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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.
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.