A non-radioactive DNA sequencing method using biotinylated dideoxynucleoside triphosphates and delta Tth DNA polymerase.
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.
Ikeda
K
K
Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., Fukui, Japan.
Inoue
H
H
Oka
M
M
Kawakami
B
B
Kawamura
Y
Y
D001710:Biotin; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000737:chemistry; D008163:Luminescent Measurements; D015394:Molecular Structure; D017422:Sequence Analysis, DNA / Q000379:methods*; D016963:Thermus thermophilus / Q000201:enzymology
D003854:Deoxyribonucleotides; D001710:Biotin; D004259:DNA-Directed DNA Polymerase; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II