isolating a full DNA sequence without a genomic library - I've got a partial sequence but what now? (Sep/21/2005 )

Hi all,

Its been awhile since I last posted here
I was having some trouble doing a genomic DNA PCR awhile back but I've managed to get it to work now
Anyway I've got a partial sequence that I can turn into a radioactive probe, but I am not able to construct a genomic DNA library ... for various reasons hehe

Does anyone know a sure-fire method to obtain a full sequence from genomic DNA?

I've thought about digesting gDNA with MCR BC restriction enzyme (cuts all methylated DNA), then run a southern blot and probe with my partial to get an estimation of where my target sequence would be on the gel. After gel extraction and topo cloning, I would then either probe clonies or do colony PCRs until I see something I like. Would a method like this work? is this normally why southern blots are done?

I really don't want to try and construct a gDNA library again, I've never been able to get the partial digestion bit right, so this will be my prefered method of isolating the full sequence unless there is something better that I am unaware of.

Thank you,

Phil

you could try a PCR based method described in this article:

Siebert, P.D., Chenchik, A., Kellogg, D.E., Lukyanov, K.A., and Lukyanov, S.A. (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 23: 1087-1088.

Its always worked well for me. Hope it helps.