using a hierarchical approach. First, each sample is amplified in one or more multiplex PCR amplification reactions. Each multiplex PCR reaction produces a mixture of amplified fragments. The sizes and amounts of these fragments are evaluated and compared to standard values reflecting the sizes and amounts of fragments produced when the same multiplex amplification is performed on the wild-type BRCA1 gene. Differences between the observed fragment sizes and/or amounts and those for the wild-type gene are indicative of a mutation with the BRCA1 gene of the sample. Next, one or more of the exons of the BRCA1 gene are sequenced, preferably only for those samples where no mutation was detected by analysis of the multiplex PCR fragments. The sequencing procedure can be performed by amplification and sequencing of the multiplex amplification mixture.

Method and reagents for testing for mutations in the BRCA1 gene

Nucleic acid polymers are sequenced by obtaining forward and reverse data sets for forward and reverse strands of a sample nucleic acid polymer. The apparent base sequences of these forward and reverse sets are determined and the apparent sequences are compared to identify any deviations from perfect complementarity. Any such deviation presents a choice between two bases, only one of which is correct. A confidence algorithm is applied to the peaks in the data sets associated with a deviation to arrive at a numerical confidence value for each of the two base choices. These confidence values are compared to each other and to a predetermined threshold, and the base represented by the peak with the better confidence value is assigned as the &ldquo;correct&rdquo; base, provided that its confidence value is better than the threshold. The confidence value takes into account at least one, and preferably more than one of several specific characteristics of the peaks in the data set that were not complementary.

Method for sequencing nucleic acids with reduced errors

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. This process is repeated until the final assay has been performed on all samples which gave negative results when tested by all less-accurate assays in the hierarchy. The test may be used for diagnosis and targeted screening for p53 mutations and mutations in the RB1 gene.

Method for testing for mutations in DNA from a patient sample

Direct determination of the sequence of an RNA sample is performed under isothermal conditions. An RNA sample containing a target nucleic acid is combined in a single reaction vessel with a reaction mixture containing two polynucleotide primers, a first primer that specifically hybridizes with a target sequence near the 3&prime; end of the target nucleic acid, and a second primer that specifically hybridizes to the 3&prime; end of an antisense copy of the target nucleic acid. At least one of the primers is labeled with a detectable label, and at least one of the first or second primer has an RNA polymerase transcription initiation signal at its 5&prime; end, which signal does not specifically hybridize to the RNA target. The reaction mixture also contains ribonucleotide triphosphates for RNA synthesis, deoxyribonucleotide triphosphates for DNA synthesis, at least one type of dideoxynucleotide triphosphate chain-terminator, and enzymes with the activity of reverse transcriptase, RNAse H, RNA Polymerase and a low discrimination DNA Polymerase such as Thermo Sequenase&trade;. The combined reactants are incubated under isothermal conditions for a length of time sufficient to generate chain-terminated reaction products, and the chain-termninated reaction products are then detected after electrophoretic separation.

Method and kit for direct isothermal sequencing of nucleic acids

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&trade; 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.

Method compositions and kit for detection

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. The method of the invention differs from the prior art, because the first and second oligonucleotide primers are each labeled with different, spectroscopically-distinguishable fluorescent labels. The method therefore obtains information about both DNA strands simultaneously while providing improved sensitivity as a result of the non-linear increase in the amount of DNA which results from the production of additional templates molecules from unterminated fragments.

Method for bi-directional sequencing of nucleic acid polymers

Method, reagents and kit for diagnosis and targeted screening for p53

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. Preferably, the amplification of the exons is multiplexed so that more than one exon is amplified in a single vessel using sets of primers which provide gene fragments of distinctive lengths when used to amplify a normal RB1 gene.

Method, reagents and kit for diagnosis and targeted screening for

Method for identification of mutations using ligation of multiple

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.

Compositions and methods for diagnosis of mutation in the von

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. For those for which an ambiguity remains, however, a final test in which the sequence of the sample is determined in both the 3' and 5-direction for all four bases is performed. To perform the method, reagents suitable for performing the three tests within the hierarchy are suitably packaged as a kit containing two or more sub-kits. The first sub-kit contains reagents for performing A and T sequencing. The additional sub-kits contains reagents for performing a four-base sequence determination on one or both strands of the target DNA.

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Scarborough, Canada

James M Dunn