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PCR not working for 5 Aza treated DNA


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#1 Vaidhi0

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Posted 16 January 2013 - 10:44 AM

I treated my cells with 5 Azacytidine, then isolated the DNA and converted it using sodium bisulfite. I have BSP specific primers that i know works because I have used it to amplify regions on bisulfite converted DNA from non 5 Aza treated cells. I have tried designing different BSP primers, used various PCS conditions but nothing is working. I know 5 Aza inhibits DNMT's but are there any other side effects of 5 Aza that affects PCR amplification? If so, any suggestions on how to solve it?

#2 Trof

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Posted 17 January 2013 - 09:29 PM

AZA causes global demethylation, so all of the cytosines will be converted into uracil. If the sequence was pretty methylated before, now it has much lower GC content that the untreated DNA. So this may be one of the problems, what PCR conditions were you using?
We amplified AZA treated DNAs, but I can't tell you details now, because my colleague did the reactions.

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#3 Trof

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Posted 17 January 2013 - 09:43 PM

So, she told me she needed to use a nested approach, and with that it wasn't eve needed to decrease the elongation temperature to 68 deg, as would be required otherwise. The properties of azacytitidine incorporation causes the DNA to be "stif" and amplifies badly.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#4 vilperte

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Posted 08 February 2013 - 03:57 AM

Try to decrease the extension temperature for 65C and increase the time for 2 minutes. It worked with my samples!
With that, there was no need for nested PCR.




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