TdT vs. Kinase

[Constantin Polychronakos]

>   bickle at ubaclu.unibas.ch (Tom Bickle) writes:
>  In article <40q8oa$cka at synapse.bms.com>, Steven Goldberg
>  <goldberg at bms.com> wrote:
>  
>  > Until recently our lab has been using T4 kinase + gamma 32P-ATP to end- 
>  > label our oligonucleotides.  However, we tried terminal deoxynucleotide 
>  > transferase and found our probes were labeled to a much higher specific 
>  > activity... are there 
>  > any known disadvantages to using probes prepared using TdT vs.kinase?
>  > 
>  > Thanks for your responses
>  > 
>  > Steve
>  
>  The main reason your probes are hotter is that you are putting poly (dA)
>  tails on your oligos- multiple hot phosphates. This might cause
>  hybridization problems in some cases....

>  Tom Bickle, Department of Microbiology, Biozentrum, Basel University
>  Klingelbergstrasse 70, CH-4056 Basel, Switzerland.


Given the extremely high specific activity of labeled dATP (you are not adding any 
cold dATP, I hope), I doubt that more than one or  two  As are added to the probe 
sequence. In my lab, we have labeled molecular size standards (very hot!)  with TdT, 
and found them to run virtually indistiguishable from unlabeled ones in 
sequencing-sized gels!
 
However, if  you are using the hybridization probes to distinguish subtle difference in 
sequence (such as allele-specific Southern or dot-blot hybridization of PCR 
fragments) even that could interfere with your method. For most other applications, 
it should be O.K.

In my mind, the main disadvantage of TdT labeled probes is that you cannot do 
primer extention with them (this is why you cannot use them as, say, sequencing 
primers.



Constantin Polychronakos MD
Department of Pediatrics
Division of Endocrinology
McGill University
Montreal, Canada