PYROSEQUENCING EXPLAINED

Описание: 454 sequencing or pyrosequencing was the first next-generation DNA sequencer on the market. It can sequence up to one billion bases in a single day, the equivalent of 1/3 of the total human genome. It works by first breaking up a sample of double stranded DNA into 400-600 bp fragments at specific points using restriction enzymes. Adaptors or small pieces of DNA tape are attached to the DNA fragments. Then tiny resin beads that contain DNA complementary to the adaptors are added. The existing double-stranded DNA fragments then start to attach to the resin beads with the help of these adaptors and when this happens, the double-stranded DNA is broken up into single-stranded DNA. This is followed by a filtration where any unbound beads or beads that contain more than one type of DNA are removed. The remaining beads are emulsified, or inserted in water-in-oil micro reactors together with PCR reagents. These complexes are then put into wells, one per well. Then waves of only one of the four nucleotide bases are added and removed to the wells in turn. Whenever a base is synthesized to the existing DNA chain it emits a light and its intensity corresponds to the amount of bases added. This makes it possible to sequence the sample of DNA since both the current type of nucleotide base added as well as the light’s intensity is known.