Epicentre Technologies Corp.

A review on the use of near-IR dyes for fluorescence detection in automated DNA sequencing systems.These dyes offer several advantages over that of visible-wavelength dyes.The biol. and chem. of the sequencing process are the same as those of any visible detection systems.Near-IR detection provides researchers with attractive alternatives to the use of visible-wavelength fluorescent dyes.

Strain engineering of bacteria has been accomplished by many methods where mobile DNA elements (transposons) are inserted into the genomic DNA of a host organism. This chapter addresses engineering with transposable elements complexed with transposase enzyme. In traditional techniques, transposon and transposase are introduced as distinct entities. The method of mobilization into cells is often unique for each class of DNA element, and for each organism. The discovery of pre-formed transposon/transposase complexes (transposomes) that can be electroporated into living cells opens a new gateway to strain mutagenesis. Described are the preparation of electrocompetent bacterial cells and their transformation with transposomes. Once within the cell, the transposome is equipped to randomly insert its DNA into chromosomes without needing additional components. Ocr, a T7 phage protein that inhibits the host restriction of electroporated DNAs, will also be discussed as an adjunct reagent that can widen the applicability of transposomes. The transposomes used in most of the applications are commercially available, but also described is the process of making custom transposon DNAs and transposomes. The techniques are not limited to bacterial strain engineering per se and may be adapted for single-cell eukaryotes as well.

DNA library preparation is a common entry point and bottleneck for next-generation sequencing. Current methods generally consist of distinct steps that often involve significant sample loss and hands-on time: DNA fragmentation, end-polishing, and adaptor-ligation. In vitro transposition with Nextera™ Transposomes simultaneously fragments and covalently tags the target DNA, thereby combining these three distinct steps into a single reaction. Platform-specific sequencing adaptors can be added, and the sample can be enriched and bar-coded using limited-cycle PCR to prepare di-tagged DNA fragment libraries. Nextera technology offers a streamlined, efficient, and high-throughput method for generating bar-coded libraries compatible with multiple next-generation sequencing platforms.