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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 polymerase (delta Tth) 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.

A Non-radioactive DNA Sequencing Method Using Biotinylated Dideoxynucleoside Triphosphates and Tth DNA Polymerase