Dear Anna

I can think of 2 things that might help you:

1. before commencing digestion try introducing a denaturation step to dissociate your heteroduplex, e.g 90C-94C for 2-5min

2. Having done that snap cool your sample on ice then dose with 1ul of Ambion Turbo DNAse 1 (AM2238); incubate for 30min; add another 1ul aliquot of DNAse I then incubate for a further 30min followed by non heat inactivation of the enzyme as recommnended in the protocol

Turbo DNAse I from ambion as detailed in the link  and similar to the recommendation above is a recombinant for of DNAse I not purified, unlike most other preparations. This means it is not contaminated with residual RNase and consequently aggressive (1 hour) incubations will not result in concomitant breakdown of your RNA in addition to digestion of your genomic DNA. The Turbo version of this ambion enzyme is an engineered version of rDNAse designed to work under difficult (high salt) conditions and is also more active. Thus, it is most likely of all preps to completely degrade your genomic DNA, irrespective of whether there is high salt or if the RNA is tied up in a heteroduplex

Hope this helps

https://www.lifetechnologies.com/order/catalog/product/AM2238

Nice question. I would bet that an excess of DNaseI should do the job. If not and if your DNA is cDNA, I suggest substituting dTTP with dUTP during the cDNA synthesis, then treat RNA-DNA hybrids with UDG (uracil-DNA glycosylase), alone or combined with endonuclease IV. This should shred your DNA strand. I admit, I never tried this, but it should work. Both UDG and endo IV are common and cheap reagents, sold by many companies (NEB, Sigma, etc.)

Dear Anna,

DNase I can under the right conditions digest the DNA from DNA:RNA hybrids. However, the activity towards the DNA-strand in a DNA-RNA hybrid is about 20 %of the activity towards dsDNA. The RNA-strand in a hybrid is not cleaved.

It is important to note that,  DNase I is often to be used at concentrations much higher than may be necessary , specially in this case. For example, you can try  2 U of Ambion DNase I (Cat. No. AM2222) for 15-min incubation. 

I hope to work with you. 

Dear Anna

I can think of 2 things that might help you:

1. before commencing digestion try introducing a denaturation step to dissociate your heteroduplex, e.g 90C-94C for 2-5min

2. Having done that snap cool your sample on ice then dose with 1ul of Ambion Turbo DNAse 1 (AM2238); incubate for 30min; add another 1ul aliquot of DNAse I then incubate for a further 30min followed by non heat inactivation of the enzyme as recommnended in the protocol

Turbo DNAse I from ambion as detailed in the link  and similar to the recommendation above is a recombinant for of DNAse I not purified, unlike most other preparations. This means it is not contaminated with residual RNase and consequently aggressive (1 hour) incubations will not result in concomitant breakdown of your RNA in addition to digestion of your genomic DNA. The Turbo version of this ambion enzyme is an engineered version of rDNAse designed to work under difficult (high salt) conditions and is also more active. Thus, it is most likely of all preps to completely degrade your genomic DNA, irrespective of whether there is high salt or if the RNA is tied up in a heteroduplex

Hope this helps

https://www.lifetechnologies.com/order/catalog/product/AM2238

Andriy, Mohamed and Laurence,

Many many thanks!

Hi Anna,

I'm having a similar problem. Have you been able to digest DNA in a DNA:RNA hybrid?

Hi Marco

• If you heat denature (90C for 5 min) or treat your DNA with 1M NaOH for 5 min at  room temp (then neautralise with 10mM Tris pH 8.0) this dissociates the hybrid heteroduplex allowing digestion of  resulting single stranded DNA with DNAse 
• Alternatively you could try the above denatitation treatment and then digest for 20min at 37C with exonuclease III

Thank you Laurence,

I'll try but I'm not optimistic. Let me explain my problem better..

I'm trying to pulldown an RNA using tiling biotinylated oligonucleotides. These multiple oligos anneal to my RNA forming an RNA:DNA hybrid (the entire lenght of the RNA is covered with oligos) that interfere with the retrotranscription (and thus PCR amplification) of my RNA of interest. 

Thanks 

Marco

Si, sono disperato :)

Marco, in your case you can probably just do a column-based RNA extraction with a high Mw cutoff (e.g. RNeasy, >200nt I think)! DNA definitely denatures in Qiazol/Tri-reagent/Trizol, so there is no reason why a DNA-RNA hybrid wouldn't.

Hi all, have you tried to use Duplex-specific nuclease?

I don't have experience with it, but I was looking for something to digest DNA in a DNA:RNA hybrid and I found that. Let me know if it works. 

http://evrogen.com/products/DSN/DSN_Description.shtml