Massively parallel DNA sequencing methods are rapidly achieving broad adoption by the life sciences research community.
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Massively parallel DNA sequencing methods are rapidly achieving broad adoption by the life sciences research community. As the productivity of these platforms continues to grow with hardware and software optimizations, the bottleneck experienced by researchers is increasingly at the front end (the construction of sequencing libraries) rather than in the sequencing itself. The conventional approaches include fragmentation of DNA (mechanical or enzymatic), end-polishing, ligation of adaptor sequences, gel-based size-selection, and PCR amplification. However, there are several limitations in using this conventional approach to generating sequencing libraries, including high requirements for labor, time and cost, as well as the low efficiency of mass conversion into sequencing-compatible material. Thus, improved automation and lowered costs in sequence library generation are needed to harness the full potential of current sequencing technology.
A great advancement in library preparation is the introduction of a hyperactive variant of the Tn5 transposase which catalyzes the fragmentation of target DNA and insertion of adaptor sequences in a 5-minute, small-volume reaction. Transposase (Tnp) Tn5 is a member of the RNase superfamily of proteins which can catalyze the movement of the transposon. Tn5-mediated transposition works through a “cut-and-paste” mechanism, where the Tn5 excises itself from the donor DNA and inserts into a target sequence, creating a 9-bp duplication of the target. Thus, Tn5 can be used in genome sequencing for fragmentation of the DNA, in the technique called ATAC-seq.
Methods for constructing in vitro fragment libraries. (a) Conventional protocol which includes mechanical or endonuclease fragmentation, end-polishing, A-tailing, adaptor ligation and PCR. (b) Transposase-mediated library construction. Adaptor insertion, fragmentation and adaptor insertion occur in a single 5-min in vitro step, followed by PCR.
Creative Biogene, as a leading biotechnology company, provides robust Tn5 transposase to accelerate your research, especially tagmentation-based sequencing library construction.
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