Hello,
I am trying to do a DNA annealing as shown in the figure. As can be seen, this is a three part annealing that involves three different oligos.
1)While there are softwares to check secondary structures of a single DNA oligo, are there softwares that will give me the most likely annealed structures to form when there is more than a single oligo? A software that takes into account the various melting temperatures involved so as to give the various possible annealed structures?
2) I am doing the annealing by adding the three components in equal amounts in a tube, heating it to 95 deg C, and gradually cooling to room temperature over 3 hours. However, my absorbance spectra indicates that the "tail" sequence (shown in blue) hasn't ligated, but the two arms have. Any reason why that could be the case?
Thank you in advance,
Regards,
Ranjit
Hi, Ranjit.
It's a little bit difficult to give you the best answer without all details like sequences and how you're performing it (dry bath, water bath, PCR machine. etc).
Anyway, my thought is you could (If you're not doing it this way) perform it on a thermal cycler and calculate what are the temps more close to the ligations you're intending to occur and after heating it up to 95ºC, do steps for the temps needed for the pther ligations in order. Of course, it will only help you If you can do it from the higher temp to the lower one.
One second thing is: The tails (in blue) are longer or shorter than the other areas to be ligated? So, They need higher or lower energy to keep up ligated? I am supposing less energy (shorter). If so, They may have difficult times to keep connected after all pair-eise reactions have occurred. Ok?
Hope it helps.
Let's see what other scientists may tell you here.
HI Adriano,
I appreciate your detailed reply. You're right, I should have given the sequences and lengths, that would make it easier for people to answer.
The exact sequences are long, but here are the other details that you will find useful: the two arms are 50% AT rich, and 30 bp each in length. Some of the nucleotides in the arms are LNAs (locked nucleic acids), which is why the melting temperature is as high as 80 deg C. The tail is 18 bp in length, 75% AT rich, with a melting temperature of around 50 deg C. All my experiments are done in a buffer ranging from 50 to 150 mM NaCl.
I am performing the ligation exactly how you said: putting it in a thermal cycler. I raise the temp to 95, stay at that temp for 5 min, and reduce by 5 deg after an interval of 15 min (ie 15 min at 90 deg, 15 min at 85 deg and so on, until I reach 25 deg).
As you say at the end, it's likely that the formation of the 18 bp tail is not energetically favourable, since the DNA has to bend quite a bit for that to happen. Instead of forming a circle, I have now designed the longer sequence such that it forms a hairpin. That may make it easier. I hope that helps.
Regards,
Ranjit
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