Hi,

 at first please take care with ACRONYM. DDAO is :

N,N-Dimethyldodecylamine –N-oxide (DDAO)

Peer reviewers were sleeping . Why?

your cited dye must have another acronym.

when you write :

Looking for alternatives to ethidium and propidum monoazide).

please note that azide acts as reactive group  in a covalent manner.

Please draw the structure of your acridine with ACL labs free  and show the 2 and 3 D structure. 

It  seems that you and your supervisor are not familiar with Google Scholar (not Google) and internet searches.

Please reformulate your question after good searches about your problem e.g. question.

JRG

Hello Jean Rene Grezes,

Thank you for your input. I already searched on Google scholar (not google) but didn't find any other use of DDAO other than staining (see "A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms", Allesen-Holm et al., 2005). There they have tested DDAO as a stain to detect extracellular DNA due to its structural similarity to acridine orange. Using the same paper as a reference, they mentioned 7-Hydroxy-9H-(1,3-Dichloro-9,9-Dimethylacridin-2-One as the IUPAC name. Since I am not a chemist I wouldn't know why the acronym and the IUPAC name don't match. Further on the authors used DDAO due to its structural similarity to Acridine orange and better fluorescence detection capacity.  Also, I didn't consult my supervisor because I first wanted to look up literature and have second opinion from other people who might have some experience. Finally,the reason of questioning this was only to see if anyone knew if this dye binds covalently to DNA as literature about use in such applications (marine sediments, extracellular DNA) is scarce. Meanwhile I still continue to search through literature.

Regards,

Ajinkya

Also as far as I have read, Acridine orange intercalates within DNA and this happens over electrostatic interactions and not covalent binding (correct me if I am wrong). Since, I would be using harsh DNA extraction downstream processes I was just considering if this interaction will stay stable or not for qPCR applications.

Hello Ajinkya,

thak you for your fast answer. I explain you the differences between A.O.  (acridin orange)  and your dye  ( icall it   HDDAO)  HDDAO.

At first  A.O is a planar dye and , according to the pKA  e.g. pKb   and pH of the solution, it exists as BASE  or CATION.

HDDAO , according to the structure, can be protonated at  N-10 ( the pKa value must be deterninated in the future.

your sentence :

Further on the authors used DDAO due to its structural similarity to Acridine orange and better fluorescence detection capacity.

the authors of the paper wrote a non sens because THERE IS NO STRUCTURAL SIMILARITY between the 2 dyes.

Have you consulted the Molecular Probes  book on line?

Please draw the structure of AO  and HDDAO   with ACD labs free or another software at the uni Bremen

HDDAO is distorded  at C- 9  (the 2 methyl groups ) and interaction (steric hindrance between  Methyl and C-1 Cl.

Finally,the reason of questioning this was only to see if anyone knew if this dye binds covalently to DNA as literature about use in such applications (marine sediments, extracellular DNA) is scarce. ....

ttps://www.thermofisher.com/order/catalog/product/D6488

The dye cannot covalent bind at   C1  or 3-Cl (Exchange affords high temperature).

However (see Thermo Fisher /above link) the hydroxyl group can be covalent bound.

http://pubs.acs.org/doi/abs/10.1021/acschembio.6b00233?journalCode=acbcct

and

https://www.thermofisher.com/order/catalog/product/H6482

https://www.thermofisher.com/de/de/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html?ICID=svtool&UID=6482p9

Can you bind  your HDDAO in this manner for qPCR?

I send the link of a publication about HDDAO and derivatives:

http://www.google.dj/patents/EP0459536B1?cl=de&hl=fr

JRG

Hello Jean,

Thank you for your detailed description and outlook on the HDDAO compound.

According to the paper you provided I assume the modified HDDAO fluoresces when the esterases act on the modified dye. In our case I just want to treat the sediment with this dye such that the binding of this dye to extracellular DNA will prevent the polymerase from amplifying it during qPCR. It seems that one will have to try out a lot of things to assess this possibility (to process covalent binding of the dye to DNA) and with the time constraint I am facing I will have to revert to using the more expensive monoazides. But with some chemical assessment it would be possible to bind the dye to DNA (I just lack the knowledge for the same at this moment of time). Thanks for your useful insight into this matter.

PS: I could, for instance, treat a known concentration of DNA with HDDAO and see if I can amplify it in qPCR to see if the dye to DNA associations are stable or not and can withstand vortexing which is inherently done during qPCR.

Hi Ajinkya, we also wondered about the use of DDAO as an eDNA stain. We didn't find that it worked well, but we do recommend TOTO-1 as an accurate and sensitive flourescent stain for visualizing eDNA. Good luck with your research! 

http://www.sciencedirect.com/science/article/pii/S016770121400195X

Hi Mira,

Thanks for your suggestion. I will surely go through this and check it out.

Cheers.

Ajinkya

Dear Jean,

I might not get an answer since this post is already 2 years old, but if you happen to see this I was wondering if you could clarify your statement for me (also keep in mind I am a biologist, not a chemist):

he authors of the paper wrote a non sens because THERE IS NO STRUCTURAL SIMILARITY between the 2 dyes.

DDAO, or as you call it HDDAO has" Dimethylacridin" in its name. Why is there no structural similarity?

I assume that most people just look at the 2D structure and that gives the impression that there is some structural similarity.

Also I am not sure if Ajinkya is to blame for acronyms used commonly in the literature. I would have asked the same question. I know that nomenclature is important, but it is not up to somebody using the dye for experiments to rename what is commonly used in the literature.

Greetings,

Jens

Dear Jens,

Thank you for your message posted 2 days ago.

I send you my answer with Infos in due course in order to clarify my statement about DDAO or HDDAO

Greetings

JRG

Jean...thanks...looking forward to learn a bit more about this.

Dear Ajinkya, Dear Jens,

I insert the part of the paper (2006)( Allesen-Holm et al.) :

3dichloro-9,9-dimethylacridin-2-one)] (Figure 8G–I). As far as we know, DDAO has not previously been used as a DNA stain, but we tested this compound due to its excellent fluorescent properties and its structural resemblance with the DNA stain acridine orange...…….

My comment:

There is NO STRUCTURAL RESEMBLANCE between Acridin Orange Base or as salt (see paper 2002 for pKb value and compare with DDAO)

and DDAO Base or as deprotonated resonance structure

(see the NMR spectrum of acridine orange)

and the structure of DDAO (part of the thesis of Dr. Tianmeng Duo/ Read the pages 28,29,35 ..)

Both as pdf files.

According to the pH and structure of DDAO , the oxygen bears a negative charge and can bind only a part of EX-DNA which bears a positive charge.

Acridine orange bears as salt a positive charge and can bind DNA with a negative charge (see papers about DNA/RNA and AO salt).

JRG

NMR of AO base

pages 28,29,35

Hi Ajinkya,

did you manage to stain with HDDAO and extract DNA and perform qPCR?

Hi Aleksandra,

Due to the way it reacts I decided not to go for it. If anything I can recommend propidium monoazide or ethidium monoazide to use it for qPCE to omit quantification of extracellular DNA.

Article Minimal Influence of Extracellular DNA on Molecular Surveys ...

Thanks a lot!