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

1 . 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.

2 . The method according to claim 1 , wherein a ribose or deoxyribose moiety of each nucleotide contains a protective group attached via a 2′ or 3′ oxygen atom, and the protective group is modified or removed after the incorporation of the nucleotide so as to expose a 3′-OH group.

3 . The method according to claim 1 , wherein the chemiluminescent label is attached to the nucleotide by affinity interaction.

4 . The method according to claim 1 , wherein all the nucleotides are allowed to come into contact with the target single-stranded polynucleotide in sequence, unincorporated nucleotides are removed before the next nucleotide is added, and the detection and removal of the chemiluminescent label are performed after the addition of each nucleotide or the addition of all the four nucleotides.

5 . The method according to claim 4 , wherein one, two, three or all of the four nucleotides are allowed to come into contact with the target single-stranded polynucleotide at the same time, unincorporated nucleotides are removed before detection, and the detection and removal of the chemiluminescent label are performed after the addition of the one, two, three or all of the four nucleotides.

6 . The method according to claim 1 , wherein the chemiluminescent label is selected from luciferases that induce different luminescence kinetics and any combination thereof, or the chemiluminescent label is selected from luciferases that induce different luminescence forms and any combination thereof.

7 . The method according to claim 1 , wherein the chemiluminescent label is a combination of two luciferases that induce different luminescence kinetics, or the chemiluminescent label is a combination of two luciferases that induce different luminescence forms.

8 . The method according to claim 2 , wherein the chemiluminescent label and the protective group are removed under the same condition.

9 . The method according to claim 3 , wherein the affinity interaction comprises antigen-antibody interaction or biotin-avidin interaction.

10 . The method according to claim 9 , wherein the avidin is streptavidin.

11 . The method according to claim 3 , wherein by attaching the chemiluminescent label to one of members participating in the affinity interaction and attaching the nucleotide to the other member participating in the affinity interaction, the chemiluminescent label is attached to the nucleotide through the affinity interaction between the members.

12 . The method according to claim 11 , wherein the member attached to the nucleotide is a biotin and the member attached to the chemiluminescent label is an avidin; or

the member attached to the nucleotide is digoxin and the member attached to the chemiluminescent label is an anti-digoxin antibody; or

the member attached to the nucleotide is digoxin and the member attached to the chemiluminescent label is an avidin, wherein the digoxin and the avidin are bound by affinity with an anti-digoxin antibody attached to the biotin.

13 . The method according to claim 12 , wherein the avidin is streptavidin.