Technology
A technique designed to rapidly enrich and amplify targets derived from samples that yield low levels of nucleic acids, such as formalin-fixed paraffin-embedded (FFPE) tissues, for use in next-generation sequencing. Briefly, total nucleic acid or genomic DNA (gDNA) is isolated from fresh or FFPE tissues. Total nucleic acid is converted to double-stranded cDNA (dscDNA) and cleaned by solid-phase reversible immobilization (SPRI). Then the dscDNA or gDNA is subjected to end repair, adenylation and ligation with a universal half-functional adapter. The modified DNA sample is then subjected to two rounds of nested low-cycle PCR, where the first round uses target gene-specific primers and the second round uses nested target gene-specific primers that are 5' tagged with a common sequencing adapter. The resulting target amplicon library is cleaned using SPRI and is suitable for use in next generation genomic assays.
Diagnostic Methods
Technology
A molecular genetic technique that isolates and sequences chromosomal regions that are rich in open chromatin. First, nuclei are harvested from a cellular sample. Then a hyperactive Tn5 transposase is added to the nuclei where it excises non-nucleosomal DNA strands and ligates co-administered high-throughput sequencing adapters (tagmentation). The tagged DNA fragments are isolated, amplified by PCR and sequenced. The number of reads for specific region of DNA correlate with increased chromatin accessibility and this method can identify regions of transcription factor and nucleosome binding.
Diagnostic Methods
Technology
A DNA sequencing technique that can differentiate cytosine from 5-methylcytosine in a DNA sample. First, a denatured DNA sample is treated with bisulfite which converts non-methylated cytosine to uracil. Next, the sample is amplified using a PCR method that does not discriminate between non-methylated and methylated sequences. The amplified DNA is subjected to nucleotide sequencing. The resulting sequence is compared to an identical control sample of DNA that was not treated with bisulfite. Unmethylated cytosines will be displayed as cytosines in the control sample and as thymines in the bisulfite-treated sample.
Diagnostic Methods
Technology
A molecular genetic technique that combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to map the binding sites of DNA-associated proteins in a sample of cells. First, crosslinked protein-DNA complexes are isolated using ChIP. Next, the crosslinks are broken, the proteins are removed and the purified DNA is modified with adaptor oligonucleotides to facilitate massively parallel DNA sequencing. Following sequencing, the DNA sequences that are obtained can be mapped to their genomic locations.
Diagnostic Methods
Technology
A DNA sequencing technique in which a mixture of deoxynucleosidetriphosphates (dNTPs) and chain-terminating dNTPs, which are radioactively or fluorescently labeled, are combined within the reaction mixture. Once the reaction is complete, the DNA strands are separated by size, and the labeled chain terminating dNTPs can be read in sequence by the investigator or by a machine.
Diagnostic Methods
Technology
The determination of the sequence of purines and pyrimidines in deoxyribonucleic acid (DNA).
Diagnostic Methods
Technology
A proprietary next-generation DNA sequencing technology from Oxford Nanopore Technologies that can directly identify and sequence a DNA molecule as it passes through a nanopore, driven by electrophoresis.
Diagnostic Methods
Technology
Technologies that facilitate the rapid determination of the nucleotide sequence of large numbers of strands or segments of DNA or RNA.
Diagnostic Methods
Technology
A technique that determines the nucleotide sequence of a pre-specified region of DNA or RNA by using primers that are specific for that region.
Diagnostic Methods
Technology
A DNA sequencing strategy where a single read is initiated from each end of a DNA fragment.
Diagnostic Methods
Technology
The process of determining the sequence of amino acids within a protein molecule.
Diagnostic Methods
Technology
A DNA sequencing method that uses a "sequencing by synthesis" strategy. The strand complementary to a template is synthesized a single base at a time through the sequential addition of individual solutions of each base. Incorporation of a base into the growing strand generates free pyrophosphate that is detected as light, as generated by a luciferase-catalyzed reaction.
Diagnostic Methods
