Материал из Zbio
Illumina relies on polymerization-based sequencing.
[править] Cycle sequencing
Sequencing reaction performed by repeating of identical cycles. During one cycle one nucleotide is determined for each cluster and sequencing primer became one nucleotide longer.
[править] Extension mixture
DNA polymerase and all four nucleotides are added to template. Each nucleotide has
-- distinguishable fluorophore (four nucleotides ⇒ four colours);
-- reversible terminator. The terminator prevents incorporation of several nucleotides one after another. Illumina does not provide information if fluorophore works as a terminator or they are two different chemical groups.
Non-incorporated fluorescent nucleotides washed out from the channel. Imaging detect incorporated nucleotides.
Fluorescent group and terminator are removed. It is possible to make a next cycle: sequencing primer became one nucleotide longer.
- 1st oligonucleotide: normal extension // no dephasing;
- 2nd oligonucleotide: nucleotide does not incorporated // negative dephasing on next cycle;
- 3rd oligonucleotide: incorporated nucleotide have no terminator // positive dephasing;
- 4th oligonucleotide: incorporated nucleotide have no fluorophore and terminator // positive dephasing;
- it is also possible, that terminator was not removed during cleavage, it will result in negative dephasing on a next cycle.