The way the Illumina next gen sequencing works is different from Sanger sequencing. In Illumina sequencing, DNA is amplified on the surface of a glass slide using primers attached to the surface. The DNA is dilute enough that each PCR is seeded by a single strand of DNA, and they are far enough apart that a spot of PCRed DNA is separated from neighboring DNA fragments.
Sequencing is done by adding fluorescently labeled nucleotides with a reversible terminator. Only one base gets added to each piece of DNA per step. A picture is taken of the surface, and each DNA fragment spot is one of four colors. Then the termintor is removed and the cycle gets repeated. The sequence of colors at a particular spot indicates the sequence of that fragment.
Jim Lund
Separate molecules in the same lane, or ligated end-on-end? Wouldn't--
parallel reactions lead to multiple peaks simultaneously, leading to
difficulty establishing which molecule has which nucleotide?*
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