A Non-radioactive DNA Sequencing Method Using Biotinylated

We synthesized a set of four biotinylated dideoxynucleoside triphosphates (biotin-9-ddNTPs) and optimized the reaction conditions for non-radioactive cycle sequencing using modified Tth DNA poly-merase (ATth) and a chemiluminescent detection system. The resulting sequencing ladders showed lower background compared to those with the conventional non-radioactive sequencing method which uses 5'-biotinylated primers, especially when PCR products were analysed. With our method, DNA sequences can be determined at any primer positions without preparing 5'-biotinylated primers for dideoxy chain-termination. Key words: non-radioactive DNA sequencing; cycle DNA sequencing; thermostable DNA polymerase; biotinylated dideoxynucleoside triphosphate 1