32P & 35S end-labelling
of Nucleoside Triphosphates
Moving outward from the 5'-C,
the
PO4 groups in a dNTP are designated alpha, beta, and gamma. In the DNA
polymerization reaction, the
beta- & gamma PO4 groups are removed, leaving the alpha PO4 to be incorporated into the
sugar-phosphate backbone. The sequencing primer is typically end-labelled
with 32P in tha alpha position; an alternative
method is a thio-susbstitution
of the O in the alpha PO4 with 35S. The sulfur label is less
radiologically hazardous than phosphorous and produces sharper bands in
the autoradiograph; autoradiographic exposure can take longer.
All text material © 2010 by Steven M. Carr