I have a dsDNA fragment that is ~ 65 bp in length (see attached figure). I wish to digest 2 nucleotides (nt) of ssDNA right in the center of this dsDNA fragment (indicated by brown arrows in the figure). Is there any restriction/nicking enzyme that will do this for me?
An important point that needs mentioning is that there will be a protein attached on either end of the dsDNA, so annealing is out of the question, since that will denature the protein. I need to be able to get this configuration at lower temperatures, where protein stability is not affected (not more than 45 deg).
'Annealing is out of the question'. I don't understand, didn't you say 'I plan to obtain this by annealing the two complementary strands'?
You need to provide more detail on what the experiment is exactly if you want to get meaningful answers. One possibility would be to design one of the strands to have a bulge and then digest with a ssDNA-specific nuclease. Another would be to anneal not two complementary strands but three, two of them leaving the desired gap. Another -perhaps- could be to use a Cas9 nickase and two guide RNAs targetting the same strand with the desired spacing. Another would be to have 2 dsDNA fragments, one of them with an 5'P overhang that hybridizes to a complementary 5'OH overhang on the other side but leaves a gap, and sealing the nick with T4 ligase.
Hope it helps!
Hi Alejandro,
Apologies for the confusion. I have changed the wording, and I hope it's clearer now.
My initial plan, like you suggested, was to anneal three complementary strands with a gap. However, with proteins attached to the two ends of my final DNA, this is not a possibility - the annealing temperature is very high, and proteins would denature at that temperature. However, your other suggestions certainly seem helpful. I would need to go over some of them carefully to understand how to go about it. I shall revert to you if I have any further queries.
Thank you so much for taking out time for a detailed response,
Regards,
Ranjit
Ranjit Gulvady I'm not sure why you say the annealing temperature is too high. DNA strands will anneal at much lower temperatures as well, usually an annealing temperature is listed as the highest temperature that permits annealing before you begin to melt the strands. You can readily create what you want using 3 oligonucleotides and annealing at room temperature or 37oC.
Michael J. Benedik : Thank you for your suggestion. Yes, I did read that annealing can also be done at a lower temperature. However, do you think it would affect the specificty of the annealing? Would the quality of annealing worsen, the further away I get from the melting temperature?
As things stand currently, the 2 shorter oligos have a high annealing temperature of 80 deg. For more efficient annealing at room temp or 37 deg, would I be better off designing the 2 shorter oligos to have a lower melting temp (as opposed to 80 deg) ?
Ranjit Gulvady: assuming there are no repeated elements in your DNA sequence, then I would not worry about specificity. Your sample is not very complex (3 oligos).
Having a high enough annealing temperature to ensure specificity is more important when you are trying to anneal oligos to their target site on a complex template such as genomic DNA. There you might have many competing partial sites. But for the experiment you are proposing, I don't believe you will have any problems annealing at a lower temperature like 37 deg C.
Michael J. Benedik Ah. This I did not know. As you mention, there are no repeat sequences in my oligo. Alright then, let me do what you suggest. That would actually solve a lot of issues I would have if I annealed at high temperatures.
Thank you!
Michael J. Benedik , I have asked another question (separately). I am unable to tag you there. If you can, could you have a look at it and help me out?
Thank you,
Ranjit
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