cycle sequencing (internal labelling)

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Thu Jun 15 09:40:09 EST 1995


Joe Cook asks:

> Does anyone know of a good cycle sequencing kit or method that incorporates
> radioactively labelled nucleotides rather than end labelling of the primer?

I think the secret is to add the label (eg. S35 dATP) on top of the cold
dATP, rather than instead of it.  You sacrifice a lot of intensity because
of the loss of specific activity; but you avoid the massive pausing
artefacts that otherwise plague this method.

Hope this helps.
Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio




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