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-   -   Chemical shearing of DNA specifically (not RNA) (http://seqanswers.com/forums/showthread.php?t=79134)

JamieWizard 11-13-2017 06:12 AM

Chemical shearing of DNA specifically (not RNA)
 
Hi,

I just wondered, are there any sequencing protocol kits around for shearing DNA by chemical means? I know that heating RNA in presence of divalent cations can be used to fragment RNA (i.e. Illumina TruSeq protocol kits). Do similar kits exist/or are methods applied for fragmenting DNA prior to sequencing?

Cheers,
Jamie

torben 11-13-2017 11:30 AM

I don't know about any kits, but DNA can be fragmented with reduced CuSO4
https://beng.soe.ucsc.edu/sites/defa...esis_Final.pdf

JamieWizard 11-13-2017 11:37 AM

Cheers for that Torben. I saw that article, but thought it looks like a masters and because it's not referenced elsewhere might therefore not be a standard method that is used in labs that do sequencing.

jwfoley 11-13-2017 04:17 PM

If enzymes count as chemicals, NEB has its "Fragmentase" mix, which appears to be a combination of a dsDNA nicking enzyme that's had its sequence specificity engineered out of it and a second enzyme that turns nicks into blunt ends.

JamieWizard 11-14-2017 05:07 AM

Thanks JW. The reason I am asking is I'm interested in the biases introduced by different fragmentation methods. Enzymatic methods, such as restriction endonucleases are a different class.

Essentially what I'm trying to establish, is that are metal cations also used to fragment DNA (I know they are used for RNA). I know the phosphate backbone of DNA/RNA carries a significant negative charge which facilitates this method. I believe RNA is slightly less stable than DNA, and I wondered if the method is also used for fragmenting DNA, but I can't seem to find reference to it online or in sequencing protocols.

Cheers


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