CN112391455A - Kit for homologous recombination repair defect detection - Google Patents
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Abstract
The invention discloses a kit for homologous recombination repair defect detection, which is used for enriching and detecting the variation condition of 25 genes by utilizing a multi-probe targeted capture technology and combining a second-generation sequencing technology, wherein the 25 genes comprise ARID1A, BRCA1, BRCA2, POLG, ATR, BRIP1, MSH2, RAD51C, ATM, MLH1, MRE11A, RAD50, ATRX, CHECK1, MUTYH, RAD51D, BAP1, CHECK2, NBN, SMARCB1, BARD1, FANCE, PALB2, WRN and BLM.
Description
Technical Field
The invention relates to the fields of molecular biology and medicine, in particular to a kit for detecting recombination repair defects.
Background
The development of cancer requires a series of pathological stages, the development process of which is dominated by genetic variation, including lesions at multiple levels of DNA, RNA, protein, etc., wherein DNA changes are important factors for the development of cancer, including single nucleotide variations, small insertions and deletions, large structural variations, and other common variant forms. With the increasing depth of cancer, the importance of the mechanism of deletion of Homologous Recombination (HRD) among different cancer species is continuously discovered. Over 50% of patients with high-grade serous ovarian cancer have significant DNA repair gene deletions, and 20-25% of prostate cancers have gene inactivating mutations involved in BRCA1/BRCA2 or other homologous recombination pathways. In the process of DNA replication, normal cells repair DNA double strand breaks through a homologous recombination mechanism so as to maintain a normal growth cycle, while in cancer cells, homologous recombination deletion occurs due to mutation of homologous recombination genes, so that DNA repair is damaged, and gene imbalance and unlimited growth of tumors are caused. For example, cells mutated in BRCA1 or BRCA2 lack the wild type gene, resulting in loss of repair by homologous recombination of DNA, resulting in genetic alteration, and thus cancer.
The development of new generation sequencing technology opens a new situation for the research of modern genomics, however, the cost of genome sequencing and the complexity of analysis are still difficult for researchers, and the emergence of target sequence capture technology alleviates the problems. Target sequence capture sequencing is sequencing after designing probes for specific genomic regions that are known. Exome capture sequencing is a special case of the above, and is a study which is specially aimed at all exome regions in the genome. The sequencing of the polygene full exons is a genome analysis method of high-throughput sequencing after capturing and enriching the DNA of the polygene full exon region by utilizing a sequence capture technology. Because the kit has the advantages of high sensitivity to common and rare variation, discovery of most disease-related variation of exon regions, sequencing of about 1% of genomes and the like, multigene whole exon sequencing is promoted to become the most effective strategy of pathogenic genes of diseases such as Mendelian diseases and the like, and the kit is also applied to research and clinical diagnosis of susceptibility genes of complex diseases.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a kit for detecting homologous recombination repair defects and application thereof.
In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions: a kit for homologous recombination repair defect detection comprising deep sequencing of 25 genes, comprising the following genes: ARID1A, BRCA1, BRCA2, POLG, ATR, BRIP1, MSH2, RAD51C, ATM, MLH1, MRE11A, RAD50, ATRX, CHECK1, MUTYH, RAD51D, BAP1, CHECK2, NBN, SMARCB1, BARD1, FANCE, PALB2, WRN, BLM; the kit comprises a library construction kit, a hybridization kit, a probe kit and a joint box.
As a further scheme of the invention: the kit comprises components and contents of a library construction kit 1 box, a hybridization kit 1 box, a probe kit 1 box and a joint kit 1 box, wherein the detection kit is applied to 16-person detection, and the storage temperature of the kit is-25 ℃ to-15 ℃.
As a further scheme of the invention: the library construction kit can be used for DNA fragmentation end repair, ligation reaction and amplification enrichment.
As a further scheme of the invention: the library construction kit comprises 300 mul of 1-tube end repair reaction solution, 500 mul of 1-tube connection reaction solution, 100 mul of 1-tube ligase, 1000 mul of 1-tube amplification reaction solution and 200 mul of 1-tube amplification primer.
As a further scheme of the invention: the hybridization kit can perform hybridization capture and cleaning after hybridization.
As a further scheme of the invention: the hybridization kit comprises 40 mu l of 1 tube of sealing solution, 100 mu l of 1 tube of Cot-1DNA, 170 mu l of 1 tube of hybridization buffer solution, 54 mu l of 1 tube of hybridization enhancement solution, 5ml of 1 bottle of 2 multiplied by magnetic bead cleaning solution, 1600 mu l of 1 tube of 10 multiplied by cleaning buffer solution, 2400 mu l of 1 tube of 10 multiplied by cleaning buffer solution, 3400 mu l of 1 tube of 10 multiplied by cleaning buffer solution and 4800 mu l of 1 tube of 10 multiplied by cleaning buffer solution.
As a further scheme of the invention: the probe kit comprises 16 tubes of probes of 25 genes, and each tube is 4 mu l.
As a further scheme of the invention: the joint box comprises 16 index joints, each joint comprises 1 tube, and each tube comprises 4 mu l.
Compared with the prior art, the invention has the beneficial effects that: the invention has reasonable design and simple structure, and realizes the purpose of simultaneously detecting 25 genes by using a capture probe technology; the invention integrates 25 genes related to homologous recombination repair defect detection into a kit, and has more convenient use and lower cost.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A kit for homologous recombination defect repair detection comprises a library building kit, a hybridization kit, a probe kit and a joint kit.
The library construction kit comprises a packaging box body, a library construction kit liner and a reagent tube, wherein the library construction kit liner is arranged inside the library construction kit packaging box body, container holes are formed in the liner, and the reagent tube comprises 300 mu l of 1-tube end repair reaction liquid, 500 mu l of 1-tube connection reaction liquid, 100 mu l of 1-tube ligase, 1000 mu l of 1-tube amplification reaction liquid and 200 mu l of 1-tube amplification primers.
The hybridization kit comprises a packaging box body, a hybridization kit liner, a reagent tube and a reagent bottle, wherein the hybridization kit liner is arranged inside the hybridization kit packaging box body, container holes are formed in the liner, the reagent tube comprises 1 tube of sealing liquid 40 mu l, 1 tube of Cot-1DNA 100 mu l, 1 tube of hybridization buffer solution 170 mu l, 1 tube of hybridization enhancing liquid 54 mu l, 1 tube of 10 multiplied by cleaning buffer solution 1600 mu l, 1 tube of 10 multiplied by cleaning buffer solution 2400 mu l, 1 tube of 10 multiplied by cleaning buffer solution 3400 mu l and 1 tube of 10 multiplied by cleaning buffer solution 4800 mu l, and the reagent bottle comprises 1 bottle of 2 multiplied by magnetic bead cleaning liquid 5 ml.
The probe kit comprises a packaging box body, a probe kit liner and reagent tubes, wherein the probe kit liner is arranged inside the packaging box body of the probe kit, container holes are formed in the liner, the reagent tubes comprise 16 tubes of 25-gene probes, and each tube is 4 mu l.
The joint kit comprises a packaging box body, a joint kit liner and reagent tubes, wherein the joint kit liner is arranged inside the joint kit packaging box body, container holes are formed in the liner, the reagent tubes comprise 16 index joints, each joint comprises 1 tube, and each tube is 4 mu l.
The specific linker in the kit refers to a specific sequence tag which can be added to both ends of the target fragment by reaction. In the kit, 16 adapters are provided, and different specific sequence tags are added to different samples. Specific sequence tags can be detected using second generation sequencing to distinguish between different samples.
The kit comprises the following components in percentage by weight:
building a library kit 1:
1 tube 300. mu.l/tube end repair reaction solution,
1 tube 500. mu.l/tube of the ligation reaction solution,
1 tube 100. mu.l/tube of ligase,
1 tube 1000. mu.l/tube of amplification reaction solution,
1 tube 200. mu.l/tube of amplification primer.
Hybridization kit 1 kit:
1 tube of 40 mul/tube of sealing liquid,
1 tube of 100. mu.l/tube of Cot-1DNA,
1 tube of 170. mu.l/tube of hybridization buffer,
1 tube of 54. mu.l/tube of hybridization enhancing solution,
1 bottle of 2 Xmagnetic bead cleaning solution with the volume of 5 ml/bottle,
1 tube 600. mu.l/tube 10 XWash buffer 1,
1 tube 400. mu.l/tube 10 XWash buffer 2,
1 tube 400. mu.l/tube 10 XWash buffer 3,
1 tube 800. mu.l/tube 10 XWash buffer 4.
Probe kit 1 kit:
16 tubes 4. mu.l/tube 25 gene probe.
1, joint kit:
16 indexes, 1 tube each, 4. mu.l of 1 tube.
The detection kit is applied to 16-person detection, and the storage temperature of the kit is-25 ℃ to-15 ℃.
Specific examples of the present invention are as follows.
1 extraction and fragmentation of genomic DNA.
1.1 extraction of genomic DNA. The tissue of the subject is extracted from the dermis layer below the epidermis, preferably including the capillaries.
1.2 fragmentation of genomic DNA. The Covaris instrument is used, and the operation is carried out according to the recommended operation flow, and the size of the target fragment is 200-300 bp.
2 fragmented DNA end filling in.
2.1 the reaction system is as follows.
2.2 the reaction sequence is as follows.
And 3, connecting by using a joint.
3.1 the reaction system is as follows.
3.2 the reaction sequence is as follows.
3.3 purifying.
Note: enough fresh 80% ethanol was prepared, ready for use, and each sample required 400. mu.l per purification.
3.3.1 Add 80. mu.l of beads to the product from the previous step and gently blow 10 times to ensure uniformity of the whole system. The library was allowed to bind to the magnetic beads by incubation for 5 minutes at room temperature.
3.3.2 centrifuge tube and put on magnetic rack to separate magnetic beads and liquid.
3.3.3 keep the centrifuge tube on the magnetic rack and after the solution is clear (about 5 minutes), carefully discard the supernatant taking care not to disturb the magnetic beads.
3.3.4 keep the centrifuge tube on a magnetic stand, add 200. mu.l of freshly prepared 80% ethanol slowly along the tube wall, take care not to disturb the magnetic beads while adding ethanol, and carefully remove the supernatant after 30 seconds of incubation.
3.3.5 repeat step 3.3.4, rinsing twice in total.
3.3.6 pipette off all residual ethanol with a 2-10. mu.l pipette. The lid was opened and dried in air for 3-5 minutes.
Note that: ensure that the beads are just dry, the beads now appear lusterless. If the beads are not completely air dried, alcohol remains in the sample, which can reduce the elution rate of DNA and may interfere with downstream reactions.
3.3.7 adding 22.5 μ l of sterilized water, mixing, and standing for 5 min.
3.3.8 centrifuge tubes briefly and place them on a magnetic rack to separate magnetic beads from liquid.
3.3.9 the tubes were held on a magnetic rack and after the solution was clear (approximately 5 minutes), 20. mu.l of the supernatant was transferred to a new 1.5mL tube.
4 Pre-Capture PCR.
4.1 the reaction system is as follows.
4.2 the reaction sequence is as follows.
4.3 purifying.
Note: enough fresh 80% ethanol was prepared, ready for use, and each sample required 400. mu.l per purification.
4.3.1 Add 45. mu.l of magnetic beads to the product from the previous step and gently blow 10 times to ensure uniformity of the whole system. The library was allowed to bind to the magnetic beads by incubation for 5 minutes at room temperature.
4.3.2 centrifuge tube and put on magnetic rack to separate magnetic beads and liquid.
4.3.3 keep the centrifuge tube on the magnetic rack and after the solution is clear (about 5 minutes), carefully discard the supernatant taking care not to disturb the magnetic beads.
4.3.4 keep the centrifuge tube on a magnetic stand, add 200. mu.l of freshly prepared 80% ethanol slowly along the tube wall, take care not to disturb the magnetic beads when adding ethanol, and carefully remove the supernatant after incubation for 30 seconds.
4.3.5 repeat step 4.3.4 for a total of two rinses.
4.3.6 pipette off all residual ethanol with a 2-10. mu.l pipette. The lid was opened and dried in air for 3-5 minutes.
Note that: ensure that the beads are just dry, the beads now appear lusterless. If the beads are not completely air-dried, alcohol remains in the sample, which can reduce the elution rate of DNA and may interfere with downstream reactions; if the beads dry out, the beads may crack, suggesting that the incubation time is extended 4.3.8 to allow sufficient hydration, which may reduce the elution of DNA and ultimately yield.
4.3.7 after the beads were dried, the centrifuge tube was removed from the magnetic rack, 22.5. mu.l of water was added to cover the beads, and the beads were pipetted and mixed well.
4.3.8 was incubated at room temperature for 2 minutes. If the magnetic beads are dry and cracked, the incubation time is suitably prolonged.
4.3.9 centrifuge the tube briefly and place in a magnetic rack, separate the beads from the liquid until the solution is clear (about 5 minutes). And if a small amount of magnetic beads are not adsorbed on the magnetic frame any more, uniformly mixing the non-adsorbed magnetic beads in the supernatant by using a liquid transfer device, resuspending the mixture, and continuing to incubate until no magnetic beads are left in the supernatant.
4.3.10 carefully pipette 21. mu.l of the supernatant into a new small PCR tube of the corresponding number.
4.3.11 Qubit quantification is performed.
5, hybridization and capture.
Note: reagents used in the experimental process need to be inverted up and down or slightly vibrated after being dissolved, instantaneous centrifugation is carried out, and only centrifugation is needed for enzyme and a probe, so that oscillation is not needed. The experiment was conducted on ice throughout the course.
5.1 preparation of the probe.
The probes were synthesized by IDT Inc, and the probe regions are shown in Table 1.
XGen lockdown probes are aqueous solutions.
a thawing the reagent at room temperature (15-25 ℃).
b, fully mixing the materials evenly, and simply rotating and mixing the materials evenly.
5.2 the following reagents were added to a 0.2mL small PCR tube.
The total amount of a Pool barcoded library was 500 ng.
| Pool barcoded library | 500ng |
| Cot-1 DNA | 5μg |
| XGen universal Blockers-Tsmix | 2μl |
b the solution was vacuumed dried at 45 ℃ for 35 minutes in a vacuum.
5.3 hybridization of the DNAs Capture probe and library.
a, unfreezing the XGen lockdown reagent; the 2Xhybridization Buffer had crystals and was heated at 65 ℃ until dissolved.
b was added to a small, drained PCR tube.
| 2×Hybridization Buffer | 8.5μl |
| Hybridization Buffer Enhancer | 2.7μl |
| Nuclease-free water | 1.8μl |
The mixture is placed at room temperature for 5 minutes, and then is placed at room temperature for 5 minutes after being blown and beaten.
c 95 ℃ for 10 minutes, and then the temperature is covered with a cover at 105 ℃. Immediately after completion, the ice cream was taken out and placed on an ice box prepared in advance, and the ice cream was allowed to stand for 2 minutes.
d immediately adding 4. mu.l XGen lockdown probe pool, quickly whipping and mixing for 10 seconds, and centrifuging for a short time to obtain a final volume of 17. mu.l.
e 65 ℃ and cover temperature 75 ℃ overnight (at least 4 hours).
5.4 post-hybridization treatment.
a prepare 1 × working solution, effective at room temperature for 4 weeks.
| For short | Reagent | Nuclease-free water |
| B | 2×Bead wash Buffer：250μl | 250μl |
| I | 10×wash Buffer I：30μl | 270μl |
| II | 10×wash Buffer II：20μl | 180μl |
| III | 10×wash Buffer III：20μl | 180μl |
| S | 10×stringent wash Buffer：40μl | 360μl |
If there are particles, wash Buffer I is heated at 65 ℃.
b, dividing the wash Buffer I and the string wash Buffer equally.
5.5 prepare Streptavidin Beads 270.
a equilibrate the Beads for 30 minutes at room temperature.
b mix Beads thoroughly with shaking for 15 seconds.
c Add 100. mu.l of Beads to a 1.5mL tube, place the tube on a magnetic rack, stratify the Beads, and discard the supernatant.
d Add 200. mu.l of 1 × Bead wash Buffer, vortex for 10 seconds, place back on magnetic stand, stratify, and discard supernatant.
e repeating d times.
f Add 100. mu.l of 1 × Bead wash Buffer and shake.
g Add resuspended 100. mu.l Beads to a 0.2ml PCR tube, place on magnetic rack, discard supernatant and proceed immediately to next step.
5.6 binding of the hybridization product to the Beads.
a, placing the treated hybridization sample into a 0.2ml PCR tube with Beads, and uniformly mixing by blowing for 10 times.
b 65 deg.C (75 deg.C) for 45 minutes.
c vortex 3 sec at 15 min intervals, 2500 rpm.
5.7 washing the beads, discarding unbound DNA.
a 65 ℃ heating, 1 × Wash Buffer I and 1 × stringent Wash Buffer.
b Add 100. mu.l of 1 × Wash Buffer I to the tube of the product, vortex briefly and transfer to a 1.5mL tube.
c placing the tube on a magnetic frame, and discarding the supernatant.
d, adding 200 mul of 1 × stringent wash Buffer, and blowing and beating up and down for 10 times; metal bath 65 deg.C for 5 min; placing on a magnetic frame, and discarding the supernatant.
e is repeated once d.
f, RT washing: adding 200 mul of 1 xwash Buffer I, whirling for 2 minutes, placing on a magnetic frame, and discarding the supernatant; adding 200 mul of 1 xwash Buffer II, whirling for 1 minute, placing on a magnetic frame, and discarding the supernatant; add 200. mu.l of 1 xwash Buffer III, vortex for 30 seconds, place on magnetic stand and discard supernatant.
g resuspending Beads: the EP tube was removed from the magnetic stand, and 20. mu.l of nucleic-free water (back band Beads for PCR) was added thereto, and the tube was blown up and down 10 times.
6 post-Capture PCR.
6.1 the reaction system is as follows.
| Stencil (with magnetic bead) | 20μl |
| PCR Primer Mix2 | 5μl |
| Amplification Mix3 | 25μl |
| Total volume | 50μl |
6.2 the reaction sequence is as follows.
6.3 the amplified product is subjected to Qubit quantification.
And 6.4, purifying.
Note: taking out the magnetic beads, uniformly mixing the magnetic beads by vortex oscillation, and standing the mixture for 30 minutes at room temperature; the beads are viscous, and a pipette is used to ensure that enough volume is obtained and slowly ejected; enough fresh 80% ethanol was prepared, ready for use, and each sample required about 400. mu.l per purification.
6.4.1 vortex the beads to mix well, add 75. mu.l of each bead to the product above, and blow gently 10 times to ensure uniformity of the whole system. The library was allowed to bind to the magnetic beads by incubation for 5 minutes at room temperature.
6.4.2 centrifuge tubes briefly and place them on magnetic rack to separate magnetic beads and liquid.
6.4.3 keep the centrifuge tube on the magnetic rack and after the solution is clear (about 5 minutes), carefully discard the supernatant taking care not to disturb the magnetic beads.
6.4.4 keep the centrifuge tube on a magnetic stand, add 200. mu.l of freshly prepared 80% ethanol slowly along the tube wall, take care not to disturb the magnetic beads while adding ethanol, and carefully remove the supernatant after 30 seconds of incubation.
6.4.5 repeat step 6.4.4, rinsing twice in total.
6.4.6 pipette off all residual ethanol with a 2-10. mu.l pipette. The lid was opened and dried in air for 3-5 minutes.
Note that: ensure that the beads are just dry, the beads now appear lusterless. If the magnetic beads are not completely dried, alcohol still remains in the sample, and the alcohol can reduce the elution efficiency of DNA and possibly interfere with downstream reaction; if the beads dry out, the beads may crack, suggesting that the incubation time is extended 6.4.8 to allow sufficient hydration, which may reduce the elution of DNA and ultimately yield.
6.4.7 after the beads were dried, the centrifuge tube was removed from the magnetic rack, 22. mu.l of IDTE was added to cover the beads, and the beads were pipetted and mixed well.
6.4.8 was incubated at room temperature for 2 minutes. If the magnetic beads are dry and cracked, the incubation time is suitably prolonged.
6.4.9 the tube was briefly centrifuged and placed in a magnetic rack to separate the beads from the liquid until the solution was clear (about 5 minutes). And if a small amount of magnetic beads are not adsorbed on the magnetic frame any more, uniformly mixing the non-adsorbed magnetic beads in the supernatant by using a liquid transfer device, resuspending the mixture, and continuing to incubate until no magnetic beads are left in the supernatant.
6.4.10 carefully pipette 20. mu.l of the supernatant into a new 1.5mL centrifuge tube of the appropriate number for the quantitation of the Qubit.
The products after hybridization capture can be stored for a long time at-80 ℃ and wait for final machine sequencing.
While the foregoing is directed to the preferred embodiment of the present invention, and not to any one of the preceding forms or essential limitations thereof, it will be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
Attached table 1 probe area.
| Target | # Regions | Size bp | Chr | Start | Stop |
| RAD50 | 26 | 3939 | chr5 | 131893017 | 131978056 |
| CHEK1 | 12 | 1431 | chr11 | 125496664 | 125525215 |
| CHEK2 | 14 | 1875 | chr22 | 29083885 | 29130709 |
| FANCE | 10 | 1611 | chr6 | 35420323 | 35434122 |
| MLH1 | 20 | 2538 | chr3 | 37035039 | 37092144 |
| MRE11A | 19 | 2267 | chr11 | 94153291 | 94225967 |
| MSH2 | 17 | 3216 | chr2 | 47630331 | 47739573 |
| MUTYH | 18 | 1879 | chr1 | 45794999 | 45805926 |
| NBN | 17 | 2761 | chr8 | 90947810 | 90996842 |
| ATM | 62 | 9188 | chr11 | 108098410 | 108236235 |
| POLG | 23 | 3720 | chr15 | 89859982 | 89876905 |
| ATR | 49 | 10597 | chr3 | 142168271 | 142297546 |
| ATRX | 37 | 8169 | chrX | 76763829 | 77041487 |
| BARD1 | 11 | 2341 | chr2 | 215593400 | 215674300 |
| RAD51C | 9 | 1322 | chr17 | 56770005 | 56811583 |
| RAD51D | 11 | 1166 | chr17 | 33427972 | 33446632 |
| BLM | 22 | 4254 | chr15 | 91290623 | 91358509 |
| SMARCB1 | 9 | 1212 | chr22 | 24129455 | 24176367 |
| WRN | 35 | 4299 | chr8 | 30915964 | 31030618 |
| PALB2 | 13 | 3561 | chr16 | 23614780 | 23652478 |
| ARID1A | 24 | 6858 | chr1 | 27022895 | 27107247 |
| BAP1 | 17 | 2259 | chr3 | 52436304 | 52443894 |
| BRIP1 | 19 | 3750 | chr17 | 59760657 | 59938900 |
| BRCA1 | 23 | 5658 | chr17 | 41197695 | 41276113 |
| BRCA2 | 26 | 10257 | chr13 | 32890598 | 32972907 |
The designed probe region covers all gene exon coding regions, and ship coverage of the designed gene can be realized.
Claims (8)
1. A kit for homologous recombination repair defect detection, characterized in that: deep sequencing of 25 genes, including the following: ARID1A, BRCA1, BRCA2, POLG, ATR, BRIP1, MSH2, RAD51C, ATM, MLH1, MRE11A, RAD50, ATRX, CHECK1, MUTYH, RAD51D, BAP1, CHECK2, NBN, SMARCB1, BARD1, FANCE, PALB2, WRN, BLM; the kit comprises a library construction kit, a hybridization kit, a probe kit and a joint box.
2. The detection kit according to claim 1, characterized in that: the kit comprises components and contents of a library construction kit 1 box, a hybridization kit 1 box, a probe kit 1 box and a joint kit 1 box, wherein the detection kit is applied to 16-person detection, and the storage temperature of the kit is-25 ℃ to-15 ℃.
3. The detection kit according to claim 1, characterized in that: the library construction kit comprises a DNA fragmentation end repair device, a ligation reaction device and an amplification enrichment device.
4. The detection kit according to claim 1, wherein the components and contents of the kit for library construction comprise 1 tube of 300 μ l/tube end repair reaction solution, 1 tube of 500 μ l/tube ligation reaction solution, 1 tube of 100 μ l/tube ligase, 1 tube of 1000 μ l/tube amplification reaction solution, 1 tube of 200 μ l/tube amplification primers, 1 outer casing, and 1 inner liner.
5. The detection kit according to claim 1, characterized in that: the hybridization kit comprises hybridization capture of DNA sequences and post-hybridization washes.
6. The detection kit according to claim 1, characterized in that: the components and contents of the hybridization kit comprise 1 tube of 40 mul/tube sealing solution, 1 tube of 100 mul/tube Cot-1DNA, 1 tube of 170 mul/tube hybridization buffer solution, 1 tube of 54 mul/tube hybridization enhancing solution, 1 bottle of 5 ml/bottle of 2X magnetic bead cleaning solution, 1 tube of 600 mul/tube 10X cleaning buffer solution 1, 1 tube of 400 mul/tube 10X cleaning buffer solution 2, 1 tube of 400 mul/tube 10X cleaning buffer solution 3, 1 tube of 800 mul/tube 10X cleaning buffer solution 4, 1 outer packing box and 1 inner lining.
7. The detection kit according to claim 1, characterized in that: the components and contents of the probe kit comprise 16 tubes of probes of 25 genes, 4 mu l of each tube, 1 outer packing box and 1 inner lining.
8. The detection kit according to claim 1, characterized in that: the components and contents of the joint kit comprise 16 index joints, 1 pipe of each joint, 4 mu l of each pipe, 1 outer packing box and 1 inner lining.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113462784A (en) * | 2021-08-31 | 2021-10-01 | 迈杰转化医学研究(苏州)有限公司 | Method for constructing target set for homologous recombination repair defect detection |
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