Abstract: 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.

Abstract: An electrophoresis cassette is made from two substrates separated by spacers, and a one-piece molded plastic edge adapter. The edge adapter provides the contoured edge to facilitate sample loading, and because it is made from molded plastic is easier to make. Furthermore, the edge adapter has vertically extending arms which define the sides of the loading region and which, when glued to the back substrate, prevent leakage of buffer solution from the region surrounding the electrophoresis origin. In one variation, a groove in the bottom edge of the edge adapter receives the top edge of the front substrate to further define the position of the edge adapter in relation to the substrate. Vertical divider fins may optionally be formed on the contoured surface of the edge adapter. These divider fins define loading wells for the introduction of sample into the lanes of the electrophoresis gel.

Abstract: Data traces from four channels of an automated electrophoresis detection apparatus are aligned by identifying peaks in each of the four data traces; optionally normalizing the data traces to achieve a uniform peak height; combining the four data traces in an initial alignment; and determining coefficients of shift and stretch for selected data points within each 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 data trace. These warp functions are applied to their respective data traces to produce four warped data traces which are aligned to form an aligned data set. The aligned data set may be displayed on a video screen of a sequencing apparatus, or may be used as the data set for a base-calling process.

Abstract: The present invention is directed to a method of determining the genotype of a human papilomavirus in a sample by amplifying a portion of the L1 open reading frame of human papilomavirus genome with the amplification primer having SEQ ID NO:2, the sequencing primer having SEQ ID NO:3 and an additional sequencing primer specific to HPV51 having SEQ ID NO:5.

Abstract: An instrument for sequencing oligonucleotides is loaded with the products of four sequencing reaction mixtures. These products are a combination of A, C, G and T reaction products for several sequencing reactions. The products of the different sequencing reactions are labeled with fluorescent tags which are distinguishable one from the other on the basis of their excitation or emission spectra. After separation of the oligonucleotides by electrophoresis, the order of the detected peaks is used to call the base sequence.

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: An instrument for sequencing oligonucleotides is loaded with the products of four sequencing reaction mixtures. These products are a combination of A, C, G and T reaction products for several sequencing reactions. The products of the different sequencing reactions are labeled with fluorescent tags which are distinguishable one from the other on the basis of their excitation or emission spectra. After separation of the oligonucleotides by electrophoresis, the order of the detected peaks is used to call the base sequence.

Abstract: 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™ 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.

Abstract: 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 “correct” 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.

Abstract: 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.

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: 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: 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™ 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.

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: 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.

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: This 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. 3).

Abstract: 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′ end of the target nucleic acid, and a second primer that specifically hybridizes to the 3′ 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′ end, which signal does not specifically hybridize to the RNA target.

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™ 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: 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.