CN117987546A - Capture probe set, library-building kit and use of multiple myeloma related genes - Google Patents

Abstract

A capture probe set of multiple myeloma related genes, a library-building kit and application. The library-building kit manufactured by the invention can detect 55 genes related to the multiple myeloma and the variation condition thereof by utilizing a hybridization capture method; only a trace amount of genome DNA is needed for constructing a DNA library, and the method has the advantages of short time consumption and high efficiency in hybridization capture and enrichment of the library.

Description

Capture probe set, library-building kit and use of multiple myeloma related genes

Technical Field

The invention belongs to the technical field of gene detection, and particularly relates to a capture probe set of multiple myeloma related genes, a library building kit and application.

Background

Methods currently in common use for detecting multiple myeloma genomic related genes include DNA sequencing, restriction fragment length polymorphism polymerase chain reaction, capillary electrophoresis, and the like. The method has the defects that one reaction can only detect one or a few gene loci related to the multiple myeloma, and tens of gene detection has the defects of long time consumption, high cost, low detection sensitivity, easiness in causing false positive or false negative and the like.

Therefore, the patent can detect 55 genes related to multiple myeloma at one time by utilizing a high-throughput sequencing technology, and develops a library kit for detecting 55 genes related to multiple myeloma genomics by utilizing a hybrid capture method.

Disclosure of Invention

In view of the above-mentioned problems, a first object of the present invention is to provide a set of capture probes for achieving the purpose of capturing multiple myeloma-related genes efficiently and rapidly.

The second object of the present invention is to provide the use of the capture probe for manufacturing a kit for detecting multiple genes of multiple myeloma, which alleviates the problem of lack of a product capable of rapidly capturing 55 genes related to multiple myeloma at one time in the prior art.

The third object of the invention is to provide a library-building kit for detecting 55 genes of multiple myeloma, which is used for achieving the purpose of quickly building a targeting library of genes related to multiple myeloma.

The fourth object of the invention is to provide a use of the kit for library construction.

In order to achieve the above object, the technical scheme of the present invention is as follows:

a capture probe comprising at least one of the base sequences SEQ ID NOS: 1 to 1000.

As a further preferred aspect of the present invention, the use of a capture probe for the manufacture of a kit for the detection of multiple myeloma-related genes.

A kit for detecting 55 genes of multiple myeloma comprises at least one group of capture probes, wherein the capture probes comprise at least one of base sequences SEQ ID NO. 1-1000.

A library construction method for capturing multiple myeloma-related genes by using the capture probe or the library-building kit.

Use of a kit for the construction of a library, comprising at least one of the following: (1) detecting a multiple myeloma-related gene; (2) Detecting a mutation in a gene associated with multiple myeloma; (3) Screening and detecting hereditary multiple myeloma related genes; (4) predicting the susceptibility of the individual to multiple myeloma; (5) monitoring the patient for minimal lesion residue.

As a further preferred aspect of the present invention, the multiple myeloma-related gene comprises ：ARID1A、ATM、ATR、BCL7A、BIRC3、BRAF、BTG1、CARD11、CCND1、CDKN1B、CDKN2A、CDKN2C、CKS1B、CRBN、CXCR4、DIS3、DNMT3A、DUSP2、EGFR、EGR1、ETV6、FAT1、FAT4、FGFR3、H1-4、HRAS、IDH1、IDH2、IKZF1、IRF4、KDM6A、KLHL6、KMT2A、KMT2C、KMT2D、KRAS、LRP1B、MET、MYC、MYD88、NF1、NRAS、NSD2、PIM1、PRDM1、PTEN、PTPN11、RB1、TENT5C、TET2、TP53、TRAF2、TRAF3、TRRAP、UBR5.

As a further preferred aspect of the present invention, the library construction method comprises the steps of:

(a) Sample extraction: collecting a sample to be tested and extracting genome DNA;

(b) DNA fragmentation: breaking the DNA to form a DNA fragment;

(c) And (3) terminal repair: performing end repair and addition of A-Tailing to the DNA fragment, and then purifying;

(d) The linker is purified;

(e) Library hybridization and capture.

The application of the library construction method in detecting the genes related to the multiple myeloma is that the library obtained by the library construction method is sequenced, and the sequencing result is compared with a database.

In summary, the invention has the following beneficial effects:

the library-building kit manufactured by the invention can detect the related genes and the variation conditions of the related genes of the multiple myeloma by utilizing a hybridization capture method; only a trace amount of genome DNA is needed for constructing a DNA library, and the method has the advantages of short time consumption and high efficiency in hybridization capture and enrichment of the library.

Drawings

FIG. 1 shows the results of the analysis of the examples.

Detailed Description

1. Library construction by enzymatic cleavage

1.1 DNA fragmentation, end repair and dA tailing.

First, the thermal cycler was turned on, and the thermal cycler thermal lid temperature was set to 105 ℃.

1.1.1 The initial amount of DNA library establishment is 50ng, and the DNA library establishment is fully and evenly mixed and then immediately separated from an ice box for standby.

1.1.2 To a reagent preparation area, a new 1.5ml centrifuge tube is taken to prepare enzyme digestion reaction mixed liquor. After Fragmentation Buffer and Fragmentation Enzyme are melted on ice, a mixed solution is prepared according to the following table, and after uniform mixing and centrifugation, the mixed solution is split into eight-joint tubes and then transferred to an amplification zone through a transfer window. (simultaneously, the DNA purification magnetic bead solution used in the next purification step can be taken out of the refrigerator, fully and uniformly mixed by shaking, and then transferred to an amplification area together, and balanced for at least 1h at room temperature)

Reagent(s)	Single reaction volume
		Fragmentation Buffer	5μL
Fragmentation Enzyme	10μL
		Total	15μL

1.1.3 Adding 35 mu L of DNA samples to be detected into eight-joint tubes prepared with reaction mixed solution, gently vibrating and mixing, rapidly placing into a thermal cycler after transient separation, determining the temperature of a thermal cover of the thermal cycler to be 105 ℃, and adopting the following procedures:

Temperature (temperature)	Time of
		30℃	25min
65℃	30min
		4℃	Hold

When the thermocycler program ended and the sample module was returned to 4 ℃, the samples were removed from the module and placed on ice.

1.2 Connection Universal adapter and purification.

1.2.1A sample of octant, each with a DNA fragment with a dA tail, was added with 3. Mu. L Universal adapter. Mixing with light vibration, centrifuging, and placing on ice for use.

1.2.2 To a reagent preparation zone, preparing a ligation reaction mixture. After DNA Ligation Buffer and DNA Ligation Mix were thawed on ice, a mixture was prepared according to the following table, mixed well, centrifuged on ice and transferred to the amplification zone for use.

Reagent(s)	Single reaction volume
		Water	17μL
DNA Ligation Buffer	20μL
		DNA Ligation Mix	10μL
Total	47μL

1.2.3 To each of the samples prepared in step 1.2.1, 47. Mu.L of the ligation reaction mixture prepared in step 1.2.2 was added. Mixing by gentle shaking, centrifuging, and rapidly placing into a thermal cycler, and incubating at 20deg.C for 15 min (with the thermal lid closed). After the completion, the sample was taken out, and immediately, the magnetic bead purification was started.

1.3 First step magnetic bead purification

1.3.1 Library purification magnetic beads need to be taken out from 4 ℃ before use, fully vortex-shake and mix uniformly, and balance at room temperature for at least 1 hour for standby.

1.3.2 The purified bead suspension, equilibrated at room temperature, was vortexed for 1min, 80 μl (0.8 times the sample volume) was added per sample split into eight vials.

1.3.3 Transferring all the connection products into the magnetic beads corresponding to the eight connecting tubes, and gently vortex and mix uniformly to avoid generating bubbles.

1.3.4 Eight connecting tubes were left to stand at room temperature for 5min, then placed on a magnetic rack for 5min until the supernatant was clear. The supernatant was aspirated and discarded.

1.3.5 Eight connecting tubes were kept on a magnetic rack, 200. Mu.L of freshly prepared 80% ethanol was carefully added to each tube to wash the magnetic bead pellet (without breaking up the pellet) and incubated for 1min at room temperature, and the supernatant was aspirated off.

1.3.6 Repeat step 1.3.5 and remove as much as possible any residual ethanol solution at the bottom of the tube with a 10. Mu.L pipette.

And 1.3.7 eight connecting pipes are left on a magnetic rack, are uncapped for 5 minutes at room temperature and are dried, so that the ethanol is volatilized completely, and excessive drying is avoided.

1.3.8 Add 18. Mu.L molecular biology grade water to each tube and blow mix with a pipette. Incubate for 2min at room temperature.

1.3.9 Each tube was placed on a magnetic rack for 3min until the supernatant was clear.

1.4 PCR amplification of UDI ConTie primers

1.4.1 Step 1.3.9 the standing period can reach a reagent preparation area, according to the number of samples, an eight-way tube is prepared on an ice box, and 25 mu L KAPA HIFI HotStart Ready Mix is taken and split-packed into each hole of the eight-way tube.

1.4.2 To each well of the octant, 10. Mu.L of the pre-set UDI primer for different index was added. And then transferred to the amplified I region through a transfer window.

1.4.3 In the order of the positions set, 15. Mu.L of the purified supernatant obtained in step 1.3.9 is sucked up and added into the corresponding tube, vortexed and mixed evenly, instantaneously separated,

1.4.4 Transfer the prepared reaction tube to the amplification zone through a transfer window for thermal cycling reaction. The reaction procedure was set up in the following table at 105℃on the hot lid:

1.5 second step magnetic bead purification

1.5.1 Library purification magnetic beads need to be taken out from 4 ℃ before use, fully vortex-shake and mix uniformly, and balance at room temperature for at least 1 hour for standby.

1.5.2 The purified bead suspension, equilibrated at room temperature, was mixed by shaking for 1min and 50. Mu.L (sample volume/sample split into 1.5ml octal tubes).

1.5.3 Taking out the product, centrifuging for 30s by a centrifuge, transferring the product to the magnetic beads corresponding to the eight connecting tubes, and gently vortex and mix uniformly to avoid generating bubbles.

1.5.4 Standing at room temperature for 5min, and placing on a magnetic separation frame for 5min until supernatant is clear. The supernatant was aspirated and discarded.

1.5.5 On a magnetic separation rack, 200. Mu.L of freshly prepared 80% ethanol was added, and the mixture was left to stand at room temperature for 1min, and the supernatant was aspirated off.

1.5.6 Step 1.5.5 was repeated and the bottom of the tube was pipetted as much as possible with a 10. Mu.L pipette to remove all remaining ethanol solution.

1.5.7 Eight connecting pipes are left on the magnetic rack, and are uncapped for 5min for drying at room temperature, so that all ethanol is ensured to volatilize completely.

1.5.8 Adding 24 μl of molecular biological water, and mixing by pipetting. Incubate for 2min at room temperature.

1.5.9 Is placed on a magnetic rack for 3min, 20 mu L of supernatant is taken (the amplified tag library is subjected to library enrichment in the next step, 1 mu L is diluted by 10 times, the concentration and fragment size of the library are respectively measured by using a Qubit and fragment analyzer, and the average fragment length of the detection library is about 350-425 bp.

2. Hybrid capture

2.1 Library hybridization

2.1.1 Library pools often contain a plurality of different sample libraries, the pool entries are calculated based on library DNA concentrations and mixed to obtain the pool. The total amount of DNA in the library pool is 1.5-4 ug, and the establishment of the library pool by using a mass sample is recommended. The amounts of DNA library used are referred to in the following table:

2.1.2 calculating the amounts of the different libraries, mixing the libraries in a 1.5ml centrifuge tube, gently shaking, and momentarily isolating for later use.

2.1.3 Adding the pre-hybridization reagents shown in the table below into the mixed sample respectively, uniformly mixing, and performing instantaneous centrifugation so as to avoid generating bubbles as much as possible.

Reagent(s)	Single reaction volume
		MM 55 probe Panel	4μL
Universal Blockers	8μL
		Blocking Solution	5μL

2.1.4 The pre-hybridization reagent tube mixed above was opened with the tube lid, then the tube was placed in a vacuum concentrator, and after trimming, dried at 27℃until the solution in the tube was completely dried. The drying process is about 2 hours.

2.1.5 Drying to the end, fast Hybridization Mix can be incubated at 65 ℃ until all the precipitate is dissolved, then according to the following table, thermal cycler program is set, thermal cover 85 ℃:

Temperature (temperature)	Time of
		95℃	5min
60℃	2h
		60℃	Hold

2.1.6 After drying, the centrifuge tube was removed from the desiccator, fast Hybridization Mix was swirled rapidly and 20. Mu.L was taken and added to the tube to resuspend the sample (do not allow the hybridization solution to return to room temperature, the fingertips flick and mix well, avoid air bubbles, stand at room temperature for 5min. All solutions in the tube were transferred rapidly and completely to a new octant tube, avoid air bubbles.

2.1.7 Add 30. Mu. L Hybridization Enhancer to the tube to cover the inner reagent surface and remove air bubbles by transient centrifugation.

2.1.8 Placing the eight-joint tube into a preheated thermal cycler for hybridization, and incubating for 15min-2h. (STREPTAVIDIN BINDING BEADS for the next capture can be taken out of the refrigerator to the reagent preparation area during this period, and equilibrated to room temperature for at least 1h after thoroughly shaking and mixing.

2.2 Library Capture and washing

2.2.1 To reagent preparation zone, the enrichment reagents (single enrichment reaction reagent dose to 1.5ml centrifuge tube, then transfer each component and equilibrated bead component (STREPTAVIDIN BINDING BEADS through transfer window) to amplification zone as follows.

Component (A)	The required volume
		Fast Binding Buffer	900μL
Fast Wash Buffer 1	450. Mu.L (60 ℃ C. Pre-heat)
		Wash Buffer 2	700 Mu L (48 ℃ C. Pre-heat)

2.2.2.2 Into an amplification zone, preheating 450 mu L FAST WASH Buffer1 at 66℃on a constant temperature metal bath; 700. Mu.L Wash Buffer 2 was preheated at 48 ℃.

2.2.3 Shaking pre-equilibrated Binding Beads until complete mixing, 100. Mu.L of magnetic Beads were taken into a 1.5mL centrifuge tube.

2.2.4 Adding 200 mu L Fast Binding Buffer to the centrifuge tube and blowing with a gun head for mixing.

2.2.5 Place the centrifuge tube on a magnetic rack for 1min or until the solution is clear, discard the supernatant and remove the centrifuge tube.

2.2.6 The above 2.2.5 washing steps were repeated 2 times for a total of 3 times.

2.2.7 After the third washing, 200 mu L Fast Binding Buffer was added and the mixture was resuspended by shaking to thoroughly mix.

2.2.8 After hybridization in step 2.1.8, the lid of the thermocycler was opened and the hybridization solution was quickly transferred to the washed bead solution. Note that: the rapid transfer of reagents from the thermocycler to the magnetic beads is a critical step, and the sample must be directly loaded onto the thermocycler without removing the hybridization tube to prevent the hybridization solution from decreasing in temperature.

2.2.9 Capping the centrifuge tube, placing on a rotary instrument, and mixing thoroughly for 30min at 25deg.C and 1200 rpm.

2.2.10 Remove the centrifuge tube from the rotator, place on a magnetic rack for 1min after rapid centrifugation, remove supernatant, remove tube, add 200. Mu.L of preheated Fast Wash Buffer 1 (do not remove from the metal bath, blow mix well).

2.2.11 The centrifuge tube was incubated at 66℃for 5min.

2.2.12 Placing the centrifuge tube on a magnetic rack for 1min after instantaneous separation, removing the supernatant, taking down the tube, adding 200 mu L of preheated Fast Wash Buffer 1 again, and blowing and uniformly mixing.

2.2.13 The centrifuge tube was incubated at 66℃for 5min.

2.2.14 Transferring all liquid in the centrifuge tube to a new 1.5ml centrifuge tube after the centrifuge tube is instantaneously detached; placing on a magnetic rack for 1min, and removing supernatant.

2.2.15 Remove the tube and add 200. Mu.L of preheated Wash Buffer 2 (without removal from the metal bath, blow mix.

2.2.16 The centrifuge tube was incubated at 48℃for 5min.

2.2.17 Placing the centrifuge tube on a magnetic rack for 1min after instantaneous separation, removing the supernatant, and taking down the tube.

2.2.18 (Steps 2.2.15 to 2.2.17 are washed 2 times, 3 times in total).

2.2.19 The centrifuge tube was then snap-removed and placed on a magnetic rack and the wash was blotted with a 10. Mu.L gun.

2.2.20 The centrifuge tube was removed from the magnet rack, 45. Mu.L of molecular biology water was added, mixed by gentle shaking, and the solution was incubated on ice (if PCR amplification was not performed temporarily, the bead mix could be frozen at-20 ℃.

2.3 PCR amplification

2.3.1 Into the reagent preparation zone, 1 octant was taken, a mixture was prepared, added to the tubes separately (25 μ L KAPA HIFI HotStartReadyMix +2.5 μ L Amplification Primers =27.5 μl), and then the octants were transferred through the transfer window to the amplification I zone (note: DNA Purification Beads can be removed from the refrigerator at this time, mixed thoroughly with shaking, transferred to the amplification zone with the octants, and equilibrated to room temperature for at least 1h.

2.3.2 Adding 22.5. Mu.L of the magnetic bead suspension obtained in the step 2.2.20 into an eight-joint tube, gently shaking, mixing and instantaneous centrifuging. The remaining 22.5. Mu.L may be stored to-20℃for further use.

2.3.3 Eight-tube was placed on a thermal cycler, thermal lid 105 ℃, amplified according to the following reaction procedure:

2.4 magnetic bead purification

2.4.1 Vortexing thoroughly mixing pre-equilibrated DNA purification magnetic beads, taking 1 clean 1.5ml centrifuge tube, adding 90 μl (1.8 x) of DNA purification magnetic beads, immediately centrifuging the eight-connecting tube after PCR is finished, transferring all the solutions in the eight-connecting tube into 1.5ml centrifuge tube, and vortexing thoroughly mixing.

2.4.2 The centrifuge tube was incubated for 5min at room temperature.

2.4.3 Place the centrifuge tube on a magnetic rack for 3min, and remove the supernatant after the solution is clarified.

2.4.4 Holding centrifuge tube on magnetic rack, adding 200 μl of freshly prepared 80% ethanol, incubating for 2min, and discarding supernatant; the 80% ethanol wash was repeated once (2 total times).

2.4.5 Placing the centrifuge tube on a magnetic rack after instantaneous centrifugation, carefully removing residual ethanol by using a 10 mu L gun head, and standing for 5-10 min at room temperature or until the magnetic beads are dried, wherein the centrifuge tube is prevented from dry cracking.

2.4.6 Remove the tube from the magnetic rack and add 32. Mu.L molecular biology water, mix with gentle shaking and incubate for 5min at room temperature.

2.4.7 Place the centrifuge tube on a magnetic rack for 2min or until the solution is clear.

2.4.8 Transfer 30 μl of supernatant to a clean 1.5ml centrifuge tube, cover the tube with the designation of library name and date of construction, and transfer to library quality control area. And (3) injection: the enriched library after purification can be stored below-20 ℃ for 30 days if it cannot be immediately sequenced on-machine.

2.4.9 Alternatively take 2. Mu.L for useDSDNA HS ASSAY KIT library concentration determination was performed, the library concentration should be greater than 2 ng/. Mu.L. At the same time, 2100 fragment analyzer can be used to detect the length distribution of library fragments, and the average fragment length should be 375-425 bp.

Examples

One example of a multiple myeloma bone marrow sample is to detect mutation of related genes. Extracting gene DNA by using a Tiangen blood genome extraction kit, constructing a library according to the method and performing hybridization capture. The results are shown in FIG. 1.

The capture probe sequence is as follows:

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Claims (6)

1. A set of capture probes, characterized in that the capture probes comprise a base sequence SEQ ID

NO 1-1000.

2. The use of a capture probe according to claim 1, wherein the capture probe is used for the manufacture of a kit for the detection of multiple myeloma-related genes.

3. The kit for constructing the pool of 55 genes of multiple myeloma is characterized by comprising at least one group of capture probes, wherein the capture probes comprise at least one of base sequences SEQ ID NO. 1-1000.

4. A library construction method, wherein the multiple myeloma-related gene is captured using the capture probe according to claim 1 or the library-building kit according to claim 3.

5. Use of a library kit according to claim 3, comprising at least one of the following:

(1) Detecting multiple myeloma related genes; (2) Detecting a mutation in a gene associated with multiple myeloma; (3) Screening and detecting hereditary multiple myeloma related genes; (4) judging prognosis of the multiple myeloma; (5) monitoring the patient for minimal lesion residue.

6. The method of claim 2 or 4, wherein the multiple myeloma-related gene comprises:

ARID1A、ATM、ATR、BCL7A、BIRC3、BRAF、BTG1、CARD11、CCND1、C DKN1B、CDKN2A、CDKN2C、CKS1B、CRBN、CXCR4、DIS3、DNMT3A、DUSP2、EGFR、EGR1、ETV6、FAT1、FAT4、FGFR3、H1-

4、HRAS、IDH1、IDH2、IKZF1、IRF4、KDM6A、KLHL6、KMT2A、KMT2C、KMT2D、KRAS、LRP1B、MET、MYC、MYD88、NF1、NRAS、NSD2、PIM1、PRDM1、PTEN、PTPN11、RB1、TENT5C、TET2、TP53、TRAF2、TRAF3、T RRAP、UBR5.