We are trying to circularize a 35 nt DNA oligo using CircLigase (epibio). The control oligo works fine although only about 50% of the oligo has reacted after 60 min. However, our oligo doesn't circularize at all. After 60 min all of the oligo has been adenylated (gel electrophoresis) but none of the DNA has circularized. Does anyone know if CircLigase has any sequence or structure specificity? Also, I am puzzled by the fact that the enzyme is able to adenylate the oligo but not complete the reaction so I would appreciate any comments about the mechanism of the enzyme that could explain this.
This may be of some help in case you haven't already seen it.
'Application of Circular Ligase to Provide Template for Rolling Circle Amplification of Low Amounts of Fragmented DNA'
This is way out of my comfort zone, but I would expect persistence length to change with sequence due to different local stacking propensities, etc. If 35 nt is close to the limit, this might imply that the fact that your control oligo circularizes does not mean that your sample oligo will.
Why don't you try adding some Mg++ (or any other divalent cation) to the reaction, to shield electrostatic repulsions among the backbone phosphates and try to coerce the oligo into bending a bit more?
there is sequence specificity for CircLigase:
5´-end: G > A >>> T >>> C
3´-end: T > A > G (3´-ends containing a Cytosine evidently did not produce any ligation products.)
Note: ">" means greater than and ">>>" means much greater than.
ref:
http://www.epibio.com/tech-support/faqs/faq-circligase-and-circligase-ii-ssdna-ligases
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