Devon Ryan k. I am just curious: how many templates are lost by this? Similar Posts. Loading Similar Posts. Content Search Users Tags Badges. Help About FAQ. Powered by the version 2. If you have any other comments or suggestions, please let us know at comment yourgenome. Can you spare minutes to tell us what you think of this website? Open survey. In: Facts Methods and Technology.
How does Illumina DNA sequencing work? The first step in this sequencing technique is to break up the DNA into more manageable fragments of around to base pairs. The DNA fragments attached to adaptors are then made single stranded. This is done by incubating the fragments with sodium hydroxide.
Once prepared, the DNA fragments are washed across the flowcell. When sequenced, each cluster of DNA molecules will emit a signal that is strong enough to be detected by a camera.
Unlabelled nucleotide bases and DNA polymerase are then added to lengthen and join the strands of DNA attached to the flowcell.
Primers and fluorescently -labelled terminators terminators are a version of nucleotide base — A, C, G or T - that stop DNA synthesis are added to the flowcell.
The primer attaches to the DNA being sequenced. The DNA polymerase then binds to the primer and adds the first fluorescently-labelled terminator to the new DNA strand.
Once a base has been added no more bases can be added to the strand of DNA until the terminator base is cut from the DNA. Lasers are passed over the flowcell to activate the fluorescent label on the nucleotide base. In contrast to the and ABI methods which use a bead-based emulsion PCR to generate "polonies", Illumina utilizes a unique "bridged" amplification reaction that occurs on the surface of the flow cell.
The flow cell surface is coated with single stranded oligonucleotides that correspond to the sequences of the adapters ligated during the sample preparation stage. Single-stranded, adapter-ligated fragments are bound to the surface of the flow cell exposed to reagents for polyermase-based extension. Repeated denaturation and extension results in localized amplification of single molecules in millions of unique locations across the flow cell surface.
This process occurs in what is referred to as Illumina's "cluster station", an automated flow cell processor. PCR is typically performed with two different oligos, often referred to as forward and reverse primers, that match some target template sequence. Normally these oligos are free in solution in a tube, and with every PCR cycle, they find a match in a template sequence and are extended to create a new template strand.
However, there's no reason why they have to be in solution. You could instead take a mixture of the two oligos and spread them out over a glass surface, allowing them to become covalently attached to the surface so they never come off the surface.
Then, when you want to do PCR, take your template molecules e. Wherever a template molecule lands, it is within reach of the forward and reverse primers bound to the surface. This is what is done for Illumina sequencing. The library is diluted such that molecules spread out across the surface at some optimum density - giving each one enough space to "build it's own house" as it were.
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