Suppose the following, I wish to ligate an ssRNA to the 3' end of a ssDNA. T4 RNA ligase should be able to do this. The ssDNA will not be phophorylated, so no issues there. The RNA will have a 5' phosphate. Now, my question is, is there a way to block the 3' OH to prevent self ligation or multiple ligations, ie, to make the desired hybrid the only possible ligation reaction? Importantly, this would need to be REVERSIBLE, ie, after the ligation I can remove the blocking group to yield a free 3' OH on the RNA. I know you can add dideoxy terminators and such, or chemically react something with the OH group, but once again I want to easily reverse this after the ligation to have a natural 3' OH group (and a specific one at that, so I don't want to add any modified nucleotides to the end). Any ideas? I haven't been able to find much online, but I may be searching with the wrong keywords.....warren...
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Michaelro
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Posted 11 February 2010 - 10:31 AM
Warren, on Feb 11 2010, 09:56 AM, said:
Suppose the following, I wish to ligate an ssRNA to the 3' end of a ssDNA. T4 RNA ligase should be able to do this. The ssDNA will not be phophorylated, so no issues there. The RNA will have a 5' phosphate. Now, my question is, is there a way to block the 3' OH to prevent self ligation or multiple ligations, ie, to make the desired hybrid the only possible ligation reaction? Importantly, this would need to be REVERSIBLE, ie, after the ligation I can remove the blocking group to yield a free 3' OH on the RNA. I know you can add dideoxy terminators and such, or chemically react something with the OH group, but once again I want to easily reverse this after the ligation to have a natural 3' OH group (and a specific one at that, so I don't want to add any modified nucleotides to the end). Any ideas? I haven't been able to find much online, but I may be searching with the wrong keywords.....warren...
Hi
I think You should consult the company/es that produce oligos like IDT.
They have different kinds of modifications for both 3' and 5' groups.
Modifications like amino blockers are widely used to prevent unwanted ligations but I'm not sure it is reversible.
Hope it is helpful.
Best
Michael
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Warren
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Posted 11 February 2010 - 10:54 AM
Michaelro, on Feb 11 2010, 01:31 PM, said:
Warren, on Feb 11 2010, 09:56 AM, said:
Suppose the following, I wish to ligate an ssRNA to the 3' end of a ssDNA. T4 RNA ligase should be able to do this. The ssDNA will not be phophorylated, so no issues there. The RNA will have a 5' phosphate. Now, my question is, is there a way to block the 3' OH to prevent self ligation or multiple ligations, ie, to make the desired hybrid the only possible ligation reaction? Importantly, this would need to be REVERSIBLE, ie, after the ligation I can remove the blocking group to yield a free 3' OH on the RNA. I know you can add dideoxy terminators and such, or chemically react something with the OH group, but once again I want to easily reverse this after the ligation to have a natural 3' OH group (and a specific one at that, so I don't want to add any modified nucleotides to the end). Any ideas? I haven't been able to find much online, but I may be searching with the wrong keywords.....warren...
Hi
I think You should consult the company/es that produce oligos like IDT.
They have different kinds of modifications for both 3' and 5' groups.
Modifications like amino blockers are widely used to prevent unwanted ligations but I'm not sure it is reversible.
Hope it is helpful.
Best
Michael
Thats not a bad idea.....
