METHOD FOR LOADING NUCLEIC ACID MOLECULE ON SOLID SUPPORT

Abstract

Provided are a method for loading a nucleic acid molecule, for example nucleic acid nanoball (e.g., DNA nanoball (DNB)), on a solid support, and a kit for the method.

Description

Claims

1-11. (canceled)

12. A method for loading a nucleic acid molecule on a solid support or for constructing a nucleic acid library that is immobilized on a solid support, comprising: (1) providing a composition comprising a nucleic acid molecule, and the solid support;(2) performing a first amplification of the nucleic acid molecule in the composition to obtain an amplification product comprising the nucleic acid molecule;(3) suspending or slowing down the first amplification;(4) loading the amplification product comprising the nucleic acid molecule onto the solid support; and(5) performing a second amplification of the nucleic acid molecule in the amplification product loaded on the solid support.

13. The method according to claim 12, wherein the method has one or more characteristics selected from the following: (1) the solid support has an array of sites for loading the amplification product; optionally, the sites are arranged on the solid support in a predetermined pattern;(2) the nucleic acid molecule is selected from the group consisting of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or an analog of DNA or RNA;(3) the composition comprises a single-stranded circular nucleic acid molecule;(4) the composition comprises a single-stranded circular DNA molecule;(5) the composition comprises at least 10, at least 20, at least 50, at least 100, at least 1000, at least 104, at least 105, at least 106, at least 107 or more nucleic acid molecules; and(6) the nucleic acid molecule comprises a target nucleic acid fragment with a length of at least 100 bp, at least 200 bp, at least 500 bp, at least 800 bp, at least 1000 bp, or at least 2000 bp.

14. The method according to claim 12, wherein in step (2), the obtained amplification product is a linear nucleic acid molecule.

15. The method according to claim 12, wherein, in step (2), the size of the obtained amplification product does not exceed a predetermined size; optionally, the predetermined size is the maximum size of the nucleic acid molecule that can be accommodated by or attached to a single site in the solid support or is the spacing of adjacent sites in the solid support.

16. The method according to claim 15, wherein the method has one or more characteristics selected from the following: (1) the size of the obtained amplification product does not exceed 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the maximum size or the spacing of adjacent sites; and(2) by controlling the time of amplification, the size of the amplification product does not exceed the predetermined size.

17. The method according to claim 12, wherein, in step (3), the first amplification is suspended or slowed down by one or more methods selected from the group consisting of: (a) controlling or reducing the amount of dNTPs in the reaction mixture for the first amplification; and(b) inhibiting the activity of the nucleic acid polymerase used in the first amplification or inactivating the nucleic acid polymerase used in the first amplification.

18. The method according to claim 17, wherein the activity of the nucleic acid polymerase is inhibited or the nucleic acid polymerase is inactivated by one or more methods selected from the group consisting of: (a) adding an activity inhibitor or denaturing agent of the nucleic acid polymerase;(b) removing a cofactor required for the nucleic acid polymerase to function;(c) adjusting the temperature of the reaction mixture for the first amplification to deviate from the working temperature range of the nucleic acid polymerase; and(d) adjusting the pH of the reaction mixture for the first amplification to deviate from the working pH range of the nucleic acid polymerase.

19. The method according to claim 17, wherein the activity of the nucleic acid polymerase is inhibited or the nucleic acid polymerase is inactivated by removing a cofactor required for the nucleic acid polymerase to function.

20. The method according to claim 19, wherein the method has one or more characteristics selected from the following: (1) the cofactor is a cation;(2) the cofactor is a magnesium ion;(3) the cofactor is removed by adding a cation chelating agent, thereby inhibiting the activity of the nucleic acid polymerase or inactivating the nucleic acid polymerase;(4) the cofactor is removed by adding a magnesium ion chelating agent; and(5) the cofactor is removed by adding a cation chelating agent which is selected from the group consisting of NTA, EDTA, HEDP, EDTMPS, DTPMPA, EDDHA, STPP, sodium dextrose and sodium metasilicate.

21. The method according to claim 17, wherein the temperature of the reaction mixture for the first amplification is adjusted to deviate from the working temperature range of the nucleic acid polymerase, thereby inhibiting the activity of the nucleic acid polymerase or inactivating the nucleic acid polymerase.

22. The method according to claim 21, wherein the method has one or more characteristics selected from the following: (1) the temperature of the reaction mixture is adjusted to be higher than the upper limit of the working temperature of the nucleic acid polymerase by at least 5° C., at least 10° C., at least 15° C. or more, or, the temperature of the reaction mixture is adjusted to be lower than the lower limit of the working temperature of the nucleic acid polymerase by at least 5° C., at least 10° C., at least 15° C. or more; and(2) the temperature of the reaction mixture is lowered to a temperature of ≤20° C., ≤10° C., ≤4° C., or lower; or, the temperature of the reaction mixture is elevated to a temperature of ≥50° C., ≥60° C., ≥ 70° C., ≥80° C., or higher.

23. The method according to claim 17, wherein the pH of the reaction mixture for the first amplification is adjusted by adding an acidic or basic buffer to deviate from the working pH range of the nucleic acid polymerase, thereby inhibiting the activity of the nucleic acid polymerase or inactivating the nucleic acid polymerase.

24. The method according to claim 23, wherein the method has one or more characteristics selected from the following: (1) the pH of the reaction mixture is adjusted to be higher than the upper limit of the working pH range of the nucleic acid polymerase by at least 1, at least 2, at least 3 or more pH units; or, the pH of the reaction mixture is adjusted to be lower than the lower limit of the working pH range of the nucleic acid polymerase by at least 1, at least 2, at least 3 or more pH units; and(2) the acidic or basic buffer is selected from the group consisting of citrate buffer, phosphate buffer, acetate buffer, carbonate buffer and Tris hydrochloride buffer.

25. The method according to claim 17, wherein the activity of the nucleic acid polymerase is inhibited or the nucleic acid polymerase is inactivated by adding an activity inhibitor or denaturing agent of the nucleic acid polymerase.

26. The method according to claim 25, wherein the method has one or more characteristics selected from the following: (1) the activity inhibitor or denaturing agent of the nucleic acid polymerase is a surfactant; and(2) the activity inhibitor or denaturing agent of the nucleic acid polymerase is selected from the group consisting of sodium deoxycholate, sodium lauryl sarcosinate, sodium lauryl sulfate, Tween-20, NP-40 and Tritox-100.

27. The method according to claim 12, wherein the method has one or more features selected from the group consisting of: (a) the solid support is selected from the group consisting of latex bead, dextran bead, polystyrene surface, polypropylene surface, polyacrylamide gel, gold surface, glass surface, chip, sensor, electrode and silicon wafer;(b) the solid support is planar, spherical or porous;(c) the maximum size of the nucleic acid nanoball that can be accommodated by or attached to a single site in the solid support is ≤ 5000 nm, ≤ 2000 nm, ≤ 1000 nm, ≤ 700 nm, ≤ 500 nm, ≤ 300 nm, or ≤ 100 nm;(d) in step (2), the first amplification of the nucleic acid molecule in the composition is carried out by rolling circle amplification;(e) in step (5), the second amplification of the nucleic acid molecule in the amplification product is carried out by rolling circle amplification;(f) the first amplification and the second amplification use the same or different nucleic acid polymerase;(g) the solid support is subjected to pretreatment prior to performing step (4);(h) the solid support is pretreated by using poloxamer, enzyme, or any combination thereof;(i) in step (5), the amplification product obtained by the second amplification is a linear nucleic acid molecule;(j) the product obtained in step (5) is used as a nucleic acid sequencing library;(k) the method further comprises, after step (5), sequencing the nucleic acid molecule loaded on the solid support; and(l) the amplification product is a nucleic acid nanoball.

28. The method according to claim 12, wherein the method has one or more features selected from the group consisting of: (a) in step (5), the second amplification of the nucleic acid molecules in the amplification product loaded on the solid support is performed by adding a reagent required for nucleic acid amplification; and(b) in step (5), the second amplification of the nucleic acid molecule in the amplification product loaded on the solid support is performed by eliminating a condition that causes the first amplification to be suspended or slowed down in step (3).

29. The method according to claim 28, wherein the method has one or more features selected from the group consisting of: (a) the reagent required for nucleic acid amplification is selected from the group consisting of nucleic acid polymerase, dNTPs, working buffer for nucleic acid polymerase, and any combination thereof;(b) in step (3), the first amplification is suspended or slowed down by inhibiting the activity of the nucleic acid polymerase used for the first amplification, and, in step (5), the second amplification is performed by restoring the activity of the nucleic acid polymerase;(c) in step (3), the first amplification is suspended or slowed down by inhibiting the activity of the nucleic acid polymerase used for the first amplification, and, in step (5), the second amplification is performed by eliminating a condition that inhibits the activity of the nucleic acid polymerase;(d) in step (3), the first amplification is suspended or slowed down by adding an activity inhibitor of the nucleic acid polymerase, and, in step (5), the activity inhibitor is removed to restore the activity of the nucleic acid polymerase, and the second amplification is performed;(e) in step (3), the first amplification is suspended or slowed down by removing a cofactor required for the nucleic acid polymerase to function, and, in step (5), the activity of the nucleic acid polymerase is restored by adding the cofactor, and the second amplification is performed;(f) in step (3), the first amplification is suspended or slowed down by adjusting the temperature of the reaction mixture to deviate from the working temperature range of the nucleic acid polymerase, and, in step (5), the activity of the nucleic acid polymerase is restored by adjusting the temperature of the reaction mixture to the working temperature range of the nucleic acid polymerase, and the second amplification is performed;(g) in step (3), the first amplification is suspended or slowed down by adjusting the pH of the reaction mixture to deviate from the working pH range of the nucleic acid polymerase, and, in step (5), the activity of the nucleic acid polymerase is restored by adjusting the pH of the reaction mixture to the working pH range of the nucleic acid polymerase, and the second amplification is performed; and(h) in step (3), the first amplification is suspended or slowed down by denaturing the nucleic acid polymerase, and, in step (5), the second amplification is performed by adding the nucleic acid polymerase.

30. A kit, which comprises: a reagent for amplifying a nucleic acid, a reagent for suspending or inhibiting nucleic acid amplification, and a reagent for loading the nucleic acid molecule onto a solid support; preferably, the kit has one or more features selected from the group consisting of: (1) the reagent for amplifying nucleic acid comprises a nucleic acid polymerase, a working buffer for the nucleic acid polymerase, a cofactor required for the nucleic acid polymerase to function, dNTPs, or any combination thereof;(2) the reagent for suspending or inhibiting nucleic acid amplification may comprise one or more selected from the group consisting of: (a) an activity inhibitor or denaturing agent of the nucleic acid polymerase;(b) a reagent capable of removing a cofactor required for the nucleic acid polymerase to function; and(c) an acidic or alkaline buffer capable of adjusting the pH of solution;(3) the reagent for loading the nucleic acid molecule onto the solid support comprises tripotassium citrate, citric acid, phi29 polymerase, or Pluronic F68; and(4) the kit further comprises a reagent for pretreating the solid support.

31. The kit according to claim 30, wherein the kit has one or more features selected from the group consisting of: (1) the nucleic acid polymerase is a DNA polymerase;(2) the cofactor is a cation;(3) the working buffer is concentrated, e.g by at least 2-fold, at least 5-fold or at least 10-fold;(4) the activity inhibitor or denaturing agent of the nucleic acid polymerase is a surfactant;(5) the reagent capable of removing the cofactor required for nucleic acid polymerase to function is a cation chelating agent; and(7) the reagent for pretreating the solid support is selected from the group consisting of poloxamer, enzyme, or any combination thereof;preferably, the kit has one or more features selected from the group consisting of: (1) the DNA polymerase is a DNA polymerase with strand displacement activity;(2) the DNA polymerase is selected from a Bst DNA polymerase, phi29 DNA polymerase or exo-Klenow;(3) the cation is a magnesium ion;(4) the surfactant is selected from the group consisting of sodium deoxycholate, sodium lauryl sarcosinate, sodium lauryl sulfate, Tween-20, NP-40 and Tritox-100;(5) the cation chelating agent is a magnesium ion chelating agent;(6) the cation chelating agent is selected from the group consisting of NTA, EDTA, HEDP, EDTMPS, DTPMPA, EDDHA, STPP, sodium dextrose and sodium metasilicate;(7) the acidic or basic buffer is selected from the group consisting of citrate buffer, phosphate buffer, acetate buffer, carbonate buffer and Tris hydrochloric acid buffer; and(8) the enzyme is selected from a DNA polymerase, T4 ligase, and BSA.