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Ligase always needed for DNA circularisation ?

DNA Techniques

Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.



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Old 09-14-2007, 11:22 PM
Pipette Filler
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Question Ligase always needed for DNA circularisation ?

Hi,

I'm wondering if a ligase (such as T4 DNA ligase) is always needed for linear DNA circularisation. I'm not an expert in biology even if I know some general things about PCR, enzymes, DNA, etc...

Let me explain myself : I want to make a circular DNA probe construction from 2 different DNA fragments...without using any ligase (in one step).

In this experiment, the hybridization of a specially designed linear single-stranded DNA probe with a target DNA brings the two ends of the probe close to each other. In general people use a ligation step of these two ends by a DNA ligase that forms a circularized probe that subsequently can be used with various proteins or enzymes etc...

I'd like to avoid the ligation step, considering that the recognition should be efficient enough (not "blunt ends" see my fig.).
1-But if I don't use any ligase protein, will my circularised probe be stable (experiments are made @room temp),
2-and moreover, wouldn't it disturb the proteins that would interact with that probe (in particular in the "gap" region ?)
3-would this result in a decreased specificity of the recognition ?

Figure available by email (I'm not allowed to post URLs to other sites cuz I have notmade 15 posts or more).
Thanks to everyone who will be interested by this problem and will answer me.


Surt
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