Composition for nucleic acid hybridization and application thereof

Abstract

The present disclosure relates to a composition for nucleic acid hybridization, belonging to the field of molecular biology. The composition comprises a trivalent cation salt. The hybridization capture system described in the present disclosure can accelerate the hybridization reaction process, shorten the hybridization time, and obtain a good hybridization capture effect.

Description

Technical Field

[0001] The present disclosure relates to the field of molecular biology, and in particular to a composition for nucleic acid hybridization and applications thereof. Background Art

[0002] Nucleic acid sequencing is the most widely used analytical tool in molecular biology. With the development of sequencing technology, it has become an important means of clinical and life science research. However, although sequencing technology has made great progress, it is still accompanied by huge costs and workloads due to the complex workflow and long duration.

[0003] In order to improve efficiency, certain target nucleic acid sequences are enriched during sequencing so that sequencing can be focused on the enriched nucleic acid sequences, thereby only sequencing the target sequences of interest, reducing costs and enabling more samples to be analyzed per sequencing.

[0004] Nucleic acid hybridization capture is a commonly used method for nucleic acid enrichment. There are many hybridization capture reagents on the market, and the hybridization time is long, which makes it impossible for some clinical detection methods based on targeted capture detection technology to produce results in a shorter time. In addition, the hybridization capture cleaning process involves too much cleaning fluid, which is inconvenient to operate.

[0005] Therefore, there is an urgent need for a system or method to reduce the complexity of the workflow, shorten the hybridization capture time, and ensure specific and uniform hybridization capture of the target sequence or region. Summary of the invention

[0006] In order to solve at least one technical problem existing in the prior art, the present disclosure provides a composition containing trivalent cations, and the application of the composition in nucleic acid hybridization capture. The application of the composition disclosed in the present disclosure can accelerate the hybridization reaction process, shorten the hybridization time, and obtain a good hybridization capture effect.

[0007] A first aspect of the present disclosure provides a composition for nucleic acid hybridization, comprising a trivalent cation salt.

[0008] By using the composition provided by the present disclosure, hybridization capture can be completed within 2 hours, and can even be completed within 1 hour.

[0009] In some embodiments, the trivalent cations include, but are not limited to, Co 3+ , Fe 3+ 、Al 3+ Cr 3+ 、Ni 3+ , B 3+ , Ir 3+ , Rh 3+Cr 3+ .

[0010] In some embodiments, the trivalent cation is in the form of a complexed trivalent cation.

[0011] In some embodiments, the complexed trivalent cation includes but is not limited to an ammine complex [X(NH3)6] 3+ , pyridine complex [X(dpy)3] 3+ , ethylenediamine complex [X(en)3] 3+ , X is a trivalent cation.

[0012] In some embodiments, the trivalent cation is an ammine-complexed trivalent cation.

[0013] In some embodiments, the trivalent cation is a complexed trivalent cation such as ammonia or ethylenediamine.

[0014] In some embodiments, the ammine complexed trivalent cation comprises a pentaammine complexed trivalent cation or a hexaammine complexed trivalent cation.

[0015] In some embodiments, the ammonia complexed trivalent cation comprises trivalent hexaammine cobalt [Co(NH3)6] 3+ 、Trivalent hexaammine iridium ([Ir(NH3)6] 3+ 、Trivalent hexaammine rhodium [Rh(NH3)6] 3+ 、Trivalent hexaammine chromium [Cr(NH3)6] 3+ etc. or their chelates.

[0016] In some specific embodiments, the ammoniated trivalent cation comprises hexaamminecobalt chloride.

[0017] In some embodiments, the trivalent cation concentration is 0.2-15 mM.

[0018] In some embodiments, the trivalent cation concentration is selected from 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM,

[0019] 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM, 2.0mM, 3.0mM, 4.0mM, 5.0mM, 6.0mM,

[0020] 7.0 mM, 8.0 mM, 9.0 mM, 10.0 mM, 11.0 mM, 12.0 mM, 13.0 mM, 14.0 mM or 15.0 mM.

[0021] In some embodiments, the trivalent cation concentration is 1.0-5.0 mM.

[0022] In some embodiments, the composition may further include an additive selected from one or more of the following substances: glycine, betaine, glycine-betaine, 7-deaza-2'-deoxyguanosine, dimethyl sulfoxide (DMSO), polyethylene glycol 400-1,000,000, glycerol, magnesium, tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC), triethylamine hydrochloride, ethylene carbonate, dextran sulfate, N-methylpyrrolidone or Denhandt's solution.

[0023] In some embodiments, the composition is selected from betaine, tetramethylammonium chloride (TMAC), N-methylpyrrolidone.

[0024] In some embodiments, the concentration of betaine is selected from 0.5 to 5M.

[0025] In some embodiments, the concentration of betaine is selected from 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M or 5M.

[0026] In some embodiments, the concentration of betaine is 1.5M.

[0027] In some embodiments, the concentration of tetramethylammonium chloride is selected from 0.5 to 1.5M.

[0028] In some embodiments, the concentration of tetramethylammonium chloride is selected from 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.4M or 1.5M.

[0029] In some embodiments, the concentration of tetramethylammonium chloride is 1M.

[0030] In some embodiments, the concentration of N-methylpyrrolidone is selected from 10% to 20% v / v.

[0031] In some embodiments, the concentration of N-methylpyrrolidone is selected from 10% v / v, 11% v / v, 12% v / v, 13% v / v, 14% v / v, 15% v / v, 16% v / v, 17% v / v, 18% v / v, 19% v / v or 20% v / v.

[0032] In some embodiments, the concentration of N-methylpyrrolidone is 16% v / v.

[0033] In some embodiments, the composition further comprises a buffer.

[0034] In some embodiments, the buffer is selected from one or more of the following: sodium chloride sodium citrate buffer (SSC), tris (hydroxymethyl) aminomethane (Tris), 2-(bis (2-hydroxyethyl) amino) acetic acid) (Bicine), (N-[tris (hydroxymethyl) methyl] glycine (Tricine), [tris (hydroxymethyl) methylamino] propane sulfonic acid (TAPS), 3-[N-tris (hydroxymethyl) methylamino] -2-hydroxypropane sulfonic acid (TAPSO), 4-(2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl) propane-2-yl] amino] ethanesulfonic acid (TES), 3-(N-morpholino) propanesulfonic acid (MOPS), piperazine-N,N'-bis (2-ethanesulfonic acid) (PIPES), 2-(N-morpholino) ethanesulfonic acid (MES), or dimethylarsonic acid (arsenic acid salt).

[0035] In some embodiments, the buffer is sodium citrate buffer (SSC).

[0036] In some embodiments, the pH of the buffer is selected from 6.0 to 9.0.

[0037] In some embodiments, the pH of the buffer is selected from 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5,

[0039] 8.6, 8.7, 8.8, 8.9 or 9.0.

[0040] In some embodiments, the pH of the buffer is selected from 8.0.

[0041] In some embodiments, the composition may further include a surfactant.

[0042] In some embodiments, the surfactant is selected from one or more of the following: sodium dodecyl sulfate (SDS), polysorbate 20 (20), octylphenol ethoxylate (TritonX-100), octylphenol polyoxyethylene ether (CA630), nonylphenol polyoxyethylene ether (NP-40), or cetrimide bromide (CTAB).

[0043] In some embodiments, the surfactant is sodium dodecyl sulfate (SDS).

[0044] In some embodiments, the concentration of the surfactant is selected from 0.005% v / v to 1% v / v.

[0045] In some embodiments, the concentration of the surfactant is selected from 0.005% v / v, 0.006% v / v, 0.007% v / v, 0.008% v / v, 0.009% v / v, 0.01% v / v, 0.02% v / v, 0.03% v / v, 0.04% v / v, 0.05% v / v, 0.06% v / v, 0.07% v / v, 0.08% v / v, 0.09% v / v, 0.1% v / v, 0.2% v / v, 0.3% v / v, 0.4% v / v, 0.5% v / v, 0.6% v / v, 0.7% v / v, 0.8% v / v, 0.9% v / v or 1% v / v.

[0046] In some embodiments, the concentration of the surfactant is 0.1% v / v.

[0047] In some embodiments, the composition comprises 1-5 mM trivalent cobalt ion, 10%-20% v / v N-methylpyrrolidone, 0.5-5 M betaine, 0.5-1.5 M tetramethylammonium chloride and 0.1 v / v% Triton X-100, pH=6.0-9.0.

[0048] In some embodiments, the composition comprises 1-5 mM trivalent cobalt ions, 16 v / v% N-methylpyrrolidone, 1.5 M betaine, 1 M tetramethylammonium chloride and 0.1 v / v% Triton X-100, pH=8.0.

[0049] According to another aspect, a method for nucleic acid hybridization is provided, wherein the method uses the above-mentioned composition disclosed herein to perform nucleic acid hybridization to obtain a hybridization product.

[0050] In some embodiments, the method comprises the following steps:

[0051] 1) In the above composition of the present disclosure, a nucleic acid sample and a nucleic acid probe are mixed to form a mixture;

[0052] 2) denaturing the mixture at a first temperature for a first period of time;

[0053] 3) Incubating the mixture at a second temperature for a second period of time to allow the nucleic acid probe to hybridize with the target nucleic acid fragment to obtain a hybridization product.

[0054] In some embodiments, the method further comprises adding a blocking agent prior to hybridization, wherein the blocking agent is complementary to a repetitive sequence or an adapter sequence of the nucleic acid sample.

[0055] In some embodiments, the method further comprises maintaining the mixture at a third temperature after step 2).

[0056] In some embodiments, the first temperature is selected from 94-98°C.

[0057] In some specific embodiments, the first temperature is selected from 94°C, 95°C, 96°C, 97°C or 98°C.

[0058] In some specific embodiments, the first temperature is selected from 95°C.

[0059] In some embodiments, the first time period is selected from 10 seconds to 15 minutes.

[0060] In some specific embodiments, the first time is selected from 10 seconds, 11 seconds, 12 seconds, 13 seconds, 14 seconds, 15 seconds, 16 seconds, 17 seconds, 18 seconds, 19 seconds, 20 seconds, 21 seconds, 22 seconds, 23 seconds, 24 seconds, 25 seconds, 26 seconds, 27 seconds, 28 seconds, 29 seconds, 30 seconds, 31 seconds, 32 seconds, 33 seconds, 34 seconds, 35 seconds, 36 seconds, 37 seconds, 38 seconds, 39 seconds. 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, or 15 minutes.

[0061] In some specific embodiments, the first time period is selected from 30 seconds.

[0062] In some embodiments, the second temperature or the third temperature is selected from 55 to 75°C.

[0063] In some specific embodiments, the second temperature or the third temperature is selected from 55°C, 56°C, 57°C, 58°C,

[0064] 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C or 75°C.

[0065] In some embodiments, the second temperature or the third temperature are the same or different.

[0066] In some specific embodiments, the second temperature or the third temperature is selected from 65°C.

[0067] In some embodiments, the second time period is selected from 30 minutes to 2 hours.

[0068] In some specific embodiments, the second time period is selected from 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 48 ​​minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 1 hour, 1.2 hours, 1.3 hours, 1.4 hours, 1.5 hours, 1.6 hours, 1.7 hours, 1.8 hours, 1.9 hours or 2 hours.

[0069] In some specific embodiments, the second time period is selected from 1 hour.

[0070] In some embodiments, the method may further include directly or indirectly connecting the hybridization product to a solid phase carrier and washing it with a washing solution.

[0071] In some embodiments, the solid support may include, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, and nylon.

[0072] In some embodiments, the solid phase carrier can be magnetic microspheres.

[0073] In some embodiments, the probe can be directly attached to a solid support.

[0074] In some embodiments, the direct connection includes, but is not limited to, covalent bonds, ionic bonds, hydrogen bonds, Coulomb forces, van der Waals forces, and hydrophobic interactions.

[0075] In some embodiments, the probe can be indirectly attached to a solid support.

[0076] In some embodiments, the probe may be linked to a first ligand.

[0077] In some embodiments, the solid support may be linked to a second ligand.

[0078] In some embodiments, the first ligand and the second ligand can bind to each other in a pair.

[0079] In some embodiments, the combination of the first ligand and the second ligand may include, but is not limited to, biotin-avidin / streptavidin, nanogold / iodacetyl-thiol, amino-aldehyde / carboxyl / isothiocyanate, and silyl-acrylamide.

[0080] In some specific embodiments, the first ligand and the second ligand may include biotin-streptavidin. In a specific embodiment, the first ligand may be biotin, and the second ligand may be streptavidin.

[0081] In some embodiments, the cleaning includes but is not limited to using a cleaning solution to remove other nucleic acid molecules and impurities that are not bound to the solid phase carrier, and retaining only one or more methods of hybridization products bound to the solid phase carrier.

[0082] In some embodiments, the cleaning solution is KS cleaning solution.

[0083] In some embodiments, the cleaning solution includes only one cleaning solution, which is KS cleaning solution, and does not include other cleaning solutions.

[0084] In some embodiments, the KS cleaning solution includes sodium citrate buffer SSC, sodium dodecyl sulfate and / or Tween 20.

[0085] In some embodiments, the KS cleaning solution includes 0.1-1.0× sodium citrate buffer, 0.005-0.02% sodium dodecyl sulfate and / or 0.005-0.02% Tween 20.

[0086] In some embodiments, the cleaning solution comprises 0.1×, 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×, 0.9× or 1.0× sodium citrate buffer.

[0087] In some embodiments, the cleaning solution comprises 0.005% v / v, 0.006% v / v, 0.007% v / v, 0.008% v / v, 0.009% v / v, 0.01% v / v, 0.011% v / v, 0.012% v / v, 0.013% v / v, 0.014% v / v, 0.015% v / v, 0.016% v / v, 0.017% v / v, 0.018% v / v, 0.019% v / v or 0.02% v / v of sodium dodecyl sulfate.

[0088] In some embodiments, the cleaning solution comprises 0.005% v / v, 0.006% v / v, 0.007% v / v, 0.008% v / v, 0.009% v / v, 0.01% v / v, 0.011% v / v, 0.012% v / v, 0.013% v / v, 0.014% v / v, 0.015% v / v, 0.016% v / v, 0.017% v / v, 0.018% v / v, 0.019% v / v or 0.02% v / v of Tween 20.

[0089] According to another aspect, the present disclosure provides a kit comprising the composition.

[0090] According to another aspect, the present disclosure provides use of the composition and / or the method and / or the kit in nucleic acid hybridization.

[0091] The present invention accelerates the hybridization reaction process by adjusting the ion valence state in the hybridization reaction solution. For the first time, a relatively low concentration of trivalent cations is used in a nucleic acid hybridization system to change the molecular state of DNA and the electrostatic potential in the hybridization capture system, thereby accelerating the renaturation process of the probe and the denatured single-stranded library. The hybridization time can be shortened while maintaining good capture efficiency and uniformity, and the capture time can be shortened to about 15 minutes. The hybridization reaction solution can be simplified into one, thereby overcoming the technical prejudice that trivalent cations cannot be used for nucleic acid hybridization reactions and achieving unexpected technical effects.

[0092] In addition, the multiple cleaning solutions used in hybridization elution can be simplified to only one cleaning solution, which can also achieve the same cleaning effect, making the operation simpler and easier to operate, further shortening the experimental time and simplifying the experimental process. DETAILED DESCRIPTION

[0093] In order to make the purpose, technical scheme and advantages of the present disclosure clearer, the present disclosure is further described in detail below in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present disclosure and are not intended to constitute any limitation to the present disclosure. In addition, in the following description, the description of the well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0094] definition

[0095] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0096] In the present disclosure, the term "nucleic acid" may refer to DNA, RNA, dsDNA, dsRNA, ssDNA, ssRNA, or DNA / RNA complexes or hybrids of sequences obtained from any source, containing target sequences and non-target sequences. For example, nucleic acid samples can be obtained from artificial sources or by chemical synthesis, or from viruses, prokaryotic cells including microorganisms, or eukaryotic cells. Biological samples can be vertebrates, invertebrates, plants, microorganisms, viruses, mycoplasmas, fungi, or archaea, including humans or non-humans.

[0097] In the present disclosure, the term "nucleic acid sample" includes a whole genome sequence, a partial genome sequence, a chromosome sequence, a mitochondrial sequence, a PCR product, a whole genome amplification product or a product of other amplification schemes, such as but not limited to a cDNA sequence, an mRNA sequence, a whole transcriptome sequence, an exon or an intron. These examples should not be construed as limiting the sample types applicable to the aspects described herein.

[0098] In the present disclosure, the term "nucleic acid probe" refers to a short single-stranded DNA or RNA fragment. The probe can be complementary to the target nucleic acid fragment. The length of the probe sequence complementary to the target nucleic acid fragment is between 60nt-150nt, preferably 120nt.

[0099] In the present disclosure, the term "target nucleic acid fragment" refers to one or more nucleotide sequences in a "nucleic acid sample" that need to be identified, detected, located, or hybridized and captured. When the probe combination of the present application is used as a method for identification, detection, location, or hybridization capture, the target nucleic acid generally includes a continuous sequence that is complementary to the probe, and the sequence may also be referred to as a target sequence. Complete complementarity between the target sequence and the probe pair is not required, and the required condition is that there is sufficient complementarity to cause hybridization and achieve recognition and location of the target nucleic acid by the probe.

[0100] In the present disclosure, the term "hybridization" or "binding" refers to the pairing (including partial complementarity) of complementary (including partially complementary) polynucleotide chains. Hybridization and the strength of hybridization (e.g., the strength of binding between polynucleotide chains) are affected by many factors well known in the art, including the degree of complementarity between polynucleotides, the stringency of related conditions, which are affected by conditions such as salt concentration, the melting temperature (Tm) of the formed hybrid, the presence of other components, the molar concentration of the hybridized chain, and the GC content of the polynucleotide chain. When a nucleotide chain is said to be "hybridized" with another polynucleotide chain, it means that there is a certain complementarity between the two polynucleotide chains, or that the two polynucleotide chains form a hybrid under high stringency conditions. When a polynucleotide chain is said not to hybridize with another polynucleotide chain, it means that there is no sequence complementarity between the two polynucleotide chains, or that the two polynucleotide chains do not form a hybrid under high stringency conditions.

[0101] In the present disclosure, the term "hybridization product" refers to the complex formed between the nucleic acid probe and the target nucleic acid sequence by means of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds can be further stabilized by base stacking interactions.

[0102] In the present disclosure, the term "complementary" refers to the concept of sequence complementarity between regions of two polynucleotide chains or between two regions of the same polynucleotide chain. It is known that the adenine base of the first region of the polynucleotide chain can form a specific hydrogen bond ("base pair") with the base of the second region of the polynucleotide chain that is reverse complementary to the first region (if the base is thymine or uracil). Similarly, it is known that the cytosine base of the first polynucleotide chain can base pair with the base of the second polynucleotide chain that is reverse complementary to the first region (if the base is guanine). If at least one nucleotide in the first region can base pair with the base of the second region when the first region of the polynucleotide is arranged in a reverse complementary manner with the second region of the same or another different polynucleotide, then the two regions are complementary. Therefore, the two complementary polynucleotide chains do not need to base pair at every nucleotide position. "Complementary" means that the first polynucleotide chain is 100% or "completely" complementary to the second polynucleotide chain, and therefore base pairing is formed at every nucleotide site. "Complementary" also means that the first polynucleotide strand is not 100% complementary (eg, 90%, or 80% or 70% complementary) and contains mismatched nucleotides at one or more nucleotide positions. In one embodiment, two complementary polynucleotide strands are capable of hybridizing to each other under high stringency hybridization conditions.

[0103] In the present disclosure, the term "additive" refers to one or more components added to the buffers described herein to enhance hybridization and improve overall specificity.

[0104] In the present disclosure, the term "stringent hybridization conditions" is defined as conditions under which a probe or primer specifically hybridizes to a target sequence but not to non-target sequences, which can be determined empirically. The term "stringent conditions" is functionally defined in terms of hybridization of a nucleic acid probe to a target nucleic acid (i.e., to a specific nucleic acid sequence of interest) by the specific hybridization method discussed in Sambrook et al., 1989, 9.52-9.55. See also Sambrook et al., 1989 9.47-9.52, 9.56-9.58; Kanehisa 1984 Nucl. Acids Res. 12: 203-213; and Wetmur et al. 1968 J. Mol. Biol. 31: 349-370. Appropriate stringent conditions that promote DNA hybridization are known to those skilled in the art.

[0105] In the present disclosure, the term "washing" or "elution" refers to an experimental technique for removing nucleic acid molecules or impurities other than the nucleic acid hybridization product connected to the solid phase support to obtain the nucleic acid hybridization product bound to the solid phase support.

[0106] In the present disclosure, the term "impurities" refers to some small molecular substances in the reaction system before elution, including but not limited to various blocking reagents, various ions in the buffer, unbound nucleic acid probes or nucleic acid samples, etc.

[0107] In the present disclosure, the term "indirect connection" means that the DNA probe is not directly connected to the solid phase carrier, but is indirectly connected to the solid phase carrier through other groups that specifically bind to the groups connected to the solid phase carrier.

[0108] In the present disclosure, the term "≥0.2× average sequencing depth" or "≥0.2× average depth" or "0.2x meandepth" refers to the proportion of sites with a sequencing depth exceeding 0.2 times the average sequencing depth, which characterizes the uniformity of sequencing coverage in the target region.

[0109] In the present disclosure, the term "≥0.5× average sequencing depth" or "≥0.5× average depth" or "0.5x meandepth" refers to the proportion of sites with a sequencing depth exceeding 0.5 times the average sequencing depth, which characterizes the uniformity of sequencing coverage in the target region.

[0110] In the present disclosure, the term "SSC" includes sodium citrate buffer.

[0111] In the present disclosure, the term "50× Denhandt's solution" contains 1% Ficoll 400, 1% polyvinylpyrrolidone (PVP) and 1% bovine serum albumin (BSA).

[0112] In the present disclosure, the term "EDTA" refers to ethylenediaminetetraacetic acid, a divalent metal ion chelator used to inhibit nuclease activity and prevent the degradation of nucleic acid molecules.

[0113] In the present disclosure, the term "dextran sulfate" can combine with water to reduce the effective volume of the hybridization solution and increase the possibility of collision and renaturation between the probe and the target, so as to achieve the purpose of increasing the hybridization rate.

[0114] In the present disclosure, the term "tetramethylammonium chloride (TMAC)" is used to reduce mismatching of DNA / DNA, RNA / DNA in hybridization reactions to improve specificity.

[0115] In this disclosure, the terms "around", "about" or "approximately" generally refer to within 20%, within 10%, within 5%, 4%, 3%, 2% or 1% of a given value or range. If not explicitly stated, the quantities given are approximate, meaning that the terms "around", "about" or "approximately" can be inferred.

[0116] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0117] The specific implementation method is described in detail below.

[0118] The nucleic acid hybridization capture experiment disclosed in the present invention is an experimental process in which a prepared nucleic acid library is placed in a composition containing a hybridization buffer and complementary bound to thousands or hundreds of thousands of probe-specific bases under certain reaction conditions. After a certain reaction time, a double-stranded nucleic acid molecule formed by complementary binding of nucleic acid probe-nucleic acid library fragments is obtained, and then the probe is directly or indirectly bound to a solid phase carrier. This binding requires certain incubation conditions, and then a cleaning solution is used to wash and elute unbound probes, unbound nucleic acid libraries and other impurities, thereby obtaining a pure hybridization product connected to the solid phase carrier.

[0119] In the present disclosure, low-concentration high-valent cations are used in the composition to replace traditional low-valent cations such as Na+ and K+, so as to accelerate the neutralization reaction and shorten the hybridization time to 1 hour.

[0120] In some embodiments, the trivalent cation is selected from cobalt trichloride hexaamine.

[0121] In some embodiments, the trivalent cation concentration is selected from ≤5 mM.

[0122] In some embodiments, the hybridization buffer comprises one or more of the following: ethylenediaminetetraacetic acid (EDTA), SSC buffer, hexaamminecobalt chloride, sodium dodecyl sulfate (SDS), betaine, tetramethylammonium chloride (TMAC), N-methylpyrrolidone, dextran sulfate, Denhandt's solution.

[0123] In some embodiments, the hybridization buffer comprises one or more of the following: 2 mM ethylenediaminetetraacetic acid (EDTA), 1× SSC buffer, 0.02% sodium dodecyl sulfate (SDS), 10% dextran sulfate, 5×

[0124] Denhandt's solution, 0-2M betaine, 0-1.5M tetramethylammonium chloride (TMAC), 10%-20% (v / v) N-methylpyrrolidone.

[0125] In some embodiments, the washing solution comprises 1×SSC buffer, 0.01%-0.1% w / v sodium dodecyl sulfate (SDS), and 0.01%-0.2% v / v Tween 20.

[0126] In some embodiments, the nucleic acid hybridization capture experiment further comprises amplifying the eluted hybridization products to obtain double-stranded nucleic acid products.

[0127] In some embodiments, the pure hybridization product is detected, and the detection includes any one or more of sequencing, PCR, qPCR, ddPCR, flight mass spectrometry processing, chip detection and methylation detection.

[0128] The present invention discloses for the first time the application of low-concentration trivalent cations in the hybridization buffer, which significantly shortens the hybridization time and achieves a good hybridization capture effect. The test results show that the capture efficiency is as high as 71.92%, and the average sequencing depth ≥0.2× is above 99%.

[0129] Examples are provided below to help understand the present disclosure. However, it should be understood that these examples are only used to illustrate the present disclosure and do not constitute any limitation. The actual protection scope of the present disclosure is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present disclosure.

[0130] Example

[0131] Example 1 Hybridization capture system test experiment

[0132] In this embodiment, a hybridization capture experiment is performed using the hybridization system disclosed in the present invention, which specifically includes hybridization of the probe with the target library, capture with streptavidin magnetic beads, thermal cleaning, room temperature cleaning, Post-PCR, on-machine sequencing, and bioinformatics analysis of off-machine data.

[0133] The specific process is as follows:

[0134] 1. Sample interruption

[0135] 1 μg of NA12878 human genome standard DNA was sheared using the Bioruptor Pico nucleic acid shearing instrument to shear the DNA into fragments of 100 bp-500 bp, with the main band ≈300 bp. The shearing reaction volume was 100 μL, and the shearing conditions were as follows: diluent: TE buffer; Cycle / burst: 30s On / 30s Off; number of cycles: 13.

[0136] 2. Disruption product purification

[0137] Take out the AMPure XP magnetic beads from the 4°C refrigerator 30 minutes in advance, equilibrate to room temperature, and shake thoroughly before use;

[0138] Pipette 60 μL AMPure XP magnetic beads into 100 μL disruption product, pipette gently 10 times to mix thoroughly, incubate at room temperature for 5 minutes, centrifuge instantly, place the centrifuge tube on a magnetic rack, let stand for 2 minutes until the liquid is completely clear, and pipette the supernatant into a new centrifuge tube;

[0139] Pipette 25 μL AMPure XP magnetic beads into 160 μL supernatant, pipette gently 10 times to mix thoroughly, incubate at room temperature for 5 minutes, centrifuge briefly, place the centrifuge tube on a magnetic rack, let stand for 2 minutes until the liquid is completely clear, pipette and discard the supernatant;

[0140] Keep the centrifuge tube on the magnetic stand, add 300 μL of freshly prepared 80% ethanol (v / v), let it stand at room temperature for 1 minute, then carefully discard the supernatant, taking care not to disturb the magnetic beads;

[0141] Repeat the above steps once and try to clean the liquid at the bottom of the centrifuge tube;

[0142] Keep the centrifuge tube lid open and dry at room temperature for 3 minutes;

[0143] Remove the centrifuge tube from the magnetic stand, add 22 μL TE buffer to elute the DNA, mix well with a pipette and dissolve at room temperature for 5 minutes;

[0144] Centrifuge briefly, place the centrifuge tube on a magnetic rack, let stand for 2 minutes until the liquid becomes clear, transfer 20 μL of the supernatant to a new centrifuge tube, and measure the concentration using a Qubit fluorometer.

[0145] 3. Library Construction

[0146] The library was constructed using the Yisheng Bio-universal DNA library construction kit (Cat No. 12201).

[0147] 3.1. End repair and dA tail addition

[0148] Take 50 ng of the sheared human genome fragment into a 0.2 mL PCR tube and prepare the reaction system according to Table 1.

[0149] Table 1. End repair / dA tailing PCR reaction system

[0150]

[0151] Use a pipette to gently pipette up and down to mix, centrifuge briefly, and then place the PCR tube on a PCR instrument to perform end repair and dA tail addition reactions according to the reaction program in Table 2.

[0152] Table 2. End repair / dA tailing PCR reaction program

[0153]

[0154] 3.2. Connector connection

[0155] In this step, the ends of the product from step 3.1 are connected to a specific MGI sequencing platform adapter. The reaction system is prepared according to Table 3.

[0156] Table 3. Adapter ligation PCR system

[0157]

[0158] Use a pipette to gently pipette up and down to mix, centrifuge briefly, place the PCR tube on a PCR instrument, and perform the adapter ligation reaction according to the reaction program in Table 4.

[0159] Table 4. Adapter ligation reaction procedure

[0160]

[0161] 3.3. Purification of ligation product

[0162] Ampure XP magnetic beads were used for purification, the amount of magnetic beads used was 0.6× (60 μL), and the resolubilization volume was 20 μL.

[0163] PCR amplification

[0164] The purified ligation product was added with 25 μL Kapa HiFi high-fidelity DNA polymerase and 5 μL primer with Index, with a total volume of 50 μL, and the following reaction was performed on a PCR instrument: 98°C for 45 s; (98°C for 15 s; 60°C for 30 s; 72°C for 30 s) 7 cycles; 72°C for 1 min, and stored at 4°C).

[0165] 3.5. Purification of amplified products

[0166] Ampure XP magnetic beads were used for purification, the amount of magnetic beads used was 0.9× (45 μL), and the resolubilization volume was 30 μL.

[0167] 4. Hybridization Capture

[0168] Using NanoDex hybrid capture reagent The above amplified and purified products were subjected to hybridization capture test using Hybrid Capture Reagents (Cat. No. 1005101) and GENE+244kb panel with a hybridization time of 1 hour and a capture time of 45 minutes.

[0169] 4.1 Library blocking and concentration

[0170] Agilent Connector Sequence Blocking Reagent Eco Universal Blocking Oligo (forMGIDI), Cat. No. C80532 and Human Genomic Repeat Sequence Blocking Reagent Human Cot-1 DNA TM (Manufacturer: Magper; Product No.: WS3101S) Take it out from -20℃, thaw it at room temperature, vortex it to mix it and centrifuge it, and put it on ice for later use. Take a new 1.5mL low-absorption centrifuge tube and add the components according to Table 5:

[0171] Table 5. Library blocking system

[0172]

[0173] Vortex to mix, centrifuge briefly, and concentrate using a vacuum concentrator (Eppendorf) at 60°C until dry.

[0174] 4.2 Hybridization of the blocked library with the probe

[0175] Place the reagents at room temperature to melt in advance. For the concentrated mixture, add other components according to Table 6:

[0176] Table 6. Hybridization system

[0177]

[0178] Vortex to mix, centrifuge briefly, and incubate at room temperature for 10 minutes. Transfer the liquid in the 1.5 mL centrifuge tube to a 0.2 mL PCR tube and centrifuge briefly to centrifuge the liquid to the bottom of the tube. Cover the tube tightly and place it in the PCR machine. Keep the PCR machine's 100°C hot lid open and run the program described in Table 7:

[0179] Table 7. Hybridization reaction procedures

[0180]

[0181] 4.3 Preparation of washing solution and streptavidin magnetic beads

[0182] use Hybrid Capture Reagents (Cat. #1005101) kit: Prepare 1× wash buffer (single reaction) according to Table 8:

[0183] Table 8. Cleaning solution preparation

[0184]

[0185] Preheat the cleaning solution using a constant temperature mixer according to the volume in Table 9 (single reaction), and preheat low-absorption 1.5 mL centrifuge tubes corresponding to the number of samples:

[0186] Table 9. Preheating volume of cleaning solution for a single reaction

[0187]

[0188] First, place Life Dynabeads TM M-270 Streptavidin magnetic beads (50 mg / mL) were taken out of the 4°C refrigerator and incubated at room temperature for at least 30 minutes. Vortex the magnetic beads for 15 seconds to completely suspend them. For each hybridization reaction, take 10 μL of Streptavidin magnetic beads in a 0.2 mL PCR tube. Add 100 μL of 1× magnetic bead washing buffer (Beads Washing Buffer), vortex for 30 seconds and centrifuge. Place the PCR tube on the magnetic stand again for 1 minute, until the solution is clear, remove the supernatant, and then wash the magnetic beads twice with 100 μL of 1× magnetic bead washing buffer (5 mM Tris-HCl (PH 7.5), 0.5 mM EDTA, 1 M NaCl, 0.1% Tween 20). After removing the supernatant, resuspend the washed streptavidin magnetic beads with magnetic bead resuspension solution. The preparation system of the magnetic bead resuspension solution is described in Table 10:

[0189] Table 10. Magnetic bead resuspension system

[0190]

[0191] Gently shake for 10 seconds to mix, and centrifuge instantly to obtain the magnetic bead mixture.

[0192] 4.4) Magnetic bead capture

[0193] Place the magnetic bead mixture on a pre-set PCR instrument (65℃, heated cover 75℃), preheat for 5 minutes, add the hybridization mixture, vortex to mix, centrifuge briefly, incubate at 65℃ for 45 minutes, and gently vortex every 10-12 minutes to ensure that the magnetic beads are completely resuspended.

[0194] 4.5) Elution

[0195] 4.5.1) Thermal elution

[0196] Add 100 μL of preheated cleaning solution 1 to the PCR tube, and then quickly transfer the liquid to a 1.5 mL low-absorption centrifuge tube preheated at 65°C. Place on a magnetic rack until the solution is clear, remove the supernatant immediately, return the EP tube to the constant temperature mixer, add 150 μL of preheated cleaning solution 2, incubate at 65°C for 5 minutes, and repeat this step again.

[0197] 4.5.2) Elution at room temperature

[0198] After hot elution, place the sample on a magnetic stand, discard the supernatant, add 150 μL of cleaning solution 1, place on a Vortex mixer with a disc (set to gear 6), shake and mix for 2 minutes, centrifuge briefly, place on a magnetic stand until the solution is clear, and remove the supernatant. Add 150 μL of cleaning solution 3, place on a Vortex mixer with a disc (set to gear 6), shake and mix for 2 minutes, centrifuge briefly, place on a magnetic stand until the solution is clear, and remove the supernatant. Add 150 μL of cleaning solution 4, place on a Vortex mixer with a disc (set to gear 6), shake and mix for 2 minutes, centrifuge briefly, place on a magnetic stand until the solution is clear, and remove the supernatant. Use 20 μL of nuclease-free water to re-dissolve, shake and mix, and centrifuge briefly.

[0199] 4.6) PCR amplification

[0200] The purified ligation product was added with 25 μL Kapa HiFi high-fidelity DNA polymerase and 5 μL MGI primers, with a total volume of 50 μL, and the following reaction was performed on a PCR instrument: 98°C for 45 s; 15 cycles of (98°C for 15 s; 60°C for 30 s; 72°C for 30 s); 72°C for 1 minute, and maintained at 4°C).

[0201] 4.7) Purification of amplified products

[0202] Ampure XP magnetic beads were used for purification. The amount of magnetic beads used was 1.2× (60 μL), the resolubilization volume was 22 μL, and the beads were placed on a magnetic stand until the solution was clear. 20 μL of the supernatant was pipetted into a new 1.5 mL EP tube.

[0203] 4.8) Library quantification and sequencing

[0204] The above library was quantitatively detected using Qubit and sent to the sequencing factory of Genenjia Medical Testing Laboratory for sequencing.

[0205] 4.9) Analysis of sequencing results

[0206] The sequencing result data show the hybridization reaction performance as shown in Table 11.

[0207] Table 11. Hybridization capture performance

[0208]

[0209] Example 2 Testing of hybridization systems containing high-valent cations

[0210] The hybridization capture experiment operation process of this embodiment is as follows:

[0211] The hybridization buffer and GENE+244kb panel of the present disclosure were used to perform a hybridization capture test on the purified product obtained from the library construction in step 3 of Example 1, with a hybridization time of 1 hour and a capture time of 45 minutes, wherein the magnetic bead washing solution and washing solutions 1 to 4 were replaced with the washing solutions prepared as shown in Table 15. The hybridization steps are as follows:

[0212] 4. Hybridization capture

[0213] 4.1 Library blocking and concentration

[0214] Combine the adapter sequence blocking reagent Universal Blocker with the human genome repeat sequence blocking reagent human Cot-1 DNA TM Take out from -20℃, thaw at room temperature, vortex to mix and centrifuge, and place on ice for later use. Take a new 1.5mL low-absorption centrifuge tube and add the components according to Table 12:

[0215] Table 12. Library blocking system

[0216]

[0217] Vortex to mix, centrifuge briefly, and concentrate using a vacuum concentrator (Eppendorf) at 60°C until dry.

[0218] 4.2 Hybridization of the blocked library with the probe

[0219] The reagents were placed at room temperature to melt in advance. For the concentrated mixture, other components were added according to Table 13:

[0220] Table 13. Hybridization system

[0221]

[0222] Vortex to mix, centrifuge briefly, and incubate at room temperature for 10 minutes. Transfer the liquid in the 1.5 mL centrifuge tube to a 0.2 mL PCR tube and centrifuge briefly to centrifuge the liquid to the bottom of the tube. Cover the tube tightly and place it in the PCR machine. Keep the PCR machine's 100°C hot lid open and run the program described in Table 14:

[0223] Table 14. Hybridization reaction program

[0224]

[0225] 4.3 Preparation of washing solution and streptavidin magnetic beads

[0226] Prepare 1× cleaning solution (single reaction) using the components according to Table 15:

[0227] Table 15. Cleaning solution preparation

[0228]

[0229] Preheat the cleaning solution using a constant temperature mixer according to the volume in Table 16 (single reaction), and preheat the corresponding number of low-absorption 1.5 mL centrifuge tubes:

[0230] Table 16. Preheating volume of cleaning solution for a single reaction

[0231]

[0232] First, place Dynabeads TM M-270 Streptavidin magnetic beads (50 mg / mL) were taken out of the 4°C refrigerator and incubated at room temperature for at least 30 minutes. Vortex the magnetic beads for 15 seconds to completely suspend them. For each hybridization reaction, take 10 μL of Streptavidin magnetic beads in a 0.2 mL PCR tube. Add 100 μL of 1× Beads Washing Buffer, vortex for 30 seconds and centrifuge. Place the PCR tube on the magnetic stand again for 1 minute, wait until the solution is clear, then aspirate the supernatant, wash the magnetic beads twice with 100 μL of 1× Beads Washing Buffer, aspirate the supernatant, and resuspend the washed streptavidin magnetic beads with magnetic bead resuspension solution. The preparation system of the magnetic bead resuspension solution is described in Table 17:

[0233] Table 17. Magnetic bead resuspension system

[0234]

[0235] Gently shake for 10 seconds to mix, and centrifuge instantly to obtain the magnetic bead mixture.

[0236] 4.4. Magnetic bead capture

[0237] Place the magnetic bead mixture on a pre-set PCR instrument (65℃, heated cover 75℃), preheat for 5 minutes, add the hybridization mixture, vortex to mix, centrifuge briefly, incubate at 65℃ for 45 minutes, and gently vortex every 10-12 minutes to ensure that the magnetic beads are completely resuspended.

[0238] 4.5. Elution

[0239] 4.5.1. Thermal elution

[0240] Add 100 μL of preheated cleaning solution 1 to the PCR tube, and then quickly transfer the liquid to a 1.5 mL low-absorption centrifuge tube preheated at 65°C. Place on a magnetic rack until the solution is clear, remove the supernatant immediately, return the EP tube to the constant temperature mixer, add 150 μL of preheated cleaning solution 2, incubate at 65°C for 5 minutes, and repeat this step again.

[0241] 4.5.2. Elution at room temperature

[0242] After hot elution, place the sample on a magnetic stand, discard the supernatant, add 150 μL of cleaning solution 1, place on a Vortex mixer with a disc (set to gear 6), shake and mix for 2 minutes, centrifuge briefly, place on a magnetic stand until the solution is clear, and remove the supernatant. Add 150 μL of cleaning solution 3, place on a Vortex mixer with a disc (set to gear 6), shake and mix for 2 minutes, centrifuge briefly, place on a magnetic stand until the solution is clear, and remove the supernatant. Add 150 μL of cleaning solution 4, place on a Vortex mixer with a disc (set to gear 6), shake and mix for 2 minutes, centrifuge briefly, place on a magnetic stand until the solution is clear, and remove the supernatant. Use 20 μL of nuclease-free water to re-dissolve, shake and mix, and centrifuge briefly.

[0243] PCR amplification

[0244] The purified ligation product was added with 25 μL Kapa HiFi high-fidelity DNA polymerase and 5 μL MGI primers, with a total volume of 50 μL, and the following reaction was performed on a PCR instrument: 98°C for 45 s; 15 cycles of (98°C for 15 s; 60°C for 30 s; 72°C for 30 s); 72°C for 1 minute, and maintained at 4°C).

[0245] 4.7. Purification of amplified products

[0246] Ampure XP magnetic beads were used for purification. The amount of magnetic beads used was 1.2× (60 μL), the resolubilization volume was 22 μL, and the beads were placed on a magnetic stand until the solution was clear. 20 μL of the supernatant was pipetted into a new 1.5 mL EP tube.

[0247] 4.8. Library quantification and sequencing

[0248] The above library was quantitatively detected using Qubit and sent to the sequencing factory of Genenjia Medical Testing Laboratory for sequencing.

[0249] Other reagents were the same as those in Example 1, except that different concentrations of monovalent, divalent or trivalent cations were used in the hybridization buffer for testing, the buffer was 1×SSC buffer, and the pH of the hybridization buffer was 8.0. The specific formula is shown in Table 18:

[0250] Table 18. Cationic salts and their concentration ratios in different hybridization buffer formulations

[0251]

[0252]

[0253] Note: Product name: 20XSSC Brand: Invitrogen, Catalog Number: 15557044;

[0254] Trade name: Magnesium chloride, hexahydrate Brand: Aladdin, Item number: M116331-500g;

[0255] Trade name: Hexaamminecobalt chloride, 99% Brand: McLean, Item number: H831444-1g;

[0256] Trade name: N-methylpyrrolidone, brand: Shenggong, item number: A500626-0100;

[0257] Trade name: Betaine, Brand: Sigma, Product number: B0300-5VL;

[0258] Trade name: Tetramethylammonium chloride, Brand: Shenggong, Item No.: A600935-0500;

[0259] Product name: Triton, brand: McLean, item number: T824275.

[0260] The capture efficiency and uniformity of this test are shown in Table 19. Formulas 2 and 3 use high concentrations of high-valent cations or their chelates, and the capture efficiency and uniformity are poor. The other hybridization schemes use lower concentrations of high-valent ions or their chelates. Formula 6 uses 1mM Co 3+ The capture efficiency reached 71.92%, which is much higher than the capture efficiency in Example 1.

[0261] Table 19. Hybridization performance of different cations

[0262] formula Cation Type Capture efficiency 0.2×average depth 0.5×average depth Recipe 1 165mM Na+ 45.10％ 99.08％ 96.92％ Recipe 2 150mM Mg2+ 18.54％ 91.60％ 76.81％ Recipe 3 15mM Co3+ 50.05％ 98.98％ 95.85％ Recipe 4 5mM Co3+ 61.27％ 98.96％ 96.50％ Recipe 5 2mM Co3+ 66.96％ 99.01％ 95.77％ Recipe 6 1mM Co3+ 71.92％ 99.00％ 97.43％

[0263] Example 3 Hybridization capture test experiment using different cleaning solutions

[0264] The experimental operation process of this embodiment is the same as that of Embodiment 2, wherein the cleaning solutions prepared in Embodiment 2 are adjusted from 5 to 1, and the capture time is adjusted from 45 minutes to 15 minutes.

[0265] The hybridization capture test of the aforementioned library was performed using the system of the present invention and the GENE+244kb panel with a hybridization time of 1 hour and a capture time of 15 minutes. The hybridization steps included:

[0266] 4. Hybridization capture

[0267] 4.1 Library blocking and concentration

[0268] Combine the adapter sequence blocking reagent Universal Blocker with the human genome repeat sequence blocking reagent human Cot-1 DNATM Take out from -20℃, thaw at room temperature, vortex to mix and centrifuge, and place on ice for later use. Take a new 1.5mL low-absorption centrifuge tube and add the components according to Table 20:

[0269] Table 20. Library blocking system

[0270]

[0271] Vortex to mix, centrifuge briefly, and concentrate using a vacuum concentrator (Eppendorf) at 60°C until dry.

[0272] 4.2 Hybridization of the blocked library with the probe

[0273] Place the reagents at room temperature to melt in advance. For the concentrated mixture, add other components according to Table 21:

[0274] Table 21. Hybridization system

[0275]

[0276] Vortex to mix, centrifuge briefly, and incubate at room temperature for 10 minutes. Transfer the liquid in the 1.5 mL centrifuge tube to a 0.2 mL PCR tube and centrifuge briefly to centrifuge the liquid to the bottom of the tube. Cover the tube tightly and place it in the PCR machine. Keep the PCR machine's 100°C hot lid open and run the program described in Table 22:

[0277] Table 22. Hybridization reaction program

[0278]

[0279] 4.3 Preparation of washing solution and streptavidin magnetic beads

[0280] Prepare 1× Cleaning Solution KS (single reaction) according to Table 23:

[0281] Table 23. Cleaning solution preparation

[0282]

[0283] Preheat the cleaning solution KS using a constant temperature mixer according to the volume in Table 24 (single reaction), and preheat the corresponding number of low-absorption 1.5 mL centrifuge tubes:

[0284] Table 24. Preheating volume of single reaction wash solution

[0285]

[0286] First, place Dynabeads TMTake out the M-270 streptavidin magnetic beads (50 mg / mL) from the 4°C refrigerator and incubate at room temperature for at least 30 minutes. Vortex the magnetic beads for 15 seconds to completely suspend them. For each hybridization reaction, take 10μL of Streptavidin magnetic beads in a 0.2mL PCR tube. Add 100μL 1× cleaning solution KS, vortex for 30 seconds and centrifuge. Place the PCR tube on the magnetic stand again for 1 minute, wait until the solution is clear, then aspirate the supernatant, wash the magnetic beads twice with 100μL 1× cleaning solution KS, aspirate the supernatant, and resuspend the washed streptavidin magnetic beads with magnetic bead resuspension solution. The preparation system of the magnetic bead resuspension solution is described in Table 25:

[0287] Table 25. Magnetic bead resuspension system

[0288]

[0289] Gently shake for 10 seconds to mix, and centrifuge instantly to obtain the magnetic bead mixture.

[0290] 4.4 Magnetic Bead Capture

[0291] Place the magnetic bead mixture on a pre-set PCR instrument (65°C, heated cover 75°C), preheat for 5 minutes, add the hybridization mixture, vortex to mix, centrifuge briefly, incubate at 65°C for 15 minutes, and gently vortex every 7.5 minutes to ensure that the magnetic beads are completely resuspended.

[0292] 4.5 Elution

[0293] 4.5.1 Thermal elution

[0294] Add 100 μL of preheated 1× cleaning solution KS to the PCR tube, and then quickly transfer the liquid to a 1.5 mL low-absorption centrifuge tube preheated at 65°C. Place on a magnetic rack until the solution is clear, remove the supernatant immediately, return the EP tube to the constant temperature mixer, add 150 μL of preheated 1× cleaning solution KS, incubate at 65°C for 5 minutes, and repeat this step again.

[0295] 4.5.2 Elution at room temperature

[0296] After hot elution, place the sample on a magnetic stand, discard the supernatant, add 150μL 1× cleaning solution KS, place it on a Vortex mixer with a disc (set to gear 6), shake and mix for 2 minutes, centrifuge briefly, place it on a magnetic stand until the solution is clear, and remove the supernatant. Add 150μL 1× cleaning solution KS, place it on a Vortex mixer with a disc (set to gear 6), shake and mix for 2 minutes, centrifuge briefly, place it on a magnetic stand until the solution is clear, and remove the supernatant. Add 150μL 1× cleaning solution KS, place it on a Vortexmixer with a disc (set to gear 6), shake and mix for 2 minutes, centrifuge briefly, place it on a magnetic stand until the solution is clear, and remove the supernatant. Use 20μL nuclease-free water to re-dissolve, shake and mix, and centrifuge briefly.

[0297] 4.6 PCR amplification

[0298] The purified ligation product was added with 25 μL Kapa HiFi high-fidelity DNA polymerase and 5 μL MGI primers, with a total volume of 50 μL, and the following reaction was performed on a PCR instrument: 98°C for 45 s; 15 cycles of (98°C for 15 s; 60°C for 30 s; 72°C for 30 s); 72°C for 1 minute, and maintained at 4°C).

[0299] 4.7 Purification of amplified products

[0300] Ampure XP magnetic beads were used for purification. The amount of magnetic beads used was 1.2× (60 μL), the resolubilization volume was 22 μL, and the beads were placed on a magnetic stand until the solution was clear. 20 μL of the supernatant was pipetted into a new 1.5 mL EP tube.

[0301] 4.8 Library Quantification and Sequencing

[0302] The above library was quantitatively detected using Qubit and sent to the sequencing factory of Genenjia Medical Testing Laboratory for sequencing.

[0303] In this example, in order to simplify the cleaning solution, the five cleaning solutions (control) of 1× cleaning solution 1, 2, 3, 4 and magnetic bead cleaning solution prepared in Example 2 were all replaced with 1×KS cleaning solution, and the hybridization capture test was performed using the hybridization buffer described in Example 2, with two replicates per group. The composition ratios of different cleaning solutions are shown in Table 26.

[0304] Table 26. Ratios of different cleaning solution components

[0305]

[0306] Note: Product name: 20X SSC Brand: Invitrogen Catalog number: 15557044;

[0307] Product Name: SDS Brand: Biotech Item No.: A500228-0250;

[0308] Product name: Tween 20 Brand: sigma Item number: P9416-50ML;

[0309] Trade Name: μLtraPure 1M Tris-HCl Buffer Brand: Invitrogen Catalog Number: 15567027L;

[0310] Trade Name: EDTA (0.5M) Brand: Invitrogen Catalog Number: AM9260G;

[0311] Trade Name: NaCl Brand: Invitrogen Catalog Number: AM9760G.

[0312] Sequencing results analysis:

[0313] The capture efficiency and uniformity of this test are shown in Table 27.

[0314] Table 27. Single eluent performance

[0315]

[0316] Compared with the control, the hybridization capture efficiency and uniformity of the single washing solution KS are highly consistent, proving that all five washing solutions, namely washing solutions 1, 2, 3, 4, and magnetic bead washing solution, can be replaced with 1×KS washing solution, thereby simplifying the elution reagents.

[0317] The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments, and any technical deformation made according to the technical solution of the present disclosure falls within the protection scope of the present disclosure. The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments, and any technical deformation made according to the technical solution of the present disclosure falls within the protection scope of the present disclosure.

Claims

1. A composition for nucleic acid hybridization, characterized in that The composition includes 1.0-5.0 mM [Co(NH3)6] 3+ , sodium citrate buffer with a pH of 6.0 to 9.0, 10% to 20% v / v N-methylpyrrolidone, 0.5M to 5M betaine, 0.5M to 1.5M tetramethylammonium chloride and 0.005% v / v to 1% v / v Triton X-100.

2. The composition according to claim 1, characterized in that The pH of the sodium citrate buffer is 7.5-8.

5.

3. A method for nucleic acid hybridization, comprising performing nucleic acid hybridization using the composition according to claim 1 or 2 to obtain a hybridization product.

4. The method according to claim 3, characterized in that The method comprises the following steps: 1) in the composition, mixing a nucleic acid sample and a nucleic acid probe to form a mixture; 2) maintaining the mixture at a first temperature for a first period of time to denature the mixture; and 3) lowering the temperature to a second temperature and maintaining it for a second period of time to allow the nucleic acid probe to hybridize with the nucleic acid fragment in the nucleic acid sample to obtain a hybridization product.

5. The method according to claim 4, characterized in that The first temperature is 94-98°C.

6. The method according to claim 4, characterized in that The first time period is 10 seconds to 15 minutes.

7. The method according to claim 4, characterized in that The second temperature is 55-75°C.

8. The method according to claim 4, characterized in that The second time period is 30 minutes to 2 hours.

9. The method according to any one of claims 3 to 8, characterized in that: The method further comprises: directly or indirectly connecting the hybridization product to a solid phase carrier, and washing it with a washing solution.

10. The method according to claim 9, characterized in that: The cleaning solution comprises 0.1-1.0× sodium citrate buffer, 0.005-0.02% v / v sodium dodecyl sulfate and 0.005-0.02% Tween 20.

11. A kit comprising the composition according to claim 1 or 2.

12. Use of the composition according to claim 1 or 2 or the kit according to claim 11 in nucleic acid hybridization.

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