Method for sequencing polynucleotides

A method for determining a sequence of a target polynucleotide, which comprises:

• (a) providing a target polynucleotide,
• (b) contacting the target polynucleotide with a primer so that the primer hybridizes to the target polynucleotide, thereby forming a partial duplex of the target polynucleotide and the primer,
• (c) contacting the partial duplex with a polymerase and a nucleotide under a condition that allows the polymerase to carry out a nucleotide polymerization reaction, so that the nucleotide is incorporated into the primer,
• wherein the nucleotide is selected from one or more of the followings:
• a first nucleotide, a second nucleotide, a third nucleotide and a fourth nucleotide, wherein the first nucleotide comprises a first nucleotide labeled with a first label and optionally an unlabeled first nucleotide, the second nucleotide comprises a second nucleotide labeled with a second label and optionally an unlabeled second nucleotide, the third nucleotide is selected from:
• (1) a third nucleotide labeled with the first label and a third nucleotide labeled with the second label, or (2) a third nucleotide simultaneously labeled with the first label and the second label, and the fourth nucleotide comprises an unlabeled fourth nucleotide,
• wherein each nucleotide has a ribose or deoxyribose moiety that contains a protecting group attached thereto via a 2′ or 3′ oxygen atom,
• (d) detecting the presence of the first label on the partial duplex of the step (c),
• (e) detecting the presence of the second label on the partial duplex of the step (c),
• (f) optionally removing the protecting group and the label on the nucleotide incorporated in the partial duplex of the step (c),
• (g) optionally repeating the steps (c) to (f) one or more times to obtain sequence information of the target polynucleotide,
• wherein the presence of the first label and the second label is detected by the same luminescence signal.