Modification of the ³²P-postlabeling method to detect a single adduct species as a single spot.

ABSTRACT

The original (32)P-postlabeling method developed by Randerath and his colleagues has been modified to detect a single type of adduct as a single spot in thin-layer chromatography (TLC), because some types of adducts gave multiple adduct spots by the original method. In the remodified methods, DNA is first digested with micrococcal nuclease and phosphodiesterase II and then labeled with [γ-(32)P]ATP under standard or adduct intensification conditions. Since the labeled digest includes adducted mono-, di-, and/or oligo-deoxynucleotides, it is further treated with phosphatase and phosphodiesterase prior to TLC. The labeled digest is treated with nuclease P1 (NP1) in Method I, with T4 polynucleotide kinase and NP1 in Method II, and then with phosphodiesterase I in both cases and subjected to TLC. The advantage of these methods is that the number of adduct species formed can be estimated by TLC.