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
As stated above.
can they replace each other in the experiment? Such as using CTP to
>do the >labelling.
>Robert Y.S. Cheng
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