Figure 5.

Analyses of α-³²P labeled triphosphates by polymerase incorporation and TLC.

A. Primer extension with unpurified reaction mixtures that contain α-³²P labeled dX³TP or dP³TP. Templates contain two K¹s or five Z²s and reactions were carried out with DNA polymerases Klenow fragment at 37 °C or KODexo- at 72 °C for 5min. Samples were resolved on 18% urea denature PAGE.

B. TLC analysis of HPLC purified α-³²P labeled AEGIS triphosphates. 10 nmol of unlabeled authentic dZ²TP, dK²TP, dP³TP and dX³TP alone, or mixed with 50-80 pmol of HPLC purified α-³²P-labeled (~1000 CPM) triphosphates was spotted on PEI cellulose plates and developed with 0.85M KH₂PO₄ pH 3.5 as the mobile phase. Both fluorescent images under UV exposure and phosphor images were recorded.

C. Primer extension assay with HPLC purified α-³²P labeled AEGIS triphosphates. Primer extension assay was carried out with Klenow large fragment at 37°C for 90s and samples were resolved on 18% PAGE with 7 M urea after mixed with 1 vol of 90% formamide and 20 mM EDTA. In left panel, lane 3, 4, 7, 8, 13 and 15 are products from 5’ ³²P -labeled primer and unlabeled AEGIS triphosphates, served as controls; lane 5, 6, 9,10, 14 and 16 are products from unlabeled primer and α-³²P labeled AEGIS triphosphates (marked with asterisk). P: 5’ ³²P -labeled primer only.