| abstract |
The present invention relates to a method for determining a nucleotide sequencenof a nucleic acid by single dye molecule detection, the method comprising:n (1) a step of immobilizing a nucleic acid molecule, or a primer which has ansequence complementary to a part of the sequence of the nucleic acid molecule,nonto the surface of a solid; (2) a step of annealing the primer or the nucleic acid molecule to the nucleic acidnmolecule or the primer, respectively; (3) a step of providing a solution which contains DNA polymerase and one type ofndye-labeled dNTP, or RNA polymerase and one type of dye-labeled NTP, to thenimmobilized nucleic acid molecule, and allowing the nucleotide to react with then3' end of the primer, whereby a nucleotide, which forms a base-pair with a basenopposed to the reaction site, is bound to the primer by action of the polymerase; (4) a step of detecting the presence of a bound, dye-labeled dNTP or NTP; (5) a step of disrupting the dye molecule of the bound, dye-labeled dNTP or NTP; (6) a step of repeating the steps (3) to (5) while changing the type of dye-labeledndNTP or NTP in turn, to sequentially bind dNTPs or NTPs which base-pair withnthe nucleotides of the nucleic acid molecule; and (7) a step of determining the nucleotide sequence of the nucleic acid molecule basednon the types of the sequentially bound dNTPs or NTPs. |