Site-directed mutagenesis studies of Tn5 transposase residues involved in synaptic complex formation

Author Posting. © American Society for Microbiology, 2007. This article is posted here by permission of American Society for Microbiology for personal use, not for redistribution. The definitive version was published in Journal of Bacteriology 189 (2007): 7436-7441, doi:10.1128/JB.00524-07.Transposition (the movement of discrete segments of DNA, resulting in rearrangement of genomic DNA) initiates when transposase forms a dimeric DNA-protein synaptic complex with transposon DNA end sequences. The synaptic complex is a prerequisite for catalytic reactions that occur during the transposition process. The transposase-DNA interactions involved in the synaptic complex have been of great interest. Here we undertook a study to verify the protein-DNA interactions that lead to synapsis in the Tn5 system. Specifically, we studied (i) Arg342, Glu344, and Asn348 and (ii) Ser438, Lys439, and Ser445, which, based on the previously published cocrystal structure of Tn5 transposase bound to a precleaved transposon end sequence, make cis and trans contacts with transposon end sequence DNA, respectively. By using genetic and biochemical assays, we showed that in all cases except one, each of these residues plays an important role in synaptic complex formation, as predicted by the cocrystal structure.This work was supported by NIH grant GM 50692

Methods for reduced cost and lower sample prep volumes for genetic analysis applications

As the cost of NGS has decreased, the library preparation cost has become a larger portion of the total expenditure. This is especially true for high-throughput applications, such as single-cell analysis. Therefore, there is a need to develop methods that can not only study the transcriptomes of single cells, but can also feasibly analyze large numbers of single cells. Miniaturizing the sample preparation volume provides the opportunity for significant cost savings. Using TTP Labtech’s mosquito liquid handlers, reagent and sample quantities can be scaled down to picogram values