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Sanger sequencing

Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. Developed by Frederick Sanger and colleagues in 1977, it was the most used sequencing method for 40 years, it was first commercialized by Applied Biosystems in 1986. More higher volume Sanger sequencing has been replaced by "Next-Gen" sequencing methods for large-scale, automated genome analyses. However, the Sanger method remains in wide use, for smaller-scale projects, for validation of Next-Gen results, it still has the advantage over short-read sequencing technologies that it can produce DNA sequence reads of > 500 nucleotides. The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleotidetriphosphates, modified di-deoxynucleotidetriphosphates, the latter of which terminate DNA strand elongation; these chain-terminating nucleotides lack a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA when a modified ddNTP is incorporated.

The ddNTPs may be radioactively or fluorescently labelled for detection in automated sequencing machines. The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides, while the other added nucleotides are ordinary ones; the dideoxynucleotide concentration should be 100-fold higher than that of the corresponding deoxynucleotide to allow enough fragments to be produced while still transcribing the complete sequence. Putting it in a more sensible order, four separate reactions are needed in this process to test all four ddNTPs. Following rounds of template DNA extension from the bound primer, the resulting DNA fragments are heat denatured and separated by size using gel electrophoresis. In the original publication of 1977, the formation of base-paired loops of ssDNA was a cause of serious difficulty in resolving bands at some locations; this is performed using a denaturing polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes.

The DNA bands may be visualized by autoradiography or UV light and the DNA sequence can be directly read off the X-ray film or gel image. In the image on the right, X-ray film was exposed to the gel, the dark bands correspond to DNA fragments of different lengths. A dark band in a lane indicates a DNA fragment, the result of chain termination after incorporation of a dideoxynucleotide; the relative positions of the different bands among the four lanes, from bottom to top, are used to read the DNA sequence. Technical variations of chain-termination sequencing include tagging with nucleotides containing radioactive phosphorus for radiolabelling, or using a primer labeled at the 5' end with a fluorescent dye. Dye-primer sequencing facilitates reading in an optical system for faster and more economical analysis and automation; the development by Leroy Hood and coworkers of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing. Chain-termination methods have simplified DNA sequencing.

For example, chain-termination-based kits are commercially available that contain the reagents needed for sequencing, pre-aliquoted and ready to use. Limitations include non-specific binding of the primer to the DNA, affecting accurate read-out of the DNA sequence, DNA secondary structures affecting the fidelity of the sequence. Dye-terminator sequencing utilizes labelling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labelled-primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which emit light at different wavelengths. Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in automated sequencing, its limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromatogram after capillary electrophoresis.

This problem has been addressed with the use of modified DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs". The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, was used for the vast majority of sequencing projects until the introduction of Next Generation Sequencing. Automated DNA-sequencing instruments can sequence up to 384 DNA samples in a single batch. Batch runs may occur up to 24 times a day. DNA sequencers separate strands by size using capillary electrophoresis, they detect and record dye fluorescence, output data as fluorescent peak trace chromatograms. Sequencing reactions, cleanup and re-suspension of samples in a buffer solution are performed separately, before loading samples onto the sequencer. A number of commercial and non-commercial software packages can trim low-quality DNA traces automatically; these programs remove low-quality base peaks. The accuracy of such algorithms is inferior to visual examination by a human operator, but is adequate for automated

Flix (TV network)

Flix is an American premium cable and satellite television network, owned by Showtime Networks, a subsidiary of ViacomCBS through its domestic networks division. Its programming consists of theatrically released feature films released from the 1970s to the present day, interspersed with some films from the 1950s and 1960s, it is the only premium television service in the United States that does not operate any multiplex channels that provide additional programming alongside the main service. Although Flix is offered as part of the Showtime multiplex, the channel's carriage varies depending on both the cable provider and market, therefore it may not be available alongside Showtime and The Movie Channel's multiplex services in all areas. Although one or both of the channels have traditionally been carried alongside the Showtime multiplex on cable and satellite providers, as of 2018, Flix as well as The Movie Channel are not presently carried by any of the over-the-top subscription television servicesHulu Live TV, PlayStation Vue, Sling TV, YouTube TV and DirecTV Now – that carry most or all of the eight Showtime multiplex channels.

The network launched on August 1992, as a single-channel "mini-pay" service. Flix featured movies from the 1960s to the 1980s, although it would begin to scatter some 1990s film titles onto the network's schedule over time. At its launch, Flix had been one of the last premium channels to restrict the broadcast of R-rated films to the nighttime hours. A notable aspect of Flix during its early days was that the channel did not advertise the channel's own prime time lineup, but ran a schedule of programs that were slated to air that evening on the other major U. S. premium channels – including HBO, Cinemax and Encore as well as sister networks Showtime and The Movie Channel – during breaks between daytime movies. Three years prior to the channel's launch, in 1989, Tele-Communications Inc. made a failed bid to acquire a 50% ownership stake in Showtime from Viacom. There was some debate as to whether Viacom or TCI conceived the idea for Encore, another "mini-pay" service, similar in format to Flix, which focused on films from the 1960s to the 1980s until a format change in 1999 in which that channel added more recent films to its schedule.

Viacom executives insisted that TCI lifted part of the idea for Encore from the company's Showtime Networks division. John Sie, the president of Encore at the time, said in a 1991 interview with Multichannel News that TCI brought up the concept of the Encore network as a way to revitalize Showtime, either by launching a new tertiary service from scratch or by overhauling the format of Showtime's existing sister network The Movie Channel. On June 14, 2005, Viacom decided to separate itself into two companies, both of which would be controlled by Viacom parent National Amusements, amid stagnation of the company's stock price; the original Viacom was restructured as CBS Corporation and acquired Showtime Networks along with CBS' broadcasting assets, Paramount Television, advertising firm Viacom Outdoor, Simon & Schuster and Paramount Parks. In 2007, Flix began to broadcast certain R-rated movies during daytime timeslots; that same year, Flix began to air movies released in 2000, including such titles as Reindeer Games and Pitch Black.

On December 4, 2019, nearly 14 years after the companies split, Viacom and CBS Corporation remerged. Flix On Demand is the channel's subscription video-on-demand service. Launched in 2005, Flix On Demand offers classic movies released between the 1950s and the 1990s, which are divided by category based on the decade of their release: 1950s and 1960s, 1970s, 1980s and 1990s; as of September 2018, Flix – through Showtime – maintains exclusive first-run film licensing agreements with network sister company CBS Films, Amblin Partners, IFC Films, Global Road Entertainment, STX Entertainment. Flix shows sub-runs – runs of films that have received broadcast or syndicated television airings – of theatrical films from Walt Disney Studios Motion Pictures, Sony Pictures, Universal Studios, Samuel Goldwyn Films, Bleecker Street, Summit Entertainment, The Weinstein Company, Anchor Bay Entertainment, Metro-Goldwyn-Mayer, Paramount Pi

Burwick, Orkney

Burwick is a small village and harbour on the island of South Ronaldsay in the Orkney Islands, Scotland. It is the closest Orkney harbour to the Scottish mainland and is the terminus of a passenger ferry which operates in the summer to John o'Groats in Caithness; the name was first recorded in about 1225 as "Bardvik", derived from the Old Norse bar vík, meaning "bay of the extremity", for its position near the southernmost point of the island. The remains of the Castle of Burwick, a defended Iron Age fort and probable secondary monastic settlement, occupy the promontory west of the harbour. Ports and Harbours of the UK - Burwick, South Ronaldsay Canmore - Three Friends: Burwick, 19th century shipwreck. Canmore - Burwick Lifeboat Station