PubMed · 13834512
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D STETTEN. 1960. Biosynthesis and pyrophosphate.. https://doi.org/10.1016/0002-9343(60)90195-9
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Genetics research has benefited tremendously from the release of the human genome sequence. Subsequent technology has been developed and adapted to accommodate the need for faster, easier throughput of genetic assays. Pyrosequencing is a unique system that allows the analysis of genetic variations including single-nucleotide polymorphisms, indels and short repeats, as well as assessing RNA allelic imbalance, DNA methylation status, and gene copy number. Advances in methodology, including multiplex and universal primer applications, have reduced assay cost and improved throughput. This chapter briefly reviews some of the many applications for Pyrosequencing technology.
A modified back-etch method is described that has been successfully used to prepare samples of thin films and nanoparticles on Si wafer substrates for examination by high-resolution transmission electron microscopy (HRTEM). This process includes ultrasonic cutting, abrasive pre-thinning and a two-stage etching procedure. Unlike previous reports of back-etching methods, tetramethyl ammonium hydroxide, which has a very high-etching selectivity of Si to SiO(2), is used for the final etching to allow removal of the Si without degradation of the SiO(2) membrane. An innovative wrapping method is also described. This novel approach reduces the preparation time for HRTEM samples to <1 h per sample for groups of 10 or more samples. As an example, the preparation of FePt nanoparticle samples for HRTEM imaging is described.
Digital transcriptomics with pyrophosphatase based ultra-high throughput DNA sequencing of di-tags provides high sensitivity and cost-effective gene expression profiling. Sample preparation and handling are greatly simplified compared to Serial Analysis of Gene Expression (SAGE). We compare DeepSAGE and LongSAGE data and demonstrate greater power of detection and multiplexing of samples derived from potato. The transcript analysis revealed a great abundance of up-regulated potato transcripts associated with stress in dormant potatoes compared to harvest. Importantly, many transcripts were detected that cannot be matched to known genes, but is likely to be part of the abiotic stress-response in potato.