Does anyone have experimental experience to block repetitive regions using probe or other synthetic sequences? I am conducting one experiment, where I need to mitigate the off-target effects. I am considering to block these off-target enriched repeat regions using synthetic probes. If anyone would have good suggestion, I would be appreciated.
Unfortunately, the statement of your question leaves considerable uncertainty regarding just what kind of experiment you are performing. You need to supply some more specific descriptions.
I am not sure how you could block off-target regions without also blocking your on target site. If I had that problem, I would probably run a blat (not blast) search with the on-target site sequence and see how many similar sequences there are genome-wide, and then try to design a probe that is as specific as possible to the on-target site. Knowing how many off-target sites there are and what do they look like, would maybe help designing off-target blockers (in addition to a better on-site binding probe). But David is certainly correct.
I agree with David that you should elaborate a bit more on what you want to do. In human or mouse Fluorescent Microscopy experiments it's common to use COT DNA to considerably reduce signal from repetitive sequences: http://www.lifetechnologies.com/order/catalog/product/15279011
There are many classes of repeats of varying length and sequence complexity. Which do you refer to?
Hi Yanfeng, the question you are asking is how does one potentially block repeats outside of a target region while potentially retaining repeats within a target. Just like Albino said, I think the easiest way is to empirically determine the repeat classes and specific locations that are being pulled down outside of the target with your specific probe set. These would first tell you if there are just a few sequences that are piling up on just a few sets of locations, or if it is generally all over the place. I typically find thousands of sequences all mostly piled up on 5-10 locations usually containing repeats. So the first step is to block off targets with PCR products amplified around these off target sites that Cot-1 is not effectively blocking. This will give you more room to go after what you are missing in the target. Second is to determine what repeats are missing within the target because Cot-1 is extremely effective (these are probably repeats with high similarity and probably larger size). Maybe a simple answer is to characterize these and adjust your "pond" fragment size accordingly along with using pre-blocked probe and immunoprecipitation with anti-biotin (streptavidin-bead capture is really bad beyond 1kb).
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