Method for sequencing polynucleotides based on optical signal kinetics of luminescent labels and secondary luminescent signals

A method for sequencing a nucleic acid molecule, comprising:

• (a) providing nucleotides A, G, C, and (T/U), wherein three of the nucleotides are respectively attached to chemiluminescent labels by linkers, wherein
• before and after cleavage of the linkers, the chemiluminescent label attached to each nucleotide shows a different luminescence kinetics or luminescence form different from that of the chemiluminescent labels attached to other nucleotides;
• wherein the first nucleotide is attached to the first molecular label through linker 1-linker 2;
• the second nucleotide is attached to the first molecular label through linker 1;
• the third nucleotide is attached to the second molecular label through linker 1;
• the fourth nucleotide is not attached to any molecular label;
• (b) incorporating a nucleotide into a complementary strand of the nucleic acid;
• (c) detecting the chemiluminescent label of the nucleotide of step (b);
• (d) removing the linker of the nucleotide of step (b);
• (e) detecting the chemiluminescent label of the nucleotide of step (b) after the treatment in step (d) to determine a type of the nucleotide incorporated;
• (f) optionally, removing the chemiluminescent label of the nucleotide of step (b);
• and
• (g) optionally, repeating steps (b) to (f) or steps (b) to (e) one or more times to sequence a target single-stranded polynucleotide;
• wherein detecting the chemiluminescent label of the nucleotide of step (b) comprises:
• making the chemiluminescent label to come into contact with a suitable substrate to trigger a chemiluminescence reaction, and detecting luminescence kinetics of light emitted therefrom;
• the chemiluminescent label is selected from biochemiluminescent labels that induce different luminescence kinetics and any combination thereof;
• or
• detecting the chemiluminescent label of the nucleotide of step (b) comprises:
• making the chemiluminescent label to come into contact with a suitable substrate to trigger the chemiluminescence reaction, and detecting a luminescence form of light emitted therefrom;
• the chemiluminescent label is selected from biochemiluminescent labels that induce different luminescence forms and any combination thereof;
• wherein the luminescence forms include flash and glow.