Reagent, method and kit for capturing target nucleic acid sequence through targeted hybridization and application of reagent, method and kit

By using a hybridization reagent containing the first and second probe groups, the low efficiency problem of the targeted hybridization capture method when the probes are small is solved, the on-target rate is improved and the uniformity and complexity of the library are maintained, the operation process is simplified and the cost is reduced.

CN120648800APending Publication Date: 2025-09-16GUANGZHOU BURNING ROCK DX CO LTD
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Patent Information

Application Number
CN202510795560.9
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2025-06-13
Publication Date
2025-09-16

AI Technical Summary

Technical Problem

Existing targeted hybridization capture methods are inefficient when the probes are small, resulting in a decrease in on-target rate. Existing improvement schemes are complex to operate or costly, making it difficult to ensure complexity and uniformity while improving the on-target rate.

Method used

A hybridization reagent comprising a first probe group and a second probe group is used. The first probe group specifically hybridizes with the target nucleic acid sequence, while the second probe group does not specifically hybridize with the target nucleic acid sequence. By increasing the concentration of the reactants, the hybridization kinetics is promoted, the on-target rate is improved, and the enrichment of the target nucleic acid sequence is achieved through capture reagent labeling.

Benefits of technology

The on-target rate of targeted hybridization capture is significantly improved while maintaining the complexity and uniformity of the library, reducing operational complexity and cost.

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Abstract

The invention provides a reagent, a method and a kit for capturing a target nucleic acid sequence through targeted hybridization and application of the reagent, the method and the kit. Wherein the reagent comprises a first probe group and a second probe group, the first probe group is composed of probes specifically hybridized with a target nucleic acid sequence, and the second probe group is composed of probes not specifically hybridized with the target nucleic acid sequence. According to the reagent, the in-target rate of a captured nucleic acid sequence is improved by combining two probe groups, and meanwhile, the complexity and uniformity of the reagent can be ensured.
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Claims

1. A reagent for targeted hybridization of a target nucleic acid sequence, characterized in that The reagent comprises a first probe group and a second probe group, wherein the first probe group consists of probes that specifically hybridize with a target nucleic acid sequence, and the second probe group consists of probes that do not specifically hybridize with the target nucleic acid sequence.

2. The reagent according to claim 1, characterized in that The second probe group comprises probes that specifically hybridize to non-target nucleic acid sequences in the pre-library.

3. The reagent according to claim 1, characterized in that The sequence of each probe in the second probe set and the flanking sequences on either or both sides thereof do not overlap with the sequence of each probe in the first probe set.

4. The reagent according to claim 1, characterized in that The size of each probe in the first probe set is no more than 100 kb, preferably 4 kb to 40 kb, more preferably 4 kb to 10 kb.

5. The reagent according to claim 1, characterized in that The size of each probe in the second probe set is 10 kb to 2000 kb, preferably 10 kb to 1000 kb, and more preferably 40 kb to 700 kb.

6. The reagent according to claim 1, characterized in that The first probe set comprises 10 to 200 probes, preferably 10 to 100 probes; The second probe set comprises 100 to 500 probes, preferably 100 to 300 probes.

7. The reagent according to claim 1, characterized in that The weight ratio of the first probe group to the second probe group is 1:2 to 1:50, preferably 1:5 to 1:

40.

8. The reagent according to claim 1, characterized in that The target nucleic acid sequence is derived from human genomic DNA.

9. The reagent according to claim 8, characterized in that The site corresponding to the target nucleic acid sequence is located in one or more of the following gene fragments: PIK3CA, RHOA, ESR1, EGFR, CEBPA, CD79B, PTEN, BCL2, BCL6, BCL7A, BCL10, PDGFRA, KIT, TP53, STAT3, STAT6, MET, BRAF, TNFRSF14, ALK, SF3B1, TET2, MYD88, EZH2, SOCS1, SDHA, NRAS , ERBB2, MYC, KRAS, CREBBP, ARID1A, FOXO1, PIM1, GATA2, ASXL1, XPO1, JAK2, SMARCA4, FGFR3, IDH1, IRF4, BTK, ERBB3, U2AF1, MAP2K1, PPM1D, EP300, DUSP2, CARD11, CALR, B2M, AKT1.

10. The reagent according to claim 2, characterized in that The non-target nucleic acid sequence is a human gene spacer sequence.

11. The reagent according to claim 10, characterized in that The non-target nucleic acid sequence is selected from SEQ ID NO: 1341-1674.

12. The reagent according to claim 1, characterized in that Each probe in the first probe group is labeled, and the label is selected from one of biotin, digoxigenin (DIG), 2,4-dinitrophenyl (DNP), amino and hydroxyl labels.

13. A method for capturing a target nucleic acid sequence by targeted hybridization, comprising the following steps: (a) providing a hybridization reagent comprising a first probe group and a second probe group, wherein the first probe group consists of probes that specifically hybridize to a target nucleic acid sequence, and the second probe group consists of probes that do not specifically hybridize to the target nucleic acid sequence; (b) contacting the first probe group and the second probe group with a prelibrary, the prelibrary being a nucleic acid library generated from a sample; (c) capturing nucleic acid sequences hybridized to the first probe set from the prelibrary; (d) Amplifying and sequencing the captured nucleic acid sequences.

14. The method according to claim 13, characterized in that The step (a) comprises: determining the sequence of the probes contained in the first probe set according to the target nucleic acid sequence, wherein the mutation site corresponding to the target nucleic acid sequence is located in one or more of the following gene fragments: PIK3CA, RHOA, ESR1, EGFR, CEBPA, CD79B, PTEN, BCL2, BCL6, BCL7A, BCL10, PDGFRA, KIT, TP53, STAT3, STAT6, MET, BRAF, TNFRSF14, ALK, SF3B1, TET2, MYD88, EZH2, SOCS1, SDHA, NRAS , ERBB2, MYC, KRAS, CREBBP, ARID1A, FOXO1, PIM1, GATA2, ASXL1, XPO1, JAK2, SMARCA4, FGFR3, IDH1, IRF4, BTK, ERBB3, U2AF1, MAP2K1, PPM1D, EP300, DUSP2, CARD11, CALR, B2M, AKT1.

15. The method according to claim 13, characterized in that The step (a) comprises: determining the sequences of the probes contained in the second probe group based on the human gene intergenic region.

16. The method according to claim 13, characterized in that Each probe in the first probe group is labeled, and the label is selected from one of biotin, digoxigenin (DIG), 2,4-dinitrophenyl (DNP), amino and hydroxyl labels; each probe in the second probe group is not labeled, and The step (d) comprises: using a capture reagent that binds to the probes contained in the first probe set to capture the nucleic acid sequence hybridized with the first probe set.

17. A kit for capturing a target nucleic acid sequence by targeted hybridization, comprising: The reagent according to any one of claims 1 to 12.

18. The kit according to claim 17, characterized in that The kit further comprises one or more of a capture reagent, an amplification reagent and a sequencing reagent, wherein the capture reagent optionally comprises streptavidin magnetic beads, the amplification reagent optionally comprises Tris, magnesium ions, dNTP and DNA polymerase, and the sequencing reagent optionally comprises a second-generation high-throughput sequencing reagent.

19. Use of the kit according to any one of claims 17 or 18 for in vitro qualitative detection of cancer, characterized in that: The cancers include: non-small cell lung cancer, small cell lung cancer, ovarian cancer, uterine cancer, breast cancer, gastric cancer, pancreatic cancer, liver cancer, colorectal cancer, bladder cancer, thyroid cancer, lymphoma, glioma, melanoma and myeloma.