Long-fragment DNA capture method, kit, and use thereof

Abstract

A long-fragment DNA capture method, a kit, and use thereof.

Description

A long-fragment DNA capture method, kit and use thereof

[0001] Cross-references

[0002] This application refers to Chinese patent application No. 202311070666.X, filed on August 23, 2023, entitled “A method, kit and use thereof for capturing long-fragment DNA”, which is incorporated into this application in its entirety by reference. Technical Field

[0003] The present application relates to the field of molecular biology, and specifically to a method for capturing long-fragment DNA, a kit, and uses thereof. Background Art

[0004] Liquid-phase hybridization capture technology is widely used in next-generation sequencing. A single-stranded DNA or RNA probe is designed for the target gene of interest. This probe is labeled with biotin and hybridized with a pre-constructed whole-genome library to form DNA-DNA or RNA-DNA hybrid products. The hybrid products are then captured by streptavidin-labeled magnetic beads. After eluting nonspecifically bound library fragments, PCR amplification is performed to obtain a library containing the target region. Finally, the resulting library is sequenced to analyze whether the target gene has mutations.

[0005] Currently, commercially available liquid phase hybridization capture kits are mainly targeted at second-generation sequencing applications. The captured library mainly contains short DNA fragments (e.g., DNA of 200bp-2000bp in size), which makes it difficult to capture and amplify longer DNA fragments.

[0006] With the development of technology, third-generation sequencing based on single-molecule sequencing technology and fourth-generation sequencing based on nanopore sequencing technology have the characteristics of ultra-long read length and high throughput, which can directly detect long DNA fragments. Therefore, new methods for capturing long DNA fragments are needed in this field.

[0007] It should be noted that the approaches described in this section are not necessarily approaches that have been previously conceived or employed. Unless otherwise indicated, it should not be assumed that any approach described in this section is prior art simply because it is included in this section. Similarly, unless otherwise indicated, the issues mentioned in this section should not be considered to have been recognized in any prior art.

[0008] Summary of the Invention

[0009] Based on this, in order to obtain long-fragment DNA for sequencing, the present application provides a long-fragment DNA capture method, which uses RNAse to treat long-fragment DNA hybridized with RNA probes, wherein the length of the long-fragment DNA is not less than 2kb, and preferably, the length of the long-fragment DNA is 4kb-10kb. The method in the present application is improved on the basis of the existing liquid-phase hybridization capture method. By adding RNAse for digestion, longer DNA fragments can be obtained, which are suitable for target gene sequencing analysis on a third-generation or fourth-generation sequencing platform. Compared with the short-fragment DNA obtained by the existing method, the long-fragment DNA obtained by the method in the present application can fully retain the information of the original genome, which is more conducive to the detection of more complex fusion gene types.

[0010] In some embodiments, a heat denaturation step is also included after RNAse digestion. The method of the present application further includes a heat denaturation step based on RNAse digestion. The two processes work together to more effectively dissociate long DNA fragments from the capture agent, resulting in higher long DNA enrichment efficiency.

[0011] According to one embodiment of the present application, a kit for implementing the method described in the present application may be provided.

[0012] According to one embodiment of the present application, a kit for capturing long-fragment DNA can be provided, characterized in that the kit includes: RNA enzyme, RNA probe, and / or capture agent, wherein the length of the long-fragment DNA is not less than 2kb, preferably, the length of the long-fragment DNA is 4kb-10kb.

[0013] According to one embodiment of the present application, a long-fragment DNA obtained according to the method described in the present application, or the use of the kit described in the present application in gene extraction, gene analysis, and gene sequencing can be provided. Preferably, the gene sequencing includes third-generation sequencing and fourth-generation sequencing.

[0014] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present application, nor is it intended to limit the scope of the present application. Other features of the present application will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings illustrate exemplary embodiments and constitute a part of the specification. Together with the description of the specification, they serve to explain exemplary implementation of the embodiments. The illustrated embodiments are for illustrative purposes only and do not limit the scope of the claims. Throughout the drawings, the same reference numerals designate similar, but not necessarily identical, elements.

[0016] FIG1 shows the fragmented DNA map of the human genome standard NA12878 in Example 1.1.

[0017] FIG2 shows the DNA map after DNA sorting in Example 1.2.

[0018] FIG3 shows the map of the 4-9 kb long fragment DNA whole genome library in Example 1.4.

[0019] FIG4 shows the library map of the 4-9 kb long-fragment DNA whole genome library after RNA probe hybridization, capture, elution, and PCR amplification in Example 2.

[0020] FIG5 shows the library map of the 4-9 kb long-fragment DNA whole genome library after RNA probe hybridization, capture, elution, heat denaturation, and PCR amplification in Example 3.

[0021] FIG6 shows the library map of the 4-9 kb long-fragment DNA whole genome library after RNA probe hybridization, capture, elution, RNase H digestion, and PCR amplification in Example 4.

[0022] FIG7 shows the library map of the 4-9 kb long-fragment DNA whole genome library after RNA probe hybridization, capture, elution, RNase H digestion, heat denaturation, and PCR amplification in Example 5.

[0023] FIG8 shows the fragmented DNA map of the human genome standard NA12878 in Example 6.1.

[0024] FIG9 shows the DNA map after DNA sorting in Example 6.2.

[0025] FIG10 shows the map of the 6-10 kb long fragment DNA whole genome library in Example 6.4.

[0026] Figure 11 shows the maps of the 6-10 kb long-fragment DNA libraries in Examples 7-10, wherein lane C1 is the map of the library after RNA probe hybridization, capture, elution, and PCR amplification in Example 7; lane D1 is the map of the library after RNA probe hybridization, capture, elution, heat denaturation, and PCR amplification in Example 8; lane E1 is the map of the library after RNA probe hybridization, capture, elution, RNase H digestion, and PCR amplification in Example 9; and lane F1 is the map of the library after RNA probe hybridization, capture, elution, RNase H digestion, heat denaturation, and PCR amplification in Example 10.

[0027] FIG12 shows the library map of the 4-9 kb long-fragment DNA whole genome library in Example 11 after hybridization with an RNA probe (T040V19), capture, elution, and PCR amplification.

[0028] Figure 13 shows the library map of the 4-9 kb long-fragment DNA whole-genome library in Example 12 after RNA probe (T040V19) hybridization, capture, elution, RNase H digestion, heat denaturation, and PCR amplification. DETAILED DESCRIPTION

[0029] Unless otherwise indicated, the terms used herein have the usual meanings understood by those skilled in the art. Unless otherwise indicated, all numbers used in this specification and claims to represent content, concentration, ratio, weight, particle size, percentage, technical effect, etc. should be understood as being modified by the term "about" or "approximately" under any circumstances. Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the appended claims are approximate values. For those skilled in the art, it can vary according to the desired properties and effects sought to be obtained through this application, and each numerical parameter should be interpreted according to the number of significant digits and conventional rounding methods or in a manner understood by those skilled in the art.

[0030] Although the numerical ranges and parameters setting forth the broad scope of this application are approximations, the numerical values ​​set forth in the specific examples are provided as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in its respective testing measurements. Every numerical range given herein will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were expressly written herein.

[0031] The "RNase" described in this application refers to an enzyme that can cut or degrade ribonucleic acid (RNA). Preferably, the RNase is RNase H (endoribonuclease H) that acts on the RNA portion of a double-stranded nucleic acid composed of DNA and RNA. Any ribonuclease with the above-mentioned activity can be preferably used in this application. RNase H can be derived from various viruses, bacteriophages, prokaryotes, or eukaryotes, for example, prokaryotic RNase H is exemplified by Escherichia coli RNase HI, and viral RNase H is exemplified by HIV-1. In some embodiments, heat-resistant ribonucleases can be used, examples of which include but are not limited to RNase H from thermophilic bacteria of the genus Bacillus, bacteria of the genus Thermophilus, bacteria of the genus Pyrococcus, bacteria of the genus Thermophilus, bacteria of the genus Archaea, and the like.

[0032] As used herein, a "probe" refers to a single-stranded nucleic acid having a known nucleotide sequence, including but not limited to DNA, RNA, derivatives thereof, or combinations thereof. A probe typically has a nucleotide sequence structure that is partially or completely complementary to the bases of a target nucleic acid and can hybridize with the target nucleic acid to form a double strand. In this application, an RNA probe is used to capture and enrich a target nucleic acid sequence, including a DNA sequence, an RNA sequence, or any other nucleic acid sequence that can form a hybrid molecule with an RNA probe.

[0033] As used herein, "target gene," "target nucleic acid," or "target molecule" are used interchangeably to refer to any nucleic acid of interest, which typically contains a target nucleic acid sequence to be identified. The target nucleic acid can be single-stranded or double-stranded and is typically DNA, RNA, a derivative thereof, or a combination thereof.

[0034] As used herein, "hybridization" refers to the process by which a nucleic acid strand binds to a complementary strand through base pairing as known in the art. In some embodiments, hybridization refers to the process by which a probe binds to a target nucleic acid. The hybridization process is affected by a variety of factors, such as the degree of complementarity between the nucleic acids, the stringency of the hybridization conditions used, the melting temperature (Tm) of the hybrid formed, and the GC content of the nucleic acids. This application is not limited to specific hybridization conditions.

[0035] As used herein, "amplification" refers to the use of a target nucleic acid as a template to generate one or more copies of the target nucleic acid. In some embodiments, amplification can be performed using polymerase chain reaction (PCR).

[0036] As used herein, "affinity tags" and "capture agents" can be covalently or non-covalently bound to each other. Examples of non-covalently bound affinity tags and capture agents include, but are not limited to, biotin / streptavidin, digoxigenin / anti-digoxigenin antibody, and fluorescein / anti-fluorescein antibody. Covalently bound affinity tags and capture agents typically include chemoselective reactive groups, examples of which include, but are not limited to, amines and active esters, as well as groups capable of reacting with each other via click chemistry, such as azide and alkyne groups.

[0037] As used herein, "sample" refers to a sample or culture obtained from any source, preferably a biological source, including but not limited to liquid, solid, or gas. Samples obtained from biological sources can be obtained from animals (including humans), such as cells, tissues, blood, etc.

[0038] As used herein, "nucleic acid sample" includes nucleic acids obtained from any source, preferably from a biological source, including but not limited to DNA, RNA, cDNA, mRNA, tRNA, and miRNA. Nucleic acid samples obtained from biological sources can preferably be obtained from animals (including humans), such as cells, tissues, and blood. Samples containing nucleic acids from any source can be used in this application.

[0039] method

[0040] According to one embodiment of the present application, a long-fragment DNA capture method may be provided, which uses RNase to treat the long-fragment DNA hybridized with an RNA probe.

[0041] In some embodiments, the method comprises the following steps: hybridizing the long fragment DNA library with the RNA probe, and then adding RNase for digestion.

[0042] In some embodiments, the method comprises the following steps: hybridizing the long-fragment DNA library with an RNA probe; capturing the long-fragment DNA hybridized with the RNA probe; and digesting the long-fragment DNA hybridized with the RNA probe using RNase.

[0043] In some embodiments, the length of the long DNA fragment is not less than 2 kb. In some embodiments, the length of the long DNA fragment can be any suitable length not less than 2 kb, such as 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, or a value in a range consisting of any two of the above points, or any suitable length greater than 10 kb. In some preferred embodiments, the length of the long DNA fragment is 4 kb-10 kb. In some preferred embodiments, the length of the long DNA fragment can be 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, or a value in a range consisting of any two of the above points.

[0044] The RNase is any suitable RNase, preferably an enzyme that can specifically degrade RNA in an RNA-DNA hybrid, such as an enzyme that can specifically degrade an RNA probe hybridized to DNA. In some embodiments, the RNase includes RNase A and / or RNase H. In some preferred embodiments, the RNase is RNase H.

[0045] The amount of RNA enzyme used is any suitable amount that can work and can be adjusted according to the instructions of different commercial enzymes. In some embodiments, the amount of the RNA enzyme used is not less than 1U. In some embodiments, the amount of the RNA enzyme used can be any suitable amount not less than 1U, such as 1U, 2U, 3U, 4U, 5U, 6U, 7U, 8U, 9U, or a value in a range consisting of any two of the above points, or any suitable amount greater than 9U. In some preferred embodiments, the amount of the RNA enzyme used is not less than 5U. In some preferred embodiments, the amount of the RNA enzyme used can be 5U, 6U, 7U, 8U, 9U, or a value in a range consisting of any two of the above points, or any suitable amount greater than 9U.

[0046] The temperature of the RNA enzyme treatment is any temperature at which the RNA enzyme is active, which depends on the type of RNA enzyme used and can be adjusted according to the instructions of different commercial enzymes. In some embodiments, the RNA enzyme used is a thermostable RNase H, which can also function at high temperatures, and the temperature of the RNA enzyme treatment can be any temperature. In some embodiments, the temperature of the RNA enzyme treatment is not higher than 65°C. In some embodiments, the temperature of the RNA enzyme treatment can be any suitable temperature not higher than 65°C, such as 5°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 65°C, or a value in the range consisting of any two of the above points. In some preferred embodiments, the temperature of the RNA enzyme treatment is 37°C.

[0047] The time for RNAse treatment is any suitable time that can play a role and can be adjusted according to the instructions of different commercial enzymes. In some embodiments, the time for RNAse treatment is not less than 20 minutes. In some embodiments, the time for RNAse treatment can be any suitable time not less than 20 minutes, such as 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, or a value within the range consisting of any two of the above points, or any suitable time greater than 60 minutes. In some preferred embodiments, the time for RNAse treatment is 20 minutes.

[0048] The RNA probe has a nucleic acid sequence complementary to the target gene so that it can hybridize with the target gene through complementary base pairing. The RNA probe and the target gene do not need to be perfectly complementary to hybridize with each other. In some embodiments, the target gene is any gene of interest. In some embodiments, the target gene is one or more. In some preferred embodiments, the target genes include BRCA1 (BRCA1 DNA repair associated, NCBI Gene ID: 672), BRCA2 (BRCA2 DNA repair associated, NCBI Gene ID: 675), STK11 (serine / threonine kinase 11, NCBI Gene ID: 6794), BARD1 (BRCA1 associated RING domain 1, NCBI Gene ID: 580), RAD50 (RAD50 double strand break repair protein, NCBI Gene ID: 10111), ATM (ATM serine / threonine kinase, NCBI Gene ID: 472), CDH1 (cadherin 1, NCBI Gene ID: 999), TP53 (tumor protein p53, NCBI Gene ID: 7157), PTEN (phosphatase and tensin homolog, NCBI Gene ID: 5728), PALB2 (partner and localizer of BRCA2, NCBI Gene ID: 79728), CHEK2 (checkpoint kinase 2, NCBI Gene ID: 11200).

[0049] The RNA probe can be modified to facilitate binding to a capture agent to capture long fragments of DNA that hybridize with the RNA probe. In some embodiments, the RNA probe is labeled with an affinity tag. Examples of suitable affinity tags and capture agents include, but are not limited to, biotin / streptavidin, digoxigenin / anti-digoxigenin antibodies, and fluorescein / anti-fluorescein antibodies. In some preferred embodiments, the affinity tag is biotin. In some preferred embodiments, the capture agent is a magnetic bead with streptavidin.

[0050] In some embodiments, after RNAse digestion, a step of heat denaturation is also included. It is well known in the art that the temperature range for denaturation of double-stranded DNA is 80-100°C, so this temperature range is selected for heat denaturation. In some embodiments, the temperature of heat denaturation is 80-100°C. In some embodiments, the temperature of heat denaturation can be 80°C, 85°C, 90°C, 95°C, 98°C, 99°C, 100°C, or a value in the range consisting of any two of the above points. In some preferred embodiments, the temperature of heat denaturation is 95°C. It is well known in the art that in PCR amplification, the commonly used denaturation conditions are 95°C and more than 30s, so this condition is used for heat denaturation. In some embodiments, the time of heat denaturation is not less than 30 seconds. In some embodiments, the thermal denaturation time can be any suitable time of not less than 30 seconds, for example 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, or a value in a range consisting of any two of the above points, or any suitable time greater than 6 minutes. In some preferred embodiments, the thermal denaturation time is 5 minutes. In some embodiments, the thermal denaturation is followed by an instantaneous cooling treatment. The instantaneous cooling treatment can be performed in any suitable manner. In some preferred embodiments, the instantaneous cooling method is to place the DNA on ice. The instantaneous cooling treatment can promptly maintain the single-stranded state after thermal denaturation in the denatured single-stranded state, effectively preventing the denatured single-stranded DNA from renaturing and annealing to a double-stranded state.

[0051] In some embodiments, before hybridizing the long-fragment DNA with the RNA probe, a step of preparing a long-fragment DNA library is further included. In some embodiments, the step of preparing the long-fragment DNA library includes: fragmenting the DNA sample to produce a fragmented DNA sample; sorting the fragmented DNA sample to produce a long-fragment DNA sample; performing end repair and adapter ligation on the long-fragment DNA sample to produce a long-fragment DNA library; and optionally, amplifying the long-fragment DNA library.

[0052] Wherein, described DNA sample can be prepared by any suitable source, preferably sample or culture obtained from biological source. In some embodiments, described DNA sample is from animal, plant or microorganism. In some preferred embodiments, described DNA sample is human genomic DNA. If the amount of DNA sample is less, described nucleic acid can also be increased before implementing the method for the application. Wherein, amplification can be carried out in any suitable manner. In some embodiments, the method for described amplification comprises polymerase chain reaction (PCR). The method for amplifying DNA by PCR is well known in the art.

[0053] In some embodiments, before using RNAse digestion, optionally further comprising the step of adding an eluent to rinse. In some embodiments, the rinse can be one or more times to remove uncaptured DNA fragments. Suitable eluents are well known in the art.

[0054] In some embodiments, after using RNA enzyme digestion, the step of amplifying the long fragment DNA is optionally included. If the amount of DNA obtained after RNA enzyme digestion is less, the nucleic acid can also be amplified before implementing the method of the present application. Wherein, amplification can be carried out in any suitable manner. In some embodiments, the method of amplification includes polymerase chain reaction (PCR). Methods for amplifying DNA by PCR are well known in the art.

[0055] Reagent test kit

[0056] According to one embodiment of the present application, a kit for implementing the method described in the present application may be provided.

[0057] According to one embodiment of the present application, a kit for capturing long-fragment DNA can be provided, characterized in that the kit includes: RNA enzyme, RNA probe, and / or capture agent, wherein the length of the long-fragment DNA is not less than 2kb, preferably, the length of the long-fragment DNA is 4kb-10kb.

[0058] The RNase is any suitable RNase, preferably an enzyme that can specifically degrade RNA in an RNA-DNA hybrid, such as an enzyme that can specifically degrade an RNA probe hybridized to DNA. In some embodiments, the RNase includes RNase A and / or RNase H. In some preferred embodiments, the RNase is RNase H.

[0059] The RNA probe has a nucleic acid sequence complementary to the target gene so that it can hybridize with the target gene through base complementary pairing. The RNA probe and the target gene do not need to be perfectly complementary to hybridize with each other. In some embodiments, the target gene is any gene of interest. In some embodiments, the target gene is one or more. In some preferred embodiments, the target genes include BRCA1 (BRCA1 DNA repair associated, NCBI Gene ID: 672), BRCA2 (BRCA2 DNA repair associated, NCBI Gene ID: 675), STK11 (serine / threonine kinase 11, NCBI Gene ID: 6794), BARD1 (BRCA1 associated RING domain 1, NCBI Gene ID: 580), RAD50 (RAD50 double strand break repair protein, NCBI Gene ID: 10111), ATM (ATM serine / threonine kinase, NCBI Gene ID: 472), CDH1 (cadherin 1, NCBI Gene ID: 999), TP53 (tumor protein p53, NCBI Gene ID: 7157), PTEN (phosphatase and tensin homolog, NCBI Gene ID: 5728), PALB2 (partner and localizer of BRCA2, NCBI Gene ID: 79728), CHEK2 (checkpoint kinase 2, NCBI Gene ID: 11200).

[0060] The RNA probe may be modified to facilitate binding to a capture agent to capture long fragments of DNA that hybridize with the RNA probe. In some embodiments, the RNA probe is affinity-tagged. In some embodiments, the capture agent is a magnetic bead. In some preferred embodiments, the magnetic bead is affinity-tagged. Examples of suitable affinity tag and capture agent pairings include, but are not limited to, biotin / streptavidin, digoxigenin / anti-digoxigenin antibody, and fluorescein / anti-fluorescein antibody. In some preferred embodiments, the RNA probe is biotin-tagged. In some preferred embodiments, the capture agent is a magnetic bead with streptavidin.

[0061] In some embodiments, the kit may further include an eluent. In some embodiments, the eluent may be one or more to remove uncaptured DNA fragments. Suitable eluents are well known in the art.

[0062] In some embodiments, the kit further optionally includes reagents for preparing a library. Suitable reagents for preparing a library are well known in the art. In some preferred embodiments, the reagents for preparing a library include, but are not limited to, sequencing adapters, ligases, end repair enzymes, and buffering agents.

[0063] In some embodiments, the kit also optionally includes an amplification reagent. In some embodiments, the amplification reagent is a PCR reagent. Suitable PCR reagents are well known in the art. In some preferred embodiments, PCR reagents include but are not limited to primers, DNA polymerase, dNTPs, and a buffering agent.

[0064] In some embodiments, the test kit also optionally includes a DNA extraction reagent. Suitable DNA extraction reagents are well known in the art. In some embodiments, a DNA sample can be prepared from any suitable source by a DNA extraction reagent, preferably from a sample or culture of biological origin. In some embodiments, the DNA sample is from an animal, a plant, or a microorganism. In some preferred embodiments, the DNA sample is from human genomic DNA.

[0065] use

[0066] According to one embodiment of the present application, the use of the long DNA fragment obtained according to the method described herein, or the kit described herein in gene extraction, gene analysis, and gene sequencing can be provided. In some embodiments, the gene sequencing includes third-generation sequencing and fourth-generation sequencing.

[0067] The long-fragment DNA obtained by the methods disclosed herein can be applied to any sequencing analysis, including but not limited to third-generation sequencing based on SMRT (single molecule real-time sequencing) technology and fourth-generation sequencing based on nanopore sequencing technology. In some preferred embodiments, the gene sequencing is nanopore sequencing. Methods for sequencing the captured DNA are well known in the art. The long-fragment DNA obtained by the methods disclosed herein can also be applied to any sequencing methods that emerge in the future.

[0068] The various embodiments and preferences for the present application described above can be combined with each other (as long as they are not inherently contradictory to each other and are applicable to the purposes of the present application), and the various embodiments formed by such combination are considered to be part of the present application.

[0069] Example

[0070] The following describes exemplary embodiments of the present application in conjunction with the accompanying drawings, including various details of the embodiments of the present application to facilitate understanding. It should be understood that they are considered merely exemplary and are in no way intended to limit the scope of protection of the present application. The scope of protection of the present application is defined solely by the claims. Therefore, it should be appreciated by those skilled in the art that various changes and modifications may be made to the embodiments described herein without departing from the scope of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0071] Unless otherwise stated, all reagents and instruments used in the following examples are commercially available conventional products. Unless otherwise stated, experiments were performed under conventional conditions or conditions recommended by the manufacturer.

[0072] Example 1: Construction of a 4-9 kb long fragment whole genome library

[0073] 1.1 Fragmentation of human genomic DNA standard NA12878

[0074] 1 μg of NA12878 standard (Coriell Institute) was fragmented using g-tubes (Covaris) and centrifuged at 9000 g for 1 min. The fragmented DNA was purified using 1.5× VAHTS DNA Clean Beads (Novozyme) and dissolved in 30 μL of nuclease-free water for later use.

[0075] The DNA fragment size was determined using an Agilent 4150 TapeStation automated electrophoresis detection system, and the results are shown in Figure 1 .

[0076] 1.2 Fragmented DNA sorting

[0077] Bluepippin (Sage Science) was used for fragment sorting, using a 0.75% agarose gel (BLF7510) and a DF3-10kb Marker S1 cassette set to sort 4-9kb DNA fragments. The sorted DNA fragments were purified and recovered using 1.5×VAHTS DNA Clean Beads for later use.

[0078] The DNA fragment size was detected using an Agilent 4150 TapeStation automated electrophoresis detection system, and the results are shown in Figure 2.

[0079] 1.3 DNA end repair and adapter ligation

[0080] The DNA fragments in step 1.2 of Example were placed in the end repair system as shown in Table 1 for end repair. The reaction conditions were set at 20°C for 30 min and 65°C for 30 min.

[0081] Table 1 End repair system

[0082] After the end-repair process, add the components listed in Table 2 to the system for adapter ligation at 20°C for 15 minutes. After the reaction, add 3 μL of USER enzyme (NEB E7500L) and digest at 37°C for 15 minutes. Purify and recover the DNA using 0.8× VAHTS DNA Clean Beads.

[0083] Table 2 Joint connection system

[0084] 1.4 PCR amplification

[0085] For long DNA amplification, LA Taq premix (Takara RR900A) was used. The amplification system is shown in Table 3.

[0086] Table 3 Amplification system

[0087] Amplification conditions were as follows: 95°C for 3 min, 12 cycles of 95°C for 20 s and 68°C for 10 min, and 72°C for 10 min, followed by storage at 12°C. The amplified product was purified and recovered using 0.8× VAHTS DNA Clean Beads.

[0088] The DNA fragment size was determined using an Agilent 4150 TapeStation automated electrophoresis detection system, and the results are shown in Figure 3.

[0089] Example 2: Liquid hybridization, capture, elution and PCR amplification

[0090] In this example, the detection panel (11 breast cancer genes: BRCA1, BRCA2, STK11, BARD1, RAD50, ATM, CDH1, TP53, PTEN, PALB2, CHEK2) was commissioned to Aigitaikang Biotechnology (Beijing) Co., Ltd. to design and prepare. It is a single-stranded RNA molecule labeled with biotin and has a size of 1401.94 Kbp.

[0091] 1.5 μg of DNA to be captured (the long fragment full gene library prepared in Example 1) was sequenced according to TargetSeq Hyb&Wash Kit v2.0 (Aijitaikang Biotechnology (Beijing) Co., Ltd.) Kit instructions for hybridization, capture, elution and PCR amplification with probes. Detailed steps are shown below. Reagents used in the following steps, unless otherwise specified, are TargetSeq Components of Hyb&Wash Kit v2.0.

[0092] 2.1 Hybridization

[0093] 1.5 μg of the DNA to be captured was concentrated by vacuum or magnetic bead purification, and then added to the system shown in Table 4. Hybridization was performed on a PCR instrument under the conditions of 80°C for 5 minutes and 50°C for 16-18 hours.

[0094] Table 4 Hybridization system

[0095] 2.2 Capture

[0096] Add 50 μL of Cap Beads to a new PCR tube and place on a magnetic rack for 1 minute. Allow the solution to clear and discard the supernatant. Rinse the Cap Beads three times with 180 μL of Binding Buffer and resuspend them in 180 μL of Binding Buffer. Add the resuspended Cap Beads to the hybridization product from Example 2.1 and mix thoroughly. Capture the hybridization product on a vertical rotary mixer at room temperature for 30 minutes.

[0097] 2.3 Elution

[0098] After capture, remove the product from Example 2.2 from the vertical rotating mixer and place it on a magnetic stand for 2 minutes. After the solution is clear, discard the supernatant. Add 150 μL of Wash Buffer 1, gently pipette to mix, resuspend the magnetic beads, place it on a vertical rotating mixer again, and wash at room temperature for 15 minutes. Then, place it on a magnetic stand and discard the supernatant. Then, use 150 μL of 50°C preheated TargetSeq Wash with Wash Buffer 2 v2 at 50°C for 10 min three times, then rinse once with 80% ethanol. After the Cap Beads have dried, add 24 μL of nuclease-free water to resuspend the beads.

[0099] 2.4 PCR amplification

[0100] The resuspended Cap Beads in Example 2.3 were used as templates and LA Taq premix (Takara RR900A) was used for amplification. The amplification system is shown in Table 5.

[0101] Table 5 Amplification system

[0102] Amplification conditions were as follows: 95°C for 3 min, 28 cycles of 95°C for 20 s, 60°C for 30 s, and 68°C for 10 min, followed by 72°C for 10 min and storage at 12°C. The amplified product was purified and recovered using 1× VAHTS DNA Clean Beads.

[0103] The DNA fragment size was detected using an Agilent 4150 TapeStation automated electrophoresis detection system, and the results are shown in Figure 4.

[0104] DNA fragment capture specificity was analyzed after sequencing on the QNome-3841 sequencing platform of Qi Carbon Technology. The results are shown in Table 6. The results show that, following the conventional liquid-phase hybridization capture process, a specific short-fragment DNA library can be obtained from a long-fragment DNA whole-genome library after RNA probe hybridization, capture, elution, and PCR amplification, but a long-fragment DNA library cannot be obtained.

[0105] Example 3: Liquid hybridization, capture, elution, thermal denaturation, and PCR amplification

[0106] The steps of this embodiment are basically the same as those of embodiment 2, except that:

[0107] 1) Add a heat denaturation step between step "2.3 Elution" and step "2.4 PCR Amplification" as follows:

[0108] Denature 24 μL of the beads suspension resuspended in nuclease-free water from Example 2.3 at 95°C for 10 min. Immediately place the denatured suspension on ice for at least 2 min. Next, place the suspension on a magnetic stand for 2 min. Allow the solution to clear, and remove the supernatant for subsequent PCR amplification (as described in Example 2.4).

[0109] 2) In the PCR amplification system, 24 μL of the "Cap Beads suspension" was replaced with 24 μL of the supernatant after heat denaturation.

[0110] The DNA fragment size was detected using an Agilent 4150 TapeStation automated electrophoresis detection system, and the results are shown in Figure 5.

[0111] The DNA fragment capture specificity was analyzed after sequencing on the QNome-3841 sequencing platform of QITRON Technologies. The analysis results are shown in Table 6.

[0112] The results showed that it was impossible to obtain a long-fragment DNA library after RNA probe hybridization, capture, elution, heat denaturation, and PCR amplification of the whole-genome library.

[0113] Example 4: Liquid hybridization, capture, elution, RNase H digestion and PCR amplification

[0114] The steps of this embodiment are basically the same as those of embodiment 2, except that:

[0115] 1) Between step 2.3 Elution and step 2.4 PCR Amplification, add RNase H digestion as follows:

[0116] To the Cap Beads resuspended in 3.5 μL of nuclease-free water in Example 2.3, 0.5 μL of 10× RNase H Reaction buffer and 1 μL of RNase H (Thermo Fisher EN0202) were added. The beads were digested at 37°C for 20 min. After the reaction, the beads were placed on a magnetic rack and the supernatant was collected for subsequent PCR amplification (as described in Example 2.4).

[0117] 2) In the PCR amplification system, replace 24 μL of the "Cap Beads suspension" with 5 μL of the digested supernatant + 19 μL of nuclease-free water.

[0118] The DNA fragment size was detected using an Agilent 4150 TapeStation automated electrophoresis detection system, and the results are shown in Figure 6.

[0119] The DNA fragment capture specificity was analyzed after sequencing on the QNome-3841 sequencing platform of QITRON Technologies. The analysis results are shown in Table 6.

[0120] The results showed that after RNA probe hybridization, capture, elution and RNase H digestion, PCR amplification of the long-fragment DNA whole-genome library could obtain the target long-fragment DNA, but the target DNA fragments were distributed more diffusely.

[0121] Example 5: Liquid hybridization, capture, elution, RNase H digestion, heat denaturation and PCR amplification

[0122] The steps of this embodiment are basically the same as those of embodiment 2, except that RNase H digestion and heat denaturation are added between step "2.3 Elution" and step "2.4 PCR Amplification" as follows:

[0123] 1) Add 0.5 μL of 10× RNase H Reaction Buffer and 1 μL of RNase H (Thermo Fisher EN0202) to 3.5 μL of Cap Beads resuspended in nuclease-free water in Example 2.3. Digest the beads at 37°C for 20 min and then at 95°C for 5 min. Immediately place on ice for at least 2 min. Then, place on a magnetic rack and remove the supernatant for subsequent PCR amplification (as described in Example 2.4).

[0124] 2) In the PCR amplification system, replace 24 μL of the "Cap Beads suspension" with 5 μL of the supernatant after digestion and heat denaturation + 19 μL of nuclease-free water.

[0125] The DNA fragment size was detected using an Agilent 4150 TapeStation automated electrophoresis detection system, and the results are shown in Figure 7.

[0126] The DNA fragment capture specificity was analyzed after sequencing on the QNome-3841 sequencing platform of QITRON Technologies. The analysis results are shown in Table 6.

[0127] The results showed that after RNA probe hybridization, capture, elution, RNase H digestion, and heat denaturation, PCR amplification of the target long DNA fragments was possible. Specifically, after RNase H digestion followed by heat denaturation, the distribution of DNA fragments obtained by PCR was more concentrated, with a higher proportion of the main peak. After sequencing, the read length N50 and total base capture efficiency were significantly higher than those obtained with RNase H digestion alone.

[0128] Table 6 Sequencing analysis results

[0129] Example 6: Construction of 6-10 kb long fragment whole genome library

[0130] 6.1 Fragmentation of Human Genomic DNA Standard NA12878

[0131] 1 μg of NA12878 standard (Coriell Institute) was fragmented using g-tubes (Covaris) and centrifuged at 8000 g for 1 min. The fragmented DNA was purified using 1.5× VAHTS DNA Clean Beads (Novozyme) and dissolved in 30 μL of nuclease-free water for later use.

[0132] The DNA fragment size was detected using an Agilent 4150 TapeStation automated electrophoresis detection system, and the results are shown in Figure 8.

[0133] 6.2 Fragmented DNA sorting

[0134] Bluepippin (Sage Science) was used for fragment sorting, using a 0.75% agarose gel (BLF7510) and a DF3-10kb Marker S1 cassette set to sort 6-10kb DNA fragments. The sorted DNA fragments were purified and recovered using 1.5×VAHTS DNA Clean Beads for later use.

[0135] The DNA fragment size was detected using an Agilent 4150 TapeStation automated electrophoresis detection system, and the results are shown in Figure 9.

[0136] 6.3 DNA end repair and adapter ligation

[0137] The DNA fragments in step 6.2 of Example were placed in the end repair system as shown in Table 7 for end repair. The reaction conditions were set at 20°C for 30 min and 65°C for 30 min.

[0138] Table 7 End repair system

[0139] After the end-repair process, add the components listed in Table 8 to the system for adapter ligation at 20°C for 15 minutes. After the reaction, add 3 μL of USER enzyme (NEB E7500L) and digest at 37°C for 15 minutes. Purify and recover the DNA using 0.8× VAHTS DNA Clean Beads.

[0140] Table 8 Joint connection system

[0141] 6.4 PCR amplification

[0142] For long DNA amplification, LA Taq premix (Takara RR900A) was used. The amplification system is shown in Table 9.

[0143] Table 9 Amplification system

[0144] Amplification conditions were as follows: 95°C for 3 min, 95°C for 20 s, 68°C for 12 min for 13 cycles, 72°C for 10 min, and storage at 12°C. The amplified product was purified and recovered using 0.8× VAHTS DNA Clean Beads.

[0145] The DNA fragment size was detected using an Agilent 4150 TapeStation automated electrophoresis detection system, and the results are shown in Figure 10.

[0146] Example 7: Liquid hybridization, capture, elution and PCR amplification

[0147] The steps of this example are basically the same as those of Example 2, except that: 1) 1.5 μg of the DNA to be captured is the long fragment full gene library prepared in Example 6; 2) the PCR amplification conditions are: 95°C for 3 minutes; 95°C for 20 seconds, 60°C for 30 seconds, 68°C for 12 minutes, 22 cycles; 72°C for 10 minutes; and storage at 12°C. The other detailed steps are the same as those of Example 2, that is, based on TargetSeq Hyb&Wash Kit v2.0 (Aijitaikang Biotechnology (Beijing) Co., Ltd.) Kit instructions for hybridization with probes, capture, elution, and PCR amplification.

[0148] The DNA fragment size was detected using an Agilent 4150 TapeStation automated electrophoresis detection system, and the results are shown in lane C1 in FIG11 .

[0149] The DNA fragment capture specificity was analyzed after sequencing on the QNome-3841 sequencing platform of QITAN Technology. The analysis results are shown in Table 10.

[0150] The results showed that by following the conventional liquid phase hybridization capture process, a specific short-fragment DNA library could be obtained from the long-fragment DNA whole genome library after RNA probe hybridization, capture, elution, and PCR amplification, but a long-fragment DNA library could not be obtained.

[0151] Example 8: Liquid hybridization, capture, elution, thermal denaturation, and PCR amplification

[0152] The steps of this example are basically the same as those of Example 3, except that: 1) 1.5 μg of the DNA to be captured is the long fragment full gene library prepared in Example 6; 2) the PCR amplification conditions are: 95°C for 3 minutes; 95°C for 20 seconds, 60°C for 30 seconds, 68°C for 12 minutes, 22 cycles; 72°C for 10 minutes; and storage at 12°C. The other detailed steps are the same as those of Example 3, that is, based on TargetSeq Hyb&Wash Kit v2.0 (Aijitaikang Biotechnology (Beijing) Co., Ltd.) Kit instructions for hybridization with probes, capture, elution, thermal denaturation, PCR amplification and other operations.

[0153] The DNA fragment size was detected using an Agilent 4150 TapeStation automated electrophoresis detection system, and the results are shown in lane D1 in FIG11 .

[0154] The DNA fragment capture specificity was analyzed after sequencing on the QNome-3841 sequencing platform of QITAN Technology. The analysis results are shown in Table 10.

[0155] The results showed that it was impossible to obtain a long-fragment DNA library after RNA probe hybridization, capture, elution, heat denaturation, and PCR amplification of the whole-genome library.

[0156] Example 9: Liquid hybridization, capture, elution, RNase H digestion and PCR amplification

[0157] The steps of this example are basically the same as those of Example 4, except that: 1) 1.5 μg of the DNA to be captured is the long fragment full gene library prepared in Example 6; 2) the PCR amplification conditions are: 95°C for 3 minutes; 95°C for 20 seconds, 60°C for 30 seconds, 68°C for 12 minutes, 22 cycles; 72°C for 10 minutes; and storage at 12°C. The other detailed steps are the same as those of Example 4, that is, based on TargetSeq The Hyb&Wash Kit v2.0 (Aijitaikang Biotechnology (Beijing) Co., Ltd.) kit instructions included hybridization with the probe, capture, elution, RNase H digestion, and PCR amplification.

[0158] The DNA fragment size was detected using an Agilent 4150 TapeStation automated electrophoresis detection system, and the results are shown in lane E1 in FIG11 .

[0159] The DNA fragment capture specificity was analyzed after sequencing on the QNome-3841 sequencing platform of QITAN Technology. The analysis results are shown in Table 10.

[0160] The results showed that after RNA probe hybridization, capture, elution and RNase H digestion, PCR amplification of the long-fragment DNA whole-genome library could obtain the target long-fragment DNA, but the target DNA fragments were distributed more diffusely.

[0161] Example 10: Liquid hybridization, capture, elution, RNase H digestion, heat denaturation and PCR amplification

[0162] The steps of this example are basically the same as those of Example 5, except that: 1) 1.5 μg of the DNA to be captured is the long fragment full gene library prepared in Example 6; 2) the PCR amplification conditions are: 95°C for 3 minutes; 95°C for 20 seconds, 60°C for 30 seconds, 68°C for 12 minutes, 22 cycles; 72°C for 10 minutes; and storage at 12°C. Other detailed steps are the same as those of Example 5, that is, based on TargetSeq The Hyb&Wash Kit v2.0 (Aijitaikang Biotechnology (Beijing) Co., Ltd.) kit instructions included hybridization with the probe, capture, elution, RNase H digestion, thermal denaturation, and PCR amplification.

[0163] The DNA fragment size was detected using an Agilent 4150 TapeStation automated electrophoresis detection system, and the results are shown in lane F1 in FIG11 .

[0164] The DNA fragment capture specificity was analyzed after sequencing on the QNome-3841 sequencing platform of QITAN Technology. The analysis results are shown in Table 10.

[0165] The results showed that after RNA probe hybridization, capture, elution, RNase H digestion, and heat denaturation, PCR amplification of the target long DNA fragments was possible. Specifically, after RNase H digestion followed by heat denaturation, the distribution of DNA fragments obtained by PCR was more concentrated, with a higher proportion of the main peak. After sequencing, the read length N50 and total base capture efficiency were significantly higher than those obtained with RNase H digestion alone.

[0166] Table 10 Sequencing analysis results

[0167] Example 11: Liquid hybridization, capture, elution, and PCR amplification

[0168] The steps of this embodiment are basically the same as those of embodiment 2, except that:

[0169] 1) The detection panel in this example (T040V19, Aijitaikang Biotechnology Co., Ltd., catalog number PT1000041) is a detection product specifically designed for the targeted drugs clearly recommended in the NCCN Guidelines for non-small cell lung cancer (NSCLC) and the eight genes involved (the entire exon region of the EGFR, KRAS, MET, and ERBB2 genes, as well as BRAF, ALK, RET, and ROS1 gene-related mutations and fusion sites). It is a biotin-labeled single-stranded RNA molecule with a size of 67.29 Kbp.

[0170] 2) PCR amplification conditions were as follows: 95°C for 3 min; 24 cycles of 95°C for 20 s, 60°C for 30 s, and 68°C for 10 min; 72°C for 10 min; and storage at 12°C.

[0171] Other detailed steps are the same as in Example 2, i.e., based on TargetSeq Hyb&Wash Kit v2.0 (Aijitaikang Biotechnology (Beijing) Co., Ltd.) Kit instructions for hybridization with probes, capture, elution, and PCR amplification.

[0172] DNA fragment size was measured using an Agilent 4150 TapeStation automated electrophoresis detection system, and the results are shown in Figure 12. The results show that following the general experimental procedure described in the hybridization capture instructions (liquid phase hybridization, capture, elution, and PCR amplification), using the commercial probe T040V19 to hybridize and capture whole-genomic DNA, the resulting DNA fragments were primarily 1-2 kb, and no longer DNA fragments were obtained.

[0173] Example 12: Liquid hybridization, capture, elution, RNase H digestion, heat denaturation and PCR amplification

[0174] In this example, the long-fragment DNA hybridization capture method provided in this application was used for experiments, which were compared with the general experimental process in the hybridization capture instructions (in Example 11) to illustrate the effectiveness of the long-fragment DNA hybridization capture method in this application and its applicability to various different RNA probe products.

[0175] The steps of this example are basically the same as those of Example 11, except that RNase H digestion and heat denaturation steps are added between step "2.3 Elution" and step "2.4 PCR Amplification", as described below:

[0176] To the resuspended Cap Beads from Example 2.3, add 0.5 μL of 10× RNase H Reaction Buffer and 1 μL of RNase H (Thermo Fisher EN0202). Digest the beads at 37°C for 20 min and then at 95°C for 5 min. Immediately after the reaction, place on ice for at least 2 min. Then, place on a magnetic rack and remove the supernatant for subsequent PCR amplification (as described in Example 2.4).

[0177] Other detailed steps are the same as in Example 2, i.e., based on TargetSeq Hyb&Wash Kit v2.0 (Aijitaikang Biotechnology (Beijing) Co., Ltd.) Kit instructions for hybridization with probes, capture, elution, and PCR amplification.

[0178] DNA fragment size was measured using an Agilent 4150 TapeStation automated electrophoresis detection system, and the results are shown in Figure 13. As can be seen from the figure, using the commercial probe T040V19 to capture whole-genomic DNA using the long-fragment DNA hybridization capture method provided in this application, the obtained DNA fragments were primarily concentrated around 7 kb, indicating that long DNA fragments could be obtained.

[0179] DNA fragment capture specificity was analyzed after sequencing on the QNome-3841 sequencing platform of Qi Carbon Technology. The analysis results are shown in Table 11 below. The results show that the long-fragment capture method provided in this application, after hybridization capture of whole-genomic DNA using the commercial probe T040V19, can achieve 99.41% coverage of eight designed gene intervals, and the obtained DNA library sequencing read length N50 can reach over 5kb, meeting the basic requirements of subsequent mutation analysis.

[0180] Table 11 Sequencing analysis results

[0181] It should be noted that the above are only preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Although specific implementation methods have been described, for the applicant or other skilled in the art, there may be or are currently no alternatives, modifications, changes, improvements and substantial equivalents of the above implementation methods. Therefore, the attached claims submitted and the claims that may be amended are intended to cover all such alternatives, modifications, changes, improvements and substantial equivalents. It is important to note that as technology evolves, many of the elements described herein may be replaced by equivalent elements that appear after the present application.

Claims

1. A method for capturing long-fragment DNA, characterized in that: Use RNase to treat long DNA fragments hybridized with RNA probes. The length of the long DNA fragment is not less than 2 kb, preferably, the length of the long DNA fragment is 4 kb-10 kb.

2. The method according to claim 1, characterized in that The method comprises the following steps: Hybridize the long-fragment DNA library with the RNA probe; Capturing long fragments of DNA that hybridize to the RNA probe; and Use RNase to digest long fragments of DNA that hybridize to the RNA probe.

3. The method according to any one of claims 1-2, wherein the RNase comprises RNase A and / or RNase H, preferably, the RNase is RNase H.

4. The method according to claim 3, wherein the amount of the RNase is not less than 1U, preferably, the amount of the RNase is not less than 5U.

5. The method according to any one of claims 1 to 3, wherein the temperature of the RNase treatment is any temperature at which the RNase is active, preferably, the temperature of the RNase treatment is not higher than 65°C, and further preferably, the temperature of the RNase treatment is 37°C.

6. The method according to any one of claims 1 to 3, wherein the time of the RNase treatment is not less than 20 minutes, preferably, the time of the RNase treatment is 20 minutes.

7. The method according to any one of claims 1 to 6, wherein the RNA probe carries an affinity tag, preferably, the affinity tag is biotin.

8. The method according to any one of claims 1 to 7, characterized in that: After RNase digestion, a heat denaturation step is also included.

9. The method according to claim 8, wherein the temperature of the thermal denaturation is 80-100°C, preferably, the temperature of the thermal denaturation is 95°C.

10. The method according to claim 8 or 9, wherein the thermal denaturation time is not less than 30 seconds, preferably, the thermal denaturation time is 5 minutes.

11. The method according to any one of claims 8 to 10, wherein the thermal denaturation is followed by an instantaneous cooling treatment.

12. The method according to any one of claims 1 to 11, characterized in that: Before hybridizing the long-fragment DNA with the RNA probe, the steps of preparing the long-fragment DNA library are also included: fragmenting the DNA sample to produce a fragmented DNA sample; sorting the fragmented DNA sample to generate a long-fragment DNA sample; Performing end repair and adapter ligation on the long-fragment DNA sample to generate a long-fragment DNA library; and Optionally, the long fragment DNA library is amplified.

13. A kit for capturing long-fragment DNA, characterized in that: The kit comprises: RNA enzyme, RNA probe, and / or capture agent, The length of the long DNA fragment is not less than 2 kb, preferably, the length of the long DNA fragment is 4 kb-10 kb.

14. The kit according to claim 13, wherein the RNase comprises RNase A and / or RNase H, preferably, the RNase is RNase H.

15. The kit according to claim 13 or 14, wherein the RNA probe carries an affinity tag, preferably, the RNA probe carries biotin.

16. The kit according to any one of claims 13 to 15, wherein the capture agent is a magnetic bead, preferably, the magnetic bead carries an affinity tag, and more preferably, the magnetic bead carries streptavidin.

17. Use of the long-fragment DNA obtained according to the method according to any one of claims 1 to 12, or the kit according to any one of claims 13 to 16 in gene extraction, gene analysis, and gene sequencing, preferably, the gene sequencing includes third-generation sequencing and fourth-generation sequencing.

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