Method and kit for enriching long-fragment DNA (deoxyribonucleic acid)

Abstract

The invention provides a method for enriching long-fragment DNA and a kit thereof, and belongs to the field of DNA enrichment. The kit comprises M270 or T1 magnetic beads, a reaction reagent A, a washing reagent B1, a washing reagent B2, a washing reagent B3, a dissolving reagent C raw material bag and a buffer solution. The kit disclosed by the invention is high in efficiency of enriching the long-fragment DNA, and has excellent application prospects in the field of enriching the long-fragment DNA.

Description

Technical Field

[0001] The present invention belongs to the field of DNA enrichment, and in particular relates to a method for enriching long-fragment DNA and a kit thereof. Background Art

[0002] Long-fragment DNA enrichment specifically refers to capturing DNA fragments with a length of 2kb-10kb in a specific genomic region. It is widely used in genome resequencing, cancer genomics research, genetic disease diagnosis and other fields, providing important tools and methods for genomics, biology and medical research.

[0003] Existing methods for enriching long DNA fragments primarily include: 1. Selectively enriching long DNA fragments using specific probes; 2. Selectively enriching long DNA fragments using streptavidin magnetic beads; and 3. Selectively enriching long DNA fragments using guide RNA (gRNA). These methods can be selected based on experimental requirements and sample characteristics to enrich long DNA fragments. The Dynabeads™ kilobaseBINDER™ Kit (containing M280 magnetic beads, Binding Solution, and Washing Solution) is commonly used for selectively enriching long DNA fragments using streptavidin magnetic beads. Streptavidin magnetic beads serve as the capture agent and detergent for long DNA enrichment. However, this kit is expensive, and its specific enrichment efficiency still needs to be improved.

[0004] There is an urgent need to find a low-priced kit with high efficiency in enriching long-fragment DNA. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for enriching long-fragment DNA and a kit thereof.

[0006] The present invention provides a long fragment DNA enrichment kit, comprising a reaction reagent A, a washing reagent B1, a washing reagent B2, a washing reagent B3, a dissolving reagent C raw material package, and a buffer solution; The reaction reagent A is an aqueous solution containing 5-20 mM Tris-HCl, 0.5-2 mM EDTA and 1-3 M NaCl; Washing reagent B1 is an aqueous solution containing 2-10 mM Tris-HCl, 0.1-1 mM EDTA, 0.5-1.5 M NaCl, and 0.01-0.1% Tween 20; Washing reagent B2 is an aqueous solution containing 0.05-0.5 M NaCl and 0.015 M sodium citrate, with a pH of 6.5-7.5; Washing reagent B3 is an aqueous solution containing 0.05-0.5 M NaOH; The raw material package of dissolving reagent C contains formamide and EDTA separately; The buffer is TE Buffer at pH 7.8-8.2.

[0007] Furthermore, the reaction reagent A is an aqueous solution of 10 mM Tris-HCl, 1 mM EDTA and 2 M NaCl; Washing reagent B1 is an aqueous solution of 5 mM Tris-HCl, 0.5 mM EDTA, 1 M NaCl, and 0.05% Tween 20; Washing reagent B2 is an aqueous solution of 0.15 M NaCl and 0.015 M sodium citrate, with NaOH added to adjust the pH to 7; Washing reagent B3 is an aqueous solution of 0.15 M NaOH.

[0008] Furthermore, the kit also includes 2.5-20 μL of streptavidin magnetic beads.

[0009] Furthermore, The streptavidin magnetic beads are 2.5-5 μL of M-270 streptavidin magnetic beads or 5 μL of T1 streptavidin magnetic beads.

[0010] The present invention also provides a method for enriching long-fragment DNA using the above kit, comprising the following steps: 1) Wash the streptavidin magnetic beads with Reagent A and discard the supernatant. The volume ratio of Reagent A to Streptavidin magnetic beads is 5-60 μL:1-10 μL. 2) Resuspend the streptavidin magnetic beads in Reagent A, add the sample to be enriched, and incubate with rotation at room temperature for 1-6 hours, mixing by rotation every 10-50 minutes for 1-20 seconds. The volume ratio of Reagent A to streptavidin magnetic beads is (5-60) μL: (1-10) μL. The sample to be enriched is 2-50 μL of a 10-500 ng / μL DNA solution in water. 3) Resuspend the streptavidin magnetic beads in washing reagent B1 and discard the supernatant. The volume ratio of washing reagent B1 to streptavidin magnetic beads is (10-80) μL: (1-10) μL; 4) Add washing reagent B2 to resuspend the magnetic beads, discard the supernatant, then add washing reagent B3 to resuspend the magnetic beads, incubate on a rotating instrument at room temperature for 1-50 minutes, and discard the supernatant; wherein the volume ratio of washing reagent B2 to streptavidin magnetic beads is (10-80) μL: (1-10) μL, and the volume ratio of washing reagent B3 to streptavidin magnetic beads is (10-80) μL: (1-10) μL; 5) Resuspend the streptavidin magnetic beads in buffer, pipette the liquid containing the magnetic beads into a clean container, and discard the supernatant. The volume ratio of buffer to streptavidin magnetic beads is (5-80) μL: (1-10) μL; 6) Add lysis reagent C at a volume ratio of 5-20 μL to 10 μL of sample to be enriched, incubate at 35-80°C for 2.5-30 minutes, and aspirate the supernatant; 7) Add PCR product purification magnetic beads to the supernatant solution from step 6), incubate at room temperature for 2-50 minutes, remove the supernatant, add 100-300 μL of 50-90% ethanol-water solution, wash 1-5 times, allow the magnetic beads to dry, add 5-40 μL of enzyme-free water, and incubate at room temperature for 2-50 minutes. The rotation incubation program is as follows: 20-60 rpm speed, 15-60° tilt, 0.1-5° vortex; The buffer solution is TE Buffer with a pH of 7.8-8.2; The dissolution reagent C is a 10 mM EDTA aqueous solution containing 95% formamide by volume.

[0011] Furthermore, In step 1), the volume ratio of reaction reagent A to streptavidin magnetic beads is (10-20) μL: (2.5-5) μL; In step 2), incubate with rotation at room temperature for 2-5 hours, mixing by rotation every 20-40 minutes for 1-6 seconds each time. The volume ratio of reaction reagent A to streptavidin magnetic beads is (10-20) μL: (2.5-5) μL. The sample to be enriched is 5-20 μL of a 50-300 ng / μL aqueous solution of DNA. In step 3), the volume ratio of washing reagent B1 to streptavidin magnetic beads is (20-40) μL: (2.5-5) μL; In step 4), the volume ratio of the washing reagent B2 to the streptavidin magnetic beads is (20-40) μL: (2.5-5) μL, and the volume ratio of the washing reagent B3 to the streptavidin magnetic beads is (20-40) μL: (2.5-5) μL.

[0012] Furthermore, In step 5), the volume ratio of buffer to streptavidin magnetic beads is (20-40) μL: (2.5-5) μL; In step 6), the volume ratio of the lysis reagent C to the sample to be enriched is (5-12) μL:10 μL, and the mixture is incubated at 55-70°C for 2.5-30 minutes. The PCR product purification magnetic beads described in step 7) are AMPure XP magnetic beads; the amount of AMPure XP magnetic beads used is 10-40 μL; after incubation at room temperature for 10 minutes, the supernatant is removed, 200 μL of 50-90% ethanol aqueous solution is added, and the beads are washed twice. After the beads are dried, 10 μL of enzyme-free water is added and incubated at room temperature for 10 minutes.

[0013] Furthermore, In step 1), the volume ratio of reaction reagent A to streptavidin magnetic beads is 10 μL:2.5 μL or 20 μL:5 μL; In step 2), the volume ratio of reaction reagent A to streptavidin magnetic beads was 10 μL:2.5 μL or 20 μL:5 μL. The mixture was incubated with rotation at room temperature for 3 h, with rotation mixing every 30 min for 3 seconds each time. The sample to be tested was 10 μL of a 100 ng / μL DNA aqueous solution.

[0014] In step 3), the volume ratio of wash reagent B1 to streptavidin magnetic beads is 20 μL:2.5 μL or 40 μL:5 μL; In step 4), the volume ratio of washing reagent B2 to streptavidin magnetic beads is 20 μL:2.5 μL or 40 μL:5 μL; the volume ratio of washing reagent B3 to streptavidin magnetic beads is 20 μL:2.5 μL or 40 μL:5 μL.

[0015] Furthermore, In step 5), the volume ratio of buffer to streptavidin magnetic beads is 20 μL:2.5 μL or 40 μL:5 μL; In step 6), the volume ratio of the lysis reagent C to the sample to be enriched is 10 μL:10 μL, and the mixture is incubated at 65°C for 5 minutes. In step 7), the amount of magnetic beads used for PCR product purification is 20 μL.

[0016] Furthermore, the program of the rotation incubation was: 30 rpm rotation speed, 30° tilt, and 1° vortex.

[0017] The long DNA fragments mentioned in the present invention specifically refer to DNA fragments with a length of 2 kb-10 kb, preferably DNA fragments with a length of 3 kb-10 kb.

[0018] The present invention provides a method for enriching long-fragment DNA and a kit thereof. The kit comprises M270 or T1 magnetic beads, a reaction reagent A, a washing reagent B1, a washing reagent B2, a washing reagent B3, a solubilization reagent C, and a buffer. The kit of the present invention has high efficiency in enriching DNA, especially long-fragment DNA. The enrichment efficiency is significantly better than that of the commercially available kit Dynabeads™ KilobaseBINDER™ Kit, and the kit has excellent application prospects.

[0019] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0020] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the relative content of the target fragments in each group. DETAILED DESCRIPTION

[0022] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0023] Company name and product number of the reagents used in the present invention:

[0024] The rotator used for the rotary incubation of the present invention is a HulaMixerTM sample mixer, item number: 15920D, and the rotation program thereof is: 30 rpm speed, 30° tilt, and 1° vortex.

[0025] Example 1: Long fragment enrichment method of the present invention 1. Experimental Methods (1) Kit composition 1. Preparation of relevant reagents (1) Reagent A: 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 2 M NaCl in water.

[0026] (2) Washing reagent B1: an aqueous solution of 5 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 1 M NaCl, and 0.05% Tween 20.

[0027] (3) Washing reagent B2: 0.15 M NaCl, 0.015 M sodium citrate aqueous solution, add NaOH to adjust the pH to 7.

[0028] (4) Washing reagent B3: 0.15M NaOH aqueous solution.

[0029] (5) Dissolution reagent C: 10 mM ethylenediaminetetraacetic acid (EDTA) aqueous solution containing 95% formamide by volume.

[0030] 2. Kit of the present invention Kit 1: 2 × 10 μL Reaction Reagent A, 20 μL Wash Reagent B1, 20 μL Wash Reagent B2, 20 μL Wash Reagent B3, 10 μL Lysis Reagent C, 20 μL TE Buffer (pH 7.8-8.2), and 2.5 μL M270 Streptavidin Magnetic Beads.

[0031] Kit 2: 2 × 20 μL Reaction Reagent A, 40 μL Wash Reagent B1, 40 μL Wash Reagent B2, 40 μL Wash Reagent B3, 10 μL Lysis Reagent C, 40 μL TE Buffer (pH 7.8-8.2), and 5 μL M270 Streptavidin Magnetic Beads.

[0032] Kit 3: 2 × 10 μL Reaction Reagent A, 20 μL Wash Reagent B1, 20 μL Wash Reagent B2, 20 μL Wash Reagent B3, 10 μL Lysis Reagent C, 40 μL TE Buffer (pH 7.8-8.2), and 5 μL T1 Streptavidin Magnetic Beads.

[0033] (2) Enrichment method 1) Place streptavidin magnetic beads in a centrifuge tube, discard the supernatant, wash the beads with Reagent A, and discard the supernatant on a magnetic stand. The volume ratio of Reagent A to streptavidin magnetic beads is 5-60 μL:1-10 μL. 2) Take 100-5000 ng of the sample to be enriched, add water to 2-50 μL, and mix well to obtain the DNA mixture to be enriched; 3) Add Reagent A to resuspend the magnetic beads, add the DNA mixture to be enriched, and incubate with rotation at room temperature for 1-6 hours. Mix thoroughly by rotation every 10-50 minutes for 1-20 seconds. The volume ratio of Reagent A to Streptavidin magnetic beads should be (5-60) μL: (1-10) μL (to reduce bead clumping). 4) Resuspend the streptavidin magnetic beads in washing reagent B1 and discard the supernatant. The volume ratio of washing reagent B1 to streptavidin magnetic beads is (10-80) μL: (1-10) μL; 5) Add washing reagent B2 to resuspend the magnetic beads, discard the supernatant, then add washing reagent B3 to resuspend the magnetic beads, incubate on a rotating instrument at room temperature for 1-50 minutes, and discard the supernatant; wherein the volume ratio of washing reagent B2 to streptavidin magnetic beads is (10-80) μL: (1-10) μL, and the volume ratio of washing reagent B3 to streptavidin magnetic beads is (10-80) μL: (1-10) μL; 6) Add TE Buffer to resuspend the magnetic beads, aspirate the liquid containing the magnetic beads and transfer it to a clean centrifuge tube (to avoid unwashed non-target DNA fragments on the wall of the old tube). The volume ratio of TE Buffer to streptavidin magnetic beads is (5-80) μL: (1-10) μL. Discard the supernatant on the magnetic stand. 7) Add lysis reagent C at a volume ratio of 5-20 μL to sample to be enriched of 10 μL. Incubate at 35-80°C for 2.5-30 minutes, and aspirate the supernatant.

[0034] 8) Add PCR product purification magnetic beads to the supernatant solution from step 7), incubate at room temperature for 2-50 minutes, remove the supernatant, add 100-300 μL of 50-90% ethanol-water solution, wash 1-5 times, allow the beads to dry, add 5-40 μL of enzyme-free water, and incubate at room temperature for 2-50 minutes.

[0035] 2. Method Validation (I) Experimental groups Group by Table 1: Table 1 Experimental groups Group Streptavidin magnetic beads and dosage Reagents Washing reagents Group 1 M280-2.5μL Binding Solution Washing Solution Group 2 M270-2.5μL Reagent A Washing Reagent B1 + Washing Reagent B2 + Washing Reagent B3 Group 3 M270-5μL Reagent A Washing Reagent B1 + Washing Reagent B2 + Washing Reagent B3 Group 4 T1-2.5 μL Reagent A Washing Reagent B1 + Washing Reagent B2 + Washing Reagent B3 Group 5 T1-5μL Reagent A Washing Reagent B1 + Washing Reagent B2 + Washing Reagent B3 Group 1 used the Dynabeads™ kilobaseBINDER™ Kit, a commonly used commercial long-fragment DNA enrichment kit.

[0036] Among them, the total amount of DNA mixture incubated with streptavidin magnetic beads in each group was 1 μg, which contained 100 ng of target fragment and 900 ng of non-target fragment, that is, the target fragment accounted for 1 / 10 in the DNA mixture.

[0037] (2) Experimental verification 1. Verify the enrichment efficiency of DNA samples Using template DNA (plasmid A synthesized by GenScript) as the target fragment, (1) 5427bp target fragment: Reagents Mass / Volume Template DNA (Plasmid A synthesized by GenScript) 30 ng 5X LongAmp Taq Reaction Solution 10 μL 10 mM dATP 1 μL 10 mM dCTP 1 μL 10 mM dGTP 1 μL 10 mM dTTP 0.875 μL 1 mM dUTP-biotin 1.25 10 µM forward primer 2 μL 10 µM reverse primer 2 μL LongAmp Taq DNA Polymerase 2 μL Nuclease-free water to 50 µL (2) 5892bp non-target fragment: Reagents Mass / Volume Template DNA (pcDNA 3.1 plasmid) 30 ng LongAmp Taq 2X Master Mix 25 μL 10 µM forward primer 2 μL 10 µM reverse primer 2 μL Nuclease-free water to 50 µL The specific qPCR reaction procedure was as follows: pre-denaturation at 94°C for 30 s; denaturation at 94°C for 30 s, annealing at 58°C for 1 min, and extension at 65°C for 8 min, for a total of 30 cycles; and final extension at 65°C for 10 min.

[0038] 2. Enrichment Experimental Steps 1) Experimental steps for group 1: (1) Place 2.5 μL of M280 streptavidin magnetic beads in a 1.5 mL centrifuge tube, discard the supernatant on a magnetic rack, wash the beads with 10 μL Binding Solution, and discard the supernatant on a magnetic rack; (2) Take 100 ng of the target DNA band with biotin and 900 ng of the non-target DNA band, add water to make up to 10 μL, and mix well; (3) Add 10 μL of Binding Solution to resuspend the magnetic beads, add 10 μL of DNA mixture, and incubate on a rotating device at room temperature for 3 h. Mix once every 30 min for 3 seconds each time (to reduce the clumping of magnetic beads); (4) Add 20 μL of washing reagent B1 to a 1.5 mL centrifuge tube to resuspend the magnetic beads, place on a magnetic stand and discard the supernatant; (5) Resuspend and wash the magnetic beads twice with 20 μL Washing Solution; (6) Add 20 μL TE Buffer to resuspend the magnetic beads, aspirate the liquid into a new 1.5 mL centrifuge tube (to avoid unwashed non-target DNA fragments on the wall of the old tube), and discard the supernatant on the magnetic rack; (7) Add 10 μL of dissolution reagent C, incubate at 65°C for 5 min, aspirate the supernatant on a magnetic rack, and place it in a new 1.5 mL centrifuge tube; (8) Add 20 μL of AMPure XP magnetic beads, incubate at room temperature for 10 minutes, then place the tube on a magnetic rack. Remove the supernatant and add 200 μL of 70% ethanol-water solution. Wash twice (do not resuspend). After the magnetic beads are dried, add 10 μL of enzyme-free water. Incubate at room temperature for 10 minutes, then place the tube on a magnetic rack. The supernatant is the enriched DNA product. (The added AMPure XP magnetic beads can purify the DNA in the dissolution reagent C and dissolve it in water for subsequent reactions.) 2) Experimental steps of group 2: (Kit 1 of the present invention) (1) Take 2.5 μL of M270 streptavidin magnetic beads in a 1.5 mL centrifuge tube, discard the supernatant on the magnetic rack, wash the beads with 10 μL of reaction reagent A, and discard the supernatant on the magnetic rack; (2) Take 100 ng of the target DNA band with biotin and 900 ng of the non-target band, add water to 10 μL, and mix to obtain a DNA mixture; (3) Add 10 μL of reaction reagent A to resuspend the magnetic beads, add 10 μL of DNA mixture, and incubate on a rotating instrument at room temperature for 3 h. Mix once every 30 min for 3 seconds each time (to reduce the clumping of magnetic beads); (4) Add 20 μL of washing reagent B1 to a 1.5 mL centrifuge tube to resuspend the magnetic beads, place on a magnetic stand and discard the supernatant; (5) Add 20 μL of washing reagent B2 to resuspend the magnetic beads, discard the supernatant on the magnetic stand, then add 20 μL of washing reagent B3 to resuspend the magnetic beads, rotate and incubate at room temperature on a rotating instrument for 10 min, and discard the supernatant on the magnetic stand; (6) Add 20 μL TE Buffer to resuspend the magnetic beads, aspirate the liquid with magnetic beads and transfer it to a new 1.5 mL centrifuge tube (to avoid unwashed non-target DNA fragments on the wall of the old tube), and discard the supernatant on the magnetic stand; (7) Add 10 μL of dissolution reagent C, incubate at 65°C for 5 min, aspirate the supernatant on a magnetic rack, and place it in a new 1.5 mL centrifuge tube; (8) Add 20 μL of AMPure XP magnetic beads, incubate at room temperature for 10 minutes, then place the tube on a magnetic rack. Remove the supernatant and add 200 μL of 70% ethanol-water solution. Wash twice (do not resuspend). After the magnetic beads are dried, add 10 μL of enzyme-free water. Incubate at room temperature for 10 minutes, then place the tube on a magnetic rack. The supernatant is the enriched DNA product. (The added AMPure XP magnetic beads can purify the DNA in the dissolution reagent C and dissolve it in water for subsequent reactions.) 3) Experimental procedures of group 3 (kit 2 of the present invention) (1)-(8) The experimental steps in group 2 were the same as (1)-(8) except that the amount of M270 streptavidin magnetic beads was 5 μL, the amount of reaction reagent A used each time was 20 μL, and the amount of washing reagent B1, washing reagent B2, washing reagent B3 and TE Buffer was 40 μL.

[0039] 4) Experimental steps for group 4: (1)-(8) The steps were the same as those in group 2 (1)-(8), except that 2.5 μL of T1 streptavidin magnetic beads were used.

[0040] 5) Experimental steps of group 5 (kit 3 of the present invention): (1)-(8) The steps were the same as those in group 2 (1)-(8), except that 5 μL of T1 streptavidin magnetic beads were used for the streptavidin magnetic beads. The amount of reaction reagent A used each time was 20 μL, and the amount of washing reagent B1, washing reagent B2, washing reagent B3, and TE Buffer was 40 μL.

[0041] 3. qPCR experiment 1) Primer sequences and plasmid sequences (1) Primer sequence

[0042] 2) Add the corresponding qPCR primers to the products obtained in step (8) of each group and operate according to the iTaq™ Universal SYBR® Green Supermix system (see the table below). Set up 3 replicates for each gene. Reagents volume Template DNA 0.5 μL 10 µM qPCR forward primer 0.2 μL 10 µM qPCR reverse primer 0.2 μL iTaq™Universal SYBR®Green Supermix 5 μL Nuclease-free water to 10 µL The specific qPCR reaction procedure was as follows: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and extension at 72 °C for 15 s, for a total of 41 cycles.

[0043] 3) Run the reaction program in the table above on a CFX96™ Real-Time System. Export the Cq values ​​for quantitative analysis. The Cq values ​​of non-target fragments were used as internal references. Group 1 served as the control group, and Groups 2, 3, 4, and 5 served as experimental groups. Graphpad Prism software was used to process the data and calculate the abundance of the target fragment in each group relative to that in Group 1.

[0044] 3. Experimental Verification Results (I) qPCR experimental results 1. Abundance of target fragments in experimental groups (Groups 2, 3, 4, and 5) relative to the control group (Group 1, i.e., Dynabeads™ KilobaseBINDER™ Kit conditions) The relative content of the target DNA fragment. The higher the relative content of the target fragment, the higher the target fragment enrichment efficiency.

[0045] The relative content of the target DNA fragment is as follows Figure 1 As shown, it can be seen that the relative contents of target DNA fragments in Groups 2, 3, and 5 are significantly higher than those in Group 1. The experimental results show that the efficiency of enriching target DNA fragments using the kit prepared by the present invention is significantly higher than that using the Dynabeads™ KilobaseBINDER™ Kit.

[0046] However, group 4, i.e., the 2.5 μL T1 magnetic bead group, was unable to enrich the target DNA fragments detectable by qPCR. Compared with group 4, group 5 only increased the amount of T1 magnetic beads, and the target fragment enrichment efficiency was significantly improved.

[0047] Comparing Group 2 and Group 3, only the amount of M270 magnetic beads was increased, and the relative content of the target fragments was almost the same. The experimental results showed that only increasing the amount of M270 magnetic beads had no significant effect on the enrichment efficiency of the target fragments.

[0048] Experimental results showed that the relative content of target fragments in Groups 2, 3, and 5 was significantly increased compared to the prior art Group 1, with Group 3 showing the most superior effect. Furthermore, the reagents used in Groups 2, 3, and 5 were low-priced, significantly reducing costs compared to Group 1.

[0049] The present invention provides a method for enriching long-fragment DNA and a kit thereof. The kit comprises M270 or T1 magnetic beads, a reaction reagent A, a washing reagent B1, a washing reagent B2, a washing reagent B3, a solubilization reagent C, and a buffer. The kit of the present invention has high efficiency in enriching DNA, especially long-fragment DNA. The enrichment efficiency is significantly better than that of the commercially available kit Dynabeads™ KilobaseBINDER™ Kit, and the kit has excellent application prospects.

Claims

1. A kit for enriching long fragment DNA, characterized in that: Contains reaction reagent A, washing reagent B1, washing reagent B2, washing reagent B3, dissolving reagent C raw material package and buffer solution; The reaction reagent A is an aqueous solution containing 5-20 mM Tris-HCl, 0.5-2 mM EDTA and 1-3 M NaCl; Washing reagent B1 is an aqueous solution containing 2-10 mM Tris-HCl, 0.1-1 mM EDTA, 0.5-1.5 M NaCl, and 0.01-0.1% Tween 20; Washing reagent B2 is an aqueous solution containing 0.05-0.5 M NaCl and 0.015 M sodium citrate, with a pH of 6.5-7.5; Washing reagent B3 is an aqueous solution containing 0.05-0.5 M NaOH; The raw material package of dissolving reagent C contains formamide and EDTA placed separately; The buffer is TE Buffer at pH 7.8-8.

2.

2. The kit for enriching long fragment DNA according to claim 1, characterized in that The reaction reagent A is an aqueous solution of 10 mM Tris-HCl, 1 mM EDTA and 2 M NaCl; Washing reagent B1 is an aqueous solution of 5 mM Tris-HCl, 0.5 mM EDTA, 1 M NaCl, and 0.05% Tween 20; Washing reagent B2 is an aqueous solution of 0.15 M NaCl and 0.015 M sodium citrate, with NaOH added to adjust the pH to 7; Washing reagent B3 is an aqueous solution of 0.15 M NaOH.

3. The kit for enriching long fragment DNA according to claim 1 or 2, characterized in that: The kit also includes 2.5-20 μL of streptavidin magnetic beads.

4. The kit for enriching long fragment DNA according to claim 3, characterized in that The streptavidin magnetic beads are 2.5-5 μL of M-270 streptavidin magnetic beads or 5 μL of T1 streptavidin magnetic beads.

5. A method for enriching long-fragment DNA using the kit according to any one of claims 1 to 4, characterized in that: The following steps are involved: 1) Wash the streptavidin magnetic beads with Reagent A and discard the supernatant. The volume ratio of Reagent A to Streptavidin magnetic beads is 5-60 μL:1-10 μL. 2) Resuspend the streptavidin magnetic beads in Reagent A, add the sample to be enriched, and incubate with rotation at room temperature for 1-6 hours. Mix thoroughly by rotation every 10-50 minutes for 1-20 seconds. The volume ratio of Reagent A to streptavidin magnetic beads is (5-60) μL: (1-10) μL. The sample to be enriched is 2-50 μL of a 10-500 ng / μL DNA solution. 3) Resuspend the streptavidin magnetic beads in washing reagent B1 and discard the supernatant. The volume ratio of washing reagent B1 to streptavidin magnetic beads is (10-80) μL: (1-10) μL; 4) Add washing reagent B2 to resuspend the magnetic beads, discard the supernatant, then add washing reagent B3 to resuspend the magnetic beads, incubate on a rotating instrument at room temperature for 1-50 minutes, and discard the supernatant; wherein the volume ratio of washing reagent B2 to streptavidin magnetic beads is (10-80) μL: (1-10) μL, and the volume ratio of washing reagent B3 to streptavidin magnetic beads is (10-80) μL: (1-10) μL; 5) Resuspend the streptavidin magnetic beads in buffer, pipette the liquid containing the magnetic beads into a clean container, and discard the supernatant. The volume ratio of buffer to streptavidin magnetic beads is (5-80) μL: (1-10) μL; 6) Add lysis reagent C at a volume ratio of 5-20 μL to 10 μL of sample to be enriched, incubate at 35-80°C for 2.5-30 minutes, and aspirate the supernatant; 7) Add PCR product purification magnetic beads to the supernatant solution from step 6), incubate at room temperature for 2-50 minutes, remove the supernatant, add 100-300 μL of 50-90% ethanol in water, wash 1-5 times, allow the beads to dry, add 5-40 μL of enzyme-free water, and incubate at room temperature for 2-50 minutes. The rotation incubation program is as follows: 20-60 rpm speed, 15-60° tilt, 0.1-5° vortex; The buffer solution is TE Buffer with a pH of 7.8-8.2; The dissolution reagent C is a 10 mM EDTA aqueous solution containing 95% formamide by volume.

6. The method according to claim 5, characterized in that In step 1), the volume ratio of reaction reagent A to streptavidin magnetic beads is (10-20) μL: (2.5-5) μL; In step 2), incubate with rotation at room temperature for 2-5 hours, mixing by rotation every 20-40 minutes for 1-6 seconds each time. The volume ratio of reaction reagent A to streptavidin magnetic beads is (10-20) μL: (2.5-5) μL. The sample to be enriched is 5-20 μL of a 50-300 ng / μL aqueous solution of DNA. In step 3), the volume ratio of washing reagent B1 to streptavidin magnetic beads is (20-40) μL: (2.5-5) μL; In step 4), the volume ratio of the washing reagent B2 to the streptavidin magnetic beads is (20-40) μL: (2.5-5) μL, and the volume ratio of the washing reagent B3 to the streptavidin magnetic beads is (20-40) μL: (2.5-5) μL.

7. The method according to claim 5 or 6, characterized in that In step 5), the volume ratio of buffer to streptavidin beads is (20-40) μL: (2.5-5) μL; In step 6), the volume ratio of the lysis reagent C to the sample to be enriched is (5-12) μL:10 μL, and the mixture is incubated at 55-70°C for 2.5-30 minutes. The PCR product purification magnetic beads described in step 7) are AMPure XP magnetic beads; the amount of AMPure XP magnetic beads used is 10-40 μL; after incubation at room temperature for 10 minutes, the supernatant is removed, 200 μL of 50-90% ethanol aqueous solution is added, and the beads are washed twice. After the magnetic beads are dried, 10 μL of enzyme-free water is added and incubated at room temperature for 10 minutes.

8. The method according to any one of claims 5 to 7, characterized in that: In step 1), the volume ratio of reaction reagent A to streptavidin magnetic beads is 10 μL:2.5 μL or 20 μL:5 μL; In step 2), the volume ratio of reaction reagent A to streptavidin magnetic beads was 10 μL:2.5 μL or 20 μL:5 μL. The mixture was incubated at room temperature for 3 h, with rotation mixing every 30 min for 3 seconds each time. The sample to be tested was 10 μL of a 100 ng / μL DNA aqueous solution. In step 3), the volume ratio of wash reagent B1 to streptavidin magnetic beads is 20 μL:2.5 μL or 40 μL:5 μL; In step 4), the volume ratio of washing reagent B2 to streptavidin magnetic beads is 20 μL:2.5 μL or 40 μL:5 μL; the volume ratio of washing reagent B3 to streptavidin magnetic beads is 20 μL:2.5 μL or 40 μL:5 μL.

9. The method according to any one of claims 5 to 8, characterized in that: In step 5), the volume ratio of buffer to streptavidin magnetic beads is 20 μL:2.5 μL or 40 μL:5 μL; In step 6), the volume ratio of the lysis reagent C to the sample to be enriched is 10 μL:10 μL, and the mixture is incubated at 65°C for 5 minutes. In step 7), the amount of magnetic beads used for PCR product purification is 20 μL.

10. The method according to any one of claims 5 to 9, characterized in that: The rotation incubation program was: 30 rpm speed, 30° tilt, and 1° vortex.

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