A non-radioactive DNA sequencing method using biotinylated dideoxynucleoside triphosphates and ΔTth DNA polymerase

Abstract

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 (ΔTth) 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.