cDNA micro-array
if i have PCR product how can I separate and isolate one strand from dsDNA?
A wide range of physicochemical conditions promote DNA denaturation, including temperature, pH and osmolarity. My understanding is that you are enquiring about the coupling reaction of a micro-array hybridization procedure. I found a protocol involving cDNA incubation with 0.5 M sodium bicarbonate at pH 9. Follow the link for the complete protocol.
http://ipmb.sinica.edu.tw/microarray/protocol.htm#Preparation_of_Labeled_cDNA
Hi Mohamad,
While I am sure Fabio's method would work well, I just had one alternative suggestion to offer (depending on what your goal with the DNA is). You could always get your primer synthesis company to 5'-end tag one of your PCR primers - the one that amplifies the strand you want - with a modification to make your strands asymmetric. This could be either a bulky blocking agent (like a m7G cap, or a triphosphate or something) or a purifiable molecule, e.g. biotin.
Doing this would allow you to differentially treat your strands, either with e.g. a 5'-3' nuclease which cannot digest the end with the bulky blocking agent (and thus would only digest the strand you don't want), or to denature then purify your tagged strain.
Just a thought - I hope it helps!
Dan
Daniel dehany Scott thanks for answering me but can you explain your technique iam don't understand your suggestion.
Hi Mohamed,
Sorry, my answer wasn't very clear.
Basically, every PCR product is amplified with two primers, each one forming the 5' end of one of the two strands that are amplified. Let's call the two strands "Watson" and "Crick", and say that you're interested in the "Crick" strand. You can synthesise your primers such that the one that primes the "Watson" strand has a normal (usually monophosphate, I think) 5' end, but the one that amplifies the "Crick" strand has a bulky adduct on the 5' end - say, a biotin. When you perform your PCR, you will now have a normal dsDNA PCR product in which the 5' end of your strand of interest - the "Crick" strand - has a biotin molecule at its 5' end.
If you were to treat this dsDNA with a dsDNA exonuclease that degrades in a 5'-->3' direction - for example, Lambda exonuclease, or T7 Exonuclease, both of which are commercially available - you would degrade the accessible "Watson" strand, but not the Biotin-blocked "Crick" strand, meaning that the "Crick" strand would remain as ssDNA. The other advantage of using a biotin addition is that biotin can be bound at very high affinity by streptavidin, meaning that you may be able to use this interaction to purify the ssDNA "Crick" strand - if you can find conditions under which the DNA is denatured but the biotin-streptavidin interaction is not.
There are of course some optimisation/clean-up steps required in both of these protocols, but hopefully they give you some ideas about how you may approach things.
Let me know if you have any other questions!
Cheers,
Dan
Dear Daniel Dehany thank so much
Your answer is very clear and perfect and I have been understood it obviously
thanks again for answering me ☺☺
No worries at all - I hope it's useful! Let us know how you get on :)
Cheers,
Dan
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