1.If I have all the four species/samples with me i.e. dsDNA, ssDNA, dsRNA and ssRNA, how can I identify and distinguish one from the other? (using spectrophotometer and running them on gel)
2. If I am loading equal amount of each species, what will be the banding pattern on a gel?
1. I dont think you can distinguish them in the spectrophotometer. They should absorb the same wavelength, but the result would be a different yield for dsDNA or ssDNA. To get the right concentration make sure you tell the Photometer (NanoDrop?) what you are loading or use the right conversion factor (in case you calculate by hand).
1. and 2. For any suggestion about gel pattern, please give some more information on the kind of DNA/RNA. Are those in vitro-products or extracted from an organism (then how did you get dsRNA)?
Hi Neelofar
In the lines of your query....In my opinon..theoretically it may be possible to identify the ssDNA, dsDNA, ssRNA and dsRNA......but it may not be practically feasible to use it on a routine basis......For example
1) The wavelength (λ) max.of the four types of molecules..may show up as slightly different...as there may be fine differences between this value if you scan the samples....because their composition (base ) are little bit different
2) The ssRNA and ssDNA will show hyperchromic shift different then the dsDNA/RNA molecules and again in each subcategory you will find fine difference between them agin due to their differen base composition
3) On gel you can very well differentiate between the ssDNA and dsDNA as the later one moves marginally slower than the former......and furthermore between DNA and RNA you can differentiate using particular gel conditons... (denaturing/non-denaturing type).....In my opinion you can also differentiate between a ssRNA and dSRNA by making use of such conditions and .....the resolution of your gel will play an important role here too....
As I told previously...these are ....possible....(in my opinion) ...you can try also in the lab...but routine or easy fesilbility....might be difficult...
bye
Hi Neelofar
A more practical solution for this problem will be colorimetric assay of RNA and DNA using spectrophtometric analysis:
1) both RNA and DNA have different colorimetric estimation methods....and
2) for example for DNA DPA method is used and in my opinion if you take same number of molecules (not amount)...and do a DPA analysis ....the ssDNA should give OD roughly half of the dsDNA or vice versa
and same will apply for ssRNA and dsRNA....
bye
Hi Neelofar, I agree with Ajay Saini's explanation. He has given a very nice practical answer. In my little experience,i amplified a ss DNA library by assymetric PCR and i could able to differentiate between ss DNA and ds DNA using agarose /Native PAGE. The ss DNA migrates bit faster,hence retard at lower position than ds DNA. Besides, you can also hyperchromic shift difference using spectrophotometer. All d Best..
If I run dsDNA, ssDNA, dsRNA and ssRNA simultaneously on a native/ non- denaturing gel, wat will b d sequence in ur opinion
Hi Neelofar, well i did not have practical experience,of running ss/ds RNA on the mentioned Gel. But,i think same apllies with the RNA as i mentioned for ss DNA or ds DNA. Well the pattern,would ofcourse depend on the size of your DNA/RNA samples, and probably you may get ds DNA/RNA at upper end and ss DNA/RNA would be at below position. Just give a try.. Cheers..
DPA method requires a deoxyribose sugar and therefore is specific to DNA as far as I know. Yes it will b gud to use dis method to distinguish DNA from RNA
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