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Frequently Asked Questions About Our Products And Services

What is the principal of the Illumina sequencing technology?

Both next generation sequencing technologies of Roche (454) and Illumina share a common approach, in, that before sequencing itself, the sequencing library has to be generated via PCR amplification of the DNA templates.

In the case of Illumina HiSeq 2000 or MiSeq, preparation of the sequencing library is done by bridge PCR, while the sequencing is done by a technology referred to as cyclic reversible termination.

In the bridge PCR primers and the DNA template are immobilised to the 2-dimensional surface. The primers are designed to target the adaptors of the DNA fragments so that the fragments bind to primers in their neighbourhood. Within each PCR cycle the fragments build so called bridges and the following denaturation leaves single stranded templates anchored to the surface. The copies remain local and form dense clusters. To sequence the clusters a universal primer is hybridized to the adaptor sequence of the DNA fragments.

FAQ 9.1

Sequencing of clusters generated via bridge PCR is done by a technique called cyclic reversible termination. Thereafter, the polymerase extension is performed with reversible terminators. These are deoxynucleotides carrying a fluorophore and a blocking group. The 4 nucleotides have different fluorophores attached. The polymerase incorporates just 1 labelled nucleotide as the blocking group terminates DNA synthesis. Unincorporated nucleotides are washed away and the array is imaged to determine the identity of the incorporated nucleotide. This is followed by a cleavage step which removes the blocking group and the fluorophore. The resulting 4-colour images are used to decode the sequence.

FAQ 9.2

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