New pyrosequencing method to analyze the function of the Klenow fragment (EXO-) for unnatural nucleic acids: pyrophosphorolysis and incorporation efficiency.
The incorporation of deoxynucleoside triphosphates (dNTPs) catalyzed by polymerases is conventionally examined using gel electrophoresis autoradiography. Here, we studied an alternative method, pyrosequencing, to verify the incorporation of dNTPs containing unnatural nucleotides by polymerases. We found that the pyrosequencing method more rapidly and easily confirmed the incorporation of dNTPs than the conventional method, especially in the presence of low-efficiency dNTP polymerases. Furthermore, the method can detect the pyrophosphorolysis reaction just before the position of the unnatural nucleic acid, and the efficiency of incorporation just after it.
Omics Science Center, RIKEN Yokohama Institute, Tsurumi-ku, Yokohama, Japan.
Takeshi
T
Hanami
Rieko
R
Oyama
Masayoshi
M
Itoh
Ayako
A
Yasunishi-Koyama
Yoshihide
Y
Hayashizaki
D004256:DNA Polymerase I / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism*; D009696:Nucleic Acids / Q000737:chemistry / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic
D003854:Deoxyribonucleotides; D009696:Nucleic Acids; D004256:DNA Polymerase I