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[76] [77] [78] High-throughput sequencing technologies are intended to lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods. [79] In ultra-high-throughput sequencing as many as 500,000 sequencing-by-synthesis operations may be run in parallel.
Dye-primer sequencing facilitates reading in an optical system for faster and more economical analysis and automation. The later development by Leroy Hood and coworkers [7] [8] of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing. Sequence ladder by radioactive sequencing compared to ...
Nevertheless, the implementation of Sanger sequencing for decoding DNA-encoded chemical libraries in high-throughput fashion was the first to be described. [18] After selection and PCR amplification of the DNA-tags of the library compounds, concatamers containing multiple coding sequences were generated and ligated into a vector.
Structure of double-stranded DNA, the product of DNA synthesis, showing individual nucleotide units and bonds. DNA synthesis is the natural or artificial creation of deoxyribonucleic acid (DNA) molecules. DNA is a macromolecule made up of nucleotide units, which are linked by covalent bonds and hydrogen bonds, in a repeating structure.
First, DNA sequencing libraries are generated by clonal amplification by PCR in vitro. Second, the DNA is sequenced by synthesis, such that the DNA sequence is determined by the addition of nucleotides to the complementary strand rather than through chain-termination chemistry. Third, the spatially segregated, amplified DNA templates are ...
The ideal size of DNA fragments for the sequencing library depends on the sequencing platform that will be used. [4] [16] DNA can first be sheared to fragments around 300–500 bp long using sonication. [4] [16] [17] Fragments of this size are suitable for high-throughput sequencing.
The technology leading to these DNA sequencers was first released by Solexa in 2006 as the Genome Analyzer. [10] Illumina purchased Solexa in 2007. The Genome Analyzer uses a sequencing by synthesis method. The first model produced 1G per run. During the year 2009 the output was increased from 20G per run in August to 50G per run in December.
The cDNA molecules generated by RACE can be sequenced using high-throughput sequencing technologies (also called, RACE-seq). High-throughput sequencing characterization of RACE fragments is highly time-efficient, more sensitive, less costly and technically feasible compared to traditional characterization of RACE fragments with molecular cloning followed by Sanger sequencing of a few clones.
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