genome amplification before cloning? - (May/02/2008 )
I'm trying to make a genomic library to analyze the diversity of a rare gene in some environmental DNA. I would like to isolate 5kb+ of the flanking sequences for a more reliable phylogeny study. I have been trying to make fosmids but have lots of problems screening (expecting perhaps 1 positive in 100,000).
I'm trying to come up with a way to enrich my gene before cloning. Has anyone tried Phi29 amplification before cloning? I am thinking about doing a Phi29 amplification primed with degenerate primers for my gene, and then try to make a mid-size clone
library. I understand whole genome amplification with Phi29 using random hexamers create lots of branch structures that aren't great for cloning, but with more specific primers, will I get around that problem?
Does anyone have experience with Phi29 and cloning? Or other ideas of enriching DNA before cloning? (PCR won't work as I want to clone the flanking unknown sequences).
You'll only get linear amplification of your genes with phi29, since the rolling circle replication or random priming won't occur. What about a degenerate primer tagged with biotin. You could do a one-sided PCR reaction (no second primer), which would amplify your fragments. Capture of the biotin on streptavidin beads would give significant enrichment of your desired sequences. You'd probably need to work with sheared genomic DNA, since the biotin tagged strand would be bound to the (uncut and unamplified) other strand.
Thanks for the reply! Biotin capturing is a good idea. What is a way to cleave off the label before cloning? May be I can add a short tail to the primer and then do an Exo digestion to get rid of the end label?
For one way PCR, I keep thinking Phi29 would still be a good option because of the possesivity and accuracy. Or am I better off with something like PFU or Vent polymerase?