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How to incorporate dUTP when using WGA2 amplification for Affymetrix’s array?


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#1 labtek

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Posted 23 November 2009 - 08:36 AM

I want to use WGA2 amplification kit for Affy’s array for CHIP-on-chip. Affy’s protocol requires incorporate dUTP during the amplification, which is essential for Affy’s fragmentation step. However, the WGA2 kit does not have dUTP. Does anyone know if dUTP can be added to WGA amplification? If yes, how much dUTP should be added in WGA amplification? (Ratio of dUTP to dTTP on Affy’s protocol is 2mM vs. 8mM). If not, then how to do fragmentation for affy’s array after WGA amplification?

Thank you very much for the help and suggestion.

#2 Clare

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Posted 26 November 2009 - 02:11 AM

Hello :(

If you're doing Chip-chip you shouldn't have to do the fragmentation step of the WGA as your DNA is already be sonicated.

We use the Sigma WGA kit and just go straight to adding the library prep buffer etc after chip.

Clare

I want to use WGA2 amplification kit for Affy’s array for CHIP-on-chip. Affy’s protocol requires incorporate dUTP during the amplification, which is essential for Affy’s fragmentation step. However, the WGA2 kit does not have dUTP. Does anyone know if dUTP can be added to WGA amplification? If yes, how much dUTP should be added in WGA amplification? (Ratio of dUTP to dTTP on Affy’s protocol is 2mM vs. 8mM). If not, then how to do fragmentation for affy’s array after WGA amplification?

Thank you very much for the help and suggestion.



#3 labtek

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Posted 27 November 2009 - 12:06 PM

thanks for reply.

I plan to use Affy’s tiling array but use WGA2 to amplify CHIP DNA.
Affy’s CHIP-on-chip protocol requires fragmentation of amplified DNA (not sonicated DNA). I believe that the sizes of fragmented DNA are smaller than 500-1000 bp (which are the size of sonicated DNA). dUTP must be incorporated during amplification so that amplified DNA can be treated with UDG/APE to fragment, followed by terminal labelling of fragmented DNA (biotin label). It is why I want to know how to incorporate dUTP if WGA2 kit is used for amplification. The ratio of dTTP to dUTP will affect the size of fragments. Does anyone have a experience about this?

Thanks.




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