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Microfluidic Sanger sequencing is a lab-on-a-chip application for DNA sequencing, in which the Sanger sequencing steps (thermal cycling, sample purification, and capillary electrophoresis) are integrated on a wafer-scale chip using nanoliter-scale sample volumes. This technology generates long and accurate sequence reads, while obviating many ...
The Sanger method became popular due to its increased efficiency and low radioactivity. The first automated DNA sequencer was the AB370A, introduced in 1986 by Applied Biosystems. The AB370A was able to sequence 96 samples simultaneously, 500 kilobases per day, and reaching read lengths up to 600 bases.
In contrast to directed sequencing, shotgun sequencing of DNA is a more rapid sequencing strategy. [6] There is a technique from the "old time" of genome sequencing. The underlying method for sequencing is the Sanger chain termination method which can have read lengths between 100 and 1000 basepairs (depending on the instruments used).
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Dideoxynucleotides are useful in the sequencing of DNA in combination with electrophoresis.A DNA sample that undergoes PCR (polymerase chain reaction) in a mixture containing all four deoxynucleotides and one dideoxynucleotide will produce strands of length equal to the position of each base of the type that complements the type having a dideoxynucleotide present.
Most high-throughput, next generation sequencing platforms produce shorter read lengths compared to Sanger sequencing.These new platforms are able to generate large quantities of data in short periods of time, but until methods were developed for de novo assembly of large genomes from short read sequences, Sanger sequencing remained the standard method of creating a reference genome. [10]
The original CAGE method (Shiraki et al., 2003) [1] was using CAP Trapper [5] for capturing the 5′ ends, oligo-dT primers for synthesizing the cDNAs, the type IIs restriction enzyme MmeI for cleaving the tags, and the Sanger method for sequencing them.
Phred then uses these parameters to look up a corresponding quality score in huge lookup tables. These lookup tables were generated from sequence traces where the correct sequence was known, and are hard coded in Phred; different lookup tables are used for different sequencing chemistries and machines.