Why dATP ?

Pete M. Muriana MURIANAP at FOODSCI.PURDUE.EDU
Fri Oct 21 09:07:44 EST 1994


In article <Cy094o.JFt at hkuxb.hku.hk
> h9390274 at hkuxa.hku.hk (MONSTER) writes:

> Can anyone tell me why we using 32P-ATP to do the 5' end labelling?

    The type of labelling that you want dictates what can be used to label.  
5'-end labelling is done enzymatically with Polynucleotide Kinase which 
transfers the gamma-P (outermost of the 3-P's) from ATP to the 5' terminus.  
Labelling at the 3' end is done with Terminal Deoxynucleotidal Transferase 
which transfers the alpha-P plus deoxynucleotide to the 3' terminus.  
Therefore, in 5'-end radio-labelling, the gamma phosphate is the isotopic 
phosphate that is transferred; in 3'-end radio-labelling, the isotopic 
phosphate is in the alpha position and it is transferred along with the 
nucleotide to the 3' terminus.  Moral of the story - you need to purchase the 
appropriate radioisotope for labelling.

>How about using CTP?  Is there any big difference between them?

   Any deoxynucleotide can be used for 3'-end labelling; I'm not sure about 
others for the 5'-end, but if you are only transferring a phosphate and not 
the nucleotide, you wouldn't need but one transfer group to do it with.

> I just wonder why those company provide different types of 32P 
>isotopes,

   As stated above.

can they replace each other in the experiment? Such as using CTP to 
>do the >labelling.

>Robert Y.S. Cheng

Regards, Peter



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