I would like to link opposite 3' and 5' termini of a blunt-ended (optionally sticky-ended) DNA duplex without having to ligate stem-loop oligonucleotides to each end. This should result in two stem-loop structures at each end of the duplex producing a single covalently closed circular DNA strand.
I've been searching high and low to see whether this is possible. So far I've only found that these stem-loop structures are formed during V(D)J recombination by RAG complexes, but I don't think this is usable for in vitro applications.
Thanks
Yes, it is doable given the length of the DNA. For short DNA strand around 15-20 bp may be difficult but for longer one it definitely works. Attach an azide residue at one end and alkyne residue at the other end. By click ligation you can get the closed structures.
Hope this will help.
Dear Suresh, thanks for the advice. However, I reckon if say you'd want to use this for rolling circle amplification, one would still have to add stem loop oligos?
Is this the same as inverse pcr where you circularise the ends using ligase enzyme ( adjusting the concentration of fragments to favour self ligation rather than fragment to fragment ligation so very dilute) then only the circles with the oligo sequences will amplify with normal PCR and presumably with rolling circle amplification?
Its comparable to circulising ssDNA. However, I was hoping an enzymatic method may exist where the dsDNA ends are ligated while the oligomer is still in its dsDNA conformation. The only method I know so far for doing this is ligating hairpin loops to both ends.
I've drawn up an (extremely crude) diagram to illustrate
I may be missing something here but dna ligase will ligate dsdna both with sticky ends or blunt ends ( which takes more ligase) and then it is a matter of distinguishing linear concatemers and circularised dna
yes just without all the clever bits that allow cloning and replication etc
Ah ok. I'm looking more for circularising the ssDNA strands of the duplex, with the ends of the sense strand ligated to the respective ends of the antisense strand. This will result in a circular ssDNA oligo with both sense and antisense strands incorporated into the sequence, while your method will preserve the sequence, but in circular format. It's a small difference, but unfortunately important for my application.
Ok I misunderstood. There is an enzyme called circ ligase ( Cambio /epicentre tech /and other companies ) may be of interest then but from your previous answer you already know about this and it does not solve your problem so hopefully another researcher will have some useful ideas.all the best
Paul
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