Pentamers

[mezzomix]

Hi,

I'm a newbie (student) and never sequenced DNA by myself so far, but I have a short question concerning SOLiD: This system uses octamers, and thus it needs 5 cycles (with 5 primers) to gain 2 colors for each nucleotide of the fragment that is being sequenced.

But what about using pentamers (i.e. XX-zzz) instead of octamers? With pentamers, only 2 cycles (with 2 primers: n as well as n-1) instead of 5 would be necessary.

Thank you very much!

[volks]

probably the ligation step wouldnt be as efficient/specific. also, you would need more consecutive ligation steps starting from the same primer, which is generally a problem. otherwise it would be easy to sequence more than 50/75 basepairs.

[mrawlins]

It would still require the same number of ligation steps, since each ligation would take the same amount of time. It's true that there would be fewer initial primers, resulting in fewer cycles, but each cycle would take longer and overall it would take roughly an equivalent amount of time.

The other factor is how efficiently the pentamers hybridize when compared to octamers. The octamers will tend to hybridize more uniformly since the distribution of octamer melting points is tighter than the distribution of pentamer melting points. There may be a loss of efficiency for certain sequences when using pentamers, which would systematically bias the results (although possibly not by much).

I would be pretty surprised if the instrument developers hadn't tested multiple lengths and determined the most efficient in terms of speed and accuracy.

[mezzomix]

Thank you for your replies.