Illumina DNA Sequencing

[Herbz]

Hi guys, i'm an undergraduate biochem student and I can't get me head round some of the illumina system.

Can anybody please put it to me in laymans language, things like:

1. Are the adapters attached on the DNA fragments used as primers??
2. Are the adapters unique for each and every library fragment??
3. What influences the formation of the bridge during the polymerization??
4. How do the reversible terminators work in this respect??
5. What is the capacity of one flow cell??

[james hadfield]

First off there is lots of info on Illumina's website and in recent publications, reading is thebest start you can give yourself.

1. Are the adapters attached on the DNA fragments used as primers??
The adapter sequencers are used in PCR amplification as the site of PCR priming.
2. Are the adapters unique for each and every library fragment??
No, the adapter sequences are the same in every molecule in a library. They are different for different applications; e.g. DNA or smallRNA. The adapters are two oligos of different sequence except for around 20bp a the 3' end of the top strand; this complementray sequence allows the two oligos to be annealed to produce a Y-shape molecule that gets two different sequences on each end of the insert DNA. Paper and pencil required to figure this out!
3. What influences the formation of the bridge during the polymerization?? Length, smaller molecules amplify more efficiently than longer ones. Greater than 600-800bp does not work well.
4. How do the reversible terminators work in this respect??
Each'cycle' of sequencing by synthesis includes; incorportion of a fluorescent blocked dNTP, imaging to detect which dNTP has been added to each DNA molecule in a cluster, cleavage of the dNTP to remove the fluorescent group and de-blocking the nucleotide so the next one can be incorporated.
5. What is the capacity of one flow cell?? Right now 1x45bp read on a good flow cell will yield amost 6GBp of data.

Please make it clear if I have not explained something correctly!