DNA Sequencing (Sanger-Based and Non-Sanger-Based)
Despite the new next-generation sequencing technologies, the demand for traditional sequencing is growing. No single method, however, can meet the needs of all studies. Therefore, along with the Illumina HiSeq 2000, a next-generation sequencing platform, the NASR offers an additional sequencing platform.
Sanger-Based DNA Sequencing
Traditional Sanger-based sequencing is strong because of genetic and epigenetic mechanisms involved in cancer development that are extensively studied at OSUCCC.
Most applications include targeted re-sequencing of genetic alterations such as mutations, deletions, duplications, larger structural rearrangements of cancer genes, animal modeling in cancer and biomarker identification. A variety of samples are processed daily such as BACs, pure plasmids, mini-prep plasmids, M13 clones, cosmids, lambda clones, PCR products, gel-isolated fragments or bacterial genomes.
The NASR operates two 48-capillary Applied Biosystems 3730 DNA Analyzers: one for long-read and one for short-read applications and all the necessary accessory equipment for Sanger-based DNA sequencing.
A modified Sanger dideoxy chemistry, the ABI Prism BigDye Terminator Cycle Sequencing Kit version 3.1 is the method of choice. Both machines use data collection software v3.0 and sequencing analysis software v5.2. Turn-around time is the same day or next morning, depending on priority, and the NASR Web site provides a sequencing request form with all the necessary information that routinely results in the highest data quality.
One thousand nucleotides of sequence data can be routinely obtained from one long-read sequencing run in the form of a raw sequence data file and a chromatogram data file. Researchers initiate the process by submitting a specific amount of template combined with the primer along with a DNA Sequence Request Sheet denoting the type of reaction to be performed and primer and sample names as outlined on the NASR Web site.
Non-Sanger-Based DNA Sequencing
The new, next-generation non-Sanger-based sequencing technologies (such as Illumina’s HiSeq 2000, which offers sequencing by synthesis with reversible terminators) were introduced based on massively parallel sequencing of millions of nucleic acid fragments. The result: novel biological applications, a new level of productivity, cost-effectiveness and accuracy. more. . .