Adapter Ligation Question

[johnmillsbro]

Hello. Looking for some help. We are using solexa to sequence a large pool of PCR products (Vent). Vent doesn't not produce overhangs. Therefore to ligate solexa adapters we will have to either enzymatically add the A overhangs or we would use adapters designed to be blunted directly to our PCR product. My question is: Are the A overhangs essential for efficient adapter ligation? Why do they use the overhangs in the first place?


Thanks many-

John

[GW_OK]

Using A tails prevents (somewhat) the formation of adapter dimers and library concatemers. Illumina protocol, though, has a specific A-tailing step after the post shearing end repair so it ought to be a moot point. Are you using some other protocol?

[johnmillsbro]

I don't have their full protocol (yet). I just knew you need an A overhang. I am curious why they add the overhang. I suppose it must be to prevent adapter:adapter ligations.

thanks

[Bruce E]

The A overhang is also needed so the adapter ligates in the correct orientation. They refer to these as adapters but it is actually one Y adapter so you need it to ligate in the correct orientation and the 3' A does this. If you were using blunt end ligation with blunt adapters as 454 uses you can get many fragments that will have adapters ligated incorrectly.