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

We synthesized a set of four biotinylated dideoxynucleosidetriphosphates (biotin-9-ddNTPs) and optimized the reaction conditionsfor non-radioactive cycle sequencing using modified Tth DNApolymerase (Tth) and a chemiluminescent detection system. Theresulting sequencing ladders showed lower background comparedto those with the conventional non-radioactive sequencing methodwhich uses 5'-biotinylated primers, especially when PCR productswere analysed. With our method, DNA sequences can be determinedat any primer positions without preparing 5'-biotinylated primersfor dideoxy chaintermination.