CN-121992072-A - Hybridization reaction solution and application thereof

Abstract

The invention belongs to the technical field of library construction, and particularly relates to hybridization reaction liquid and application thereof. The invention provides a hybridization reaction liquid, which has better capturing efficiency than the existing hybridization reaction liquid, can be compatible with panels with different sizes and different hybridization durations, has higher and relatively stable capturing efficiency by small panels, has selectable hybridization time of 1-20h, has unified fast and slow miscellaneous reagents, and is convenient for production and simultaneous operation of various products.

Inventors

• WU JINFENG
• LIU JIAHUI
• FENG QIANQIAN
• ZHANG HONGZHI
• WANG BO
• GUAN YANFANG
• HUANG YI

Assignees

• 吉因加生物医学科技(绍兴)有限公司
• 长沙吉因加生物科技有限公司
• 苏州吉因加生物医学工程有限公司

Dates

Publication Date

20260508

Application Date

20251226

Claims (14)

1. 1. A hybridization reaction solution comprises a hybridization reaction solution 1 and a hybridization reaction solution 2, wherein the hybridization reaction solution 1 comprises betaine, tetramethyl ammonium chloride, mg 2+ and Triton X-100, and the hybridization reaction solution 2 comprises formamide and N-methylpyrrolidone.
2. 2. The hybridization reaction solution according to claim 1, wherein, The hybridization reaction liquid 1 and the hybridization reaction liquid 2 exist independently or in a mixed mode; preferably, the concentration of Mg 2+ in the hybridization reaction solution 1 is 1-100 mM; Preferably, the concentration of the betaine in the hybridization reaction solution 1 is 1-10M; Preferably, the concentration of the tetramethyl ammonium chloride in the hybridization reaction solution 1 is 1-10M; preferably, the concentration of Triton X-100 in the hybridization reaction solution 1 is 0.01-1% by volume percent; Preferably, the concentration of the formamide in the hybridization reaction liquid 2 is 50-90% by volume percent; Preferably, the concentration of the N-methyl pyrrolidone in the hybridization reaction liquid 2 is 10-50% by volume percent; preferably, when the hybridization reaction solution 1 and the hybridization reaction solution 2 are mixed, the volume ratio of the hybridization reaction solution 1 to the hybridization reaction solution 2 is (1-10): 1.
3. 3. A hybridization capture kit comprising the hybridization reaction solution according to any one of claims 1 to 2.
4. 4. The hybridization capture kit according to claim 3, wherein, The hybridization capture kit further comprises a magnetic bead suspension; preferably, the magnetic bead suspension comprises the hybridization reaction liquid 1 and the hybridization reaction liquid 2 described in any one of claims 1 to 2; preferably, the hybridization capture kit further comprises a library blocking solution; Preferably, the hybridization capture kit further comprises a capture probe; preferably, the hybridization capture kit further comprises a wash solution combination; preferably, the cleaning solution combination comprises a magnetic bead cleaning solution, an elution reaction solution I, an elution reaction solution S, an elution reaction solution II and an elution reaction solution III; Preferably, the magnetic bead cleaning solution comprises Tris-Hcl, EDTA, naCl and Tween 20; Preferably, the elution reaction solution I comprises SSC buffer and SDS; Preferably, the elution reaction solution S comprises SSC buffer and Tween20; preferably, the elution reaction solution II comprises SSC buffer and Tween20; preferably, the elution reaction III comprises SSC buffer; preferably, the hybrid capture kit further comprises a combination of reagents for capturing amplification of the library; preferably, the reagent combination for amplification of the capture library comprises amplification primers and a DNA polymerase reaction solution.
5. 5. A DNA-targeted library construction kit comprising the hybridization capture kit of any one of claims 3-4.
6. 6. The DNA targeting library construction kit according to claim 5, wherein, The DNA targeting library construction kit further comprises a DNA library construction reagent combination; Preferably, the DNA library construction reagent combination comprises at least one of a reagent combination for linker ligation, a reagent combination for end repair and addition of "A", a reagent combination for library amplification, a substance for breaking DNA, and a nucleic acid extraction reagent combination.
7. 7. A hybridization method comprising the step of using the hybridization reaction solution according to any one of claims 1 to 2.
8. 8. The method of claim 7, wherein the step of determining the position of the probe is performed, Mixing a DNA library, a hybridization reaction solution, a capture probe and a library sealing solution, and reacting; preferably, the concentration of Mg 2+ in the reaction system is 0.5-50mM; preferably, the concentration of the betaine in the reaction system is 0.5-5M; preferably, the concentration of the tetramethyl ammonium chloride in the reaction system is 0.5-5M; preferably, the concentration of Triton X-100 in the reaction system is 0.005-0.5% by volume percent; preferably, the concentration of the formamide in the reaction system is 5-20% by volume percent; Preferably, the concentration of the N-methyl pyrrolidone in the reaction system is 1-10% by volume percent; preferably, the reaction time is 1-20 hours; preferably, the library sealing solution is the library sealing solution of claim 4.
9. 9. A hybridization capture method comprising the step of employing the hybridization capture kit according to any one of claims 3 to 4.
10. 10. The method of claim 9, wherein the step of determining the position of the substrate comprises, The method comprises the following steps: b1 Hybridization; b2 Capturing; b3 Eluting; b4 Amplification of the capture library; The method of hybridization is the method of any one of claims 7-8; Preferably, the method of capturing is by mixing magnetic beads with the product of b 1), reacting; preferably, the eluting comprises thermal eluting and room temperature eluting; preferably, the method for amplifying the capture library comprises the steps of mixing the product obtained in the step b 3) and the reagent combination for amplifying the capture library, and performing PCR reaction.
11. 11. A DNA targeting library construction method comprising the step of using the DNA targeting library construction kit of any one of claims 5 to 6.
12. 12. The method of claim 11, wherein the step of determining the position of the probe is performed, The method comprises the following steps: 1) Constructing a DNA library; 2) Hybridization capturing; the method of hybrid capture is the method of any one of claims 9-10; Preferably, the method of DNA library construction comprises the step of employing a combination of DNA library construction reagents as described in claim 6.
13. 13. A DNA-targeted library obtained by the method of any one of claims 11-12 of the present invention.
14. 14. A sequencing method for sequencing the DNA targeting library of claim 13.

Description

Hybridization reaction solution and application thereof Technical Field The invention belongs to the technical field of library construction, and particularly relates to hybridization reaction liquid and application thereof. Background The high-throughput sequencing technology (NGS) has wide application in the fields of basic research, prenatal diagnosis, genetic disease diagnosis, tumor diagnosis, accurate medical treatment and the like by virtue of the advantages of high throughput, high accuracy, rich information and the like. However, not only is whole genome sequencing time and effort consuming, but it also generates much redundant and clinically worthless data, so capturing specific regions for detection is the mainstream treatment protocol in clinical use today. Compared with whole genome sequencing, the targeted hybridization capture sequencing can separate and enrich specific areas, so that the detection sensitivity is high, and the subsequent data analysis work is greatly reduced. Therefore, efficient capture of reagents is of paramount importance. Disclosure of Invention An object of the first aspect of the present invention is to provide a hybridization reaction solution. The object of the second aspect of the present invention is to provide a hybridization capture kit. The object of the third aspect of the present invention is to provide a DNA targeting library construction kit. The fourth aspect of the present invention is directed to a hybridization method. The fifth aspect of the present invention is directed to a hybridization capture method. The sixth aspect of the present invention is directed to a method for constructing a DNA targeting library. The object of the seventh aspect of the present invention is to provide a DNA targeted library. An object of the eighth aspect of the present invention is to provide a sequencing method. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: In a first aspect of the present invention, there is provided a hybridization reaction solution comprising a hybridization reaction solution 1 and a hybridization reaction solution 2, wherein the hybridization reaction solution 1 comprises betaine, tetramethylammonium chloride, mg 2+, and Triton X-100, and the hybridization reaction solution 2 comprises formamide, and N-methylpyrrolidone. In some embodiments, the hybridization reaction solution 1 and the hybridization reaction solution 2 exist independently or in a mixture, and further exist independently. In some embodiments, the concentration of Mg 2+ in the hybridization reaction solution 1 is 1-100 mM, further 10-100 mM, still further 20-40 mM, and still further 25-35 mM. In some embodiments, the magnesium ion is from magnesium acetate. In some embodiments, the betaine is present in hybridization reaction solution 1 at a concentration of 1 to 10M, further 2 to 5M, and still further 2.5 to 3.5M. In some embodiments, the concentration of tetramethyl ammonium chloride in hybridization reaction solution 1 is 1-10M, further 1-3M, and still further 1.5-2.5M. In some embodiments, the concentration of Triton X-100 in hybridization reaction solution 1 is 0.01% to 1%, further 0.01% to 0.1%, and still further 0.01% to 0.02% by volume. In some embodiments, the concentration of formamide in hybridization reaction solution 2 is 50% to 90%, further 60% to 80%, and still further 75% to 80% by volume. In some embodiments, the concentration of the N-methylpyrrolidone in the hybridization reaction solution 2 is 10% to 50%, further 20% to 40%, and still further 20% to 25% by volume. In some embodiments, the hybridization reaction solution 1 and the hybridization reaction solution 2 are mixed, and the volume ratio of the hybridization reaction solution 1 to the hybridization reaction solution 2 is (1-10): 1, further (2-5): 1, further (3-4): 1. In a second aspect of the present invention, there is provided a hybridization capture kit comprising the hybridization reaction solution of the first aspect of the present invention. In some embodiments, the hybrid capture kit further comprises a magnetic bead suspension. In some embodiments, the magnetic bead suspension comprises hybridization reaction solution 1 and hybridization reaction solution 2 in the first aspect of the present invention. In some embodiments, the volume ratio of hybridization reaction solution 1 to hybridization reaction solution 2 is (2-4): 1. In some embodiments, the hybridization capture kit further comprises a library blocking solution. In some embodiments, the library blocking solution comprises Human Cot DNA or Salmon SpermDNA, and A Blocker (preferably a universal Blocker). The Human Cot DNA is genomic DNA extracted from Human placenta, is a highly repeated sequence with the length of about 50 to 300bp in Human genome, can conveniently and effectively block the repeated sequence in the nucleic acid sequence of a probe or a sample to be detected, shields the re