It is easy to produce linearized ends with sticky ends using restriction enzymes and these sticky ends are readily ligated by DNA ligase enzyme alone without the need for DNA end-processing.
How do I make such linearized DNA behave like the DNA breaks created by ionizing radiation where DNA breaks need end-processing before DNA ligation can proceed.
In simple words, how do I create a linearized DNA with sticky ends resembling radio damaged 'non-ligatable' ends.
Not interested in 'dephosphorylation'.
Can someone direct me to one such method, if it exists, please.
You could use a phosphatase enzyme (available from a number of companies) to remove the 5' phosphates from your DNA ends. The sticky ends would still be able to hydrogen bond between base pairs but the backbone will no longer be able to be ligated.
Hi Raj,
What's your downstream experiment? Is keeping the ends sticky essential?
Bill
Thanks for your answers. I am aware of enzymatic dephosphorylation but not interested in that.
The question was more directed to those working on chemical modification of DNA to mimic certain chemistry observed during radiolysis of DNA.
The interest is to generate DNA substrate with known sequence and precise ends that would 'mimic' ionizing radiation induced DNA breaks.
Hi,
following article mentions synthetic oligonucleotide models for DSB ends in the introduction and give references (no 5 and 35):
Nucleic Acids Res. 2012 Nov;40(21):10821-31. doi: 10.1093/nar/gks879. Epub 2012 Sep 24.
The dynamics of Ku70/80 and DNA-PKcs at DSBs induced by ionizing radiation is dependent on the complexity of damage.
Reynolds P1, Anderson JA, Harper JV, Hill MA, Botchway SW, Parker AW, O'Neill P.
Maybe this is what you are looking for.
Best wishes,
GAD
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