WO2017193833A1 - Method and kit comprising 4,000 human pathogenic target genes - Google Patents

Abstract

A method and kit comprising 4,000 human pathogenic target genes. The method comprises the following steps: fragmenting a DNA to obtain a DNA fragment, performing end repair, performing A-tailing, adding a linker, then performing pre-capture LM-PCR; mixing 4,000 human pathogenic target genes with a pre-capture LM-PCR product to obtain a mixed library sample, mixing and hybridizing a probe library specific to the 4,000 human pathogenic target genes with the mixed library sample, then performing magnetic bead capturing and washing of a DNA; and performing post-capture LM-PCR. The kit comprises a DNA end repairing reagent, A-tailing reagent, linker-adding reagent, LM-PCR reagent, and capture reagent. 4,000 human pathogenic genes are selected as targets and combined in the invention. A biotin probe capture technology is then used to perform one-time amplification and high-throughput sequencing at the same time, thereby allowing simultaneous screening for multiple mutant forms of the genes. The testing method has a high level of sensitivity, and can use 4,000 pathogenic genes as a primary clinical diagnostic tool. The method can reduce cost, reduce the likelihood of misdiagnosis, improve true positive rate of testing, and can be promoted in clinical settings.

Description

Method and kit for enriching 4000 human pathogenic target genes Technical field

The invention relates to the field of gene detection, in particular to a method and a kit for enriching 4000 human pathogenic target genes.

Background technique

The human genome is composed of 23 pairs of chromosomes and contains about 3 billion DNA base pairs. The human genome project investigates that the true chromatin gene sequence in the human genome contains approximately 20,000 to 25,000 protein-coding genes. The protein coding sequence (ie, the exon) is less than 1.5% in the human genome. In addition to genes and regulatory sequences, there are still many regions of unknown function, including many repeats, transposons, or pseudogenes. When one or more genes are abnormally expressed, it may cause some clinical symptoms in a corresponding phenotype. The causes of genetic abnormalities include genetic mutations and abnormal chromosome numbers. If the damaged gene is inherited from the parent to the offspring, it becomes a hereditary disease. There are currently about 7,000 genetic diseases known, but only nearly 4,000 genes have well-defined human pathogenic mechanisms and can be transmitted in families.

Gene detection is the detection of a human DNA sequence from blood, other body fluids or cells, and the sequence encoding the gene encoded by the subject is measured and aligned. However, how to effectively and cost-effectively identify several mutations in human pathogenic genes that cause specific diseases is very challenging. This is because when 20,000 human genes are detected, 16,000 genes are unclear, and the large use of sequencing data results in low coverage of the exposed area, resulting in a large degree of missed diagnosis. If high-throughput sequencing methods are used for sequencing detection of exons of all 20,000 genes, the cost is very high, which is not conducive to its clinical promotion.

High-throughput sequencing technology, also known as second-generation sequencing, next-generation sequencing, deep sequencing, or massively parallel sequencing, is a revolutionary change to traditional sequencing, sequencing hundreds of thousands to millions of DNA molecules at a time. . The core idea is Sequencing by Synthesis, which determines the sequence of DNA by capturing the newly synthesized ends of the markers. High-throughput sequencing makes one It is possible to perform detailed analysis of the transcriptome and genome of each species. Currently, there are three main types of sequencing platforms: Illumina's Hiseq platform, life technologies' PGM platform, and Roche's 454 platform.

High-throughput sequencing technology is extremely important in research and clinical applications due to its ultra-high sequencing capabilities. However, although second-generation sequencing greatly reduces the cost of DNA analysis, the sample preparation process is still cumbersome and has become an obstacle to its widespread application in the future. Therefore, a lot of research has been done on its DNA library construction method, in order to simplify the preparation steps of the sample and obtain high quality test samples.

Summary of the invention

In view of the above, it is necessary to address the above problems, and the present invention provides a method and kit for enriching 4000 human target genes. The present invention selects and combines 4000 targets from OMIM, HGMD, UniProt, HUGO, NCBI and Refseq databases and currently known human pathogenic genes.

In order to achieve the above object, the present invention adopts the following technical solutions:

A method for enriching 4000 human pathogenic target genes, comprising the following steps:

S1, extracting total DNA and breaking it to obtain a DNA fragment;

S2, end-repairing the DNA fragment to obtain a blunt-ended DNA fragment;

S3, adding A to the 3' end of the blunt-ended DNA fragment to obtain a DNA fragment with A added;

S4, adding a linker at the 3' end of the DNA fragment added with A to obtain a DNA fragment to which a linker is added;

S5, performing Pre-capture LM-PCR on the DNA fragment of the adaptor;

S6, mixing a Pre-capture LM-PCR product of 4000 human pathogenic target genes to obtain a mixed library sample, hybridizing with a mixed library sample using a 4000 human pathogenic target gene-specific probe library, and capturing the DNA and eluting the magnetic beads;

S7, DNA samples captured by magnetic beads were subjected to Post-capture LM-PCR.

Preferably, the reaction system of Pre-capture LM-PCR is as follows:

Preferably, the reaction conditions of Pre-Capture LM-PCR are as follows: 98 ° C 45 sec; 98 ° C 15 sec, 60 ° C 30 sec, 72 ° C 30 sec, 9 cycles; 72 ° C 1 min; 4 ° C.

Preferably, the system of the Post-capture LM-PCR reaction is as follows:

Preferably, the conditions of the Post-Capture LM-PCR reaction are as follows: 98 ° C 45 sec; 98 ° C 15 sec, 60 ° C 30 sec, 72 ° C 30 sec, 14 cycles; 72 ° C 1 min; 4 ° C.

Preferably, in the present invention, the step of hybridizing the 4000 human pathogenic target gene-specific probe to the mixed library sample is as follows: adding COT DNA and sample library to the centrifuge tube to obtain a mixture 1; and adding SeqCap HE Universal Oligo and SeqCap to the mixture 1 respectively. HE Index Oligos, the mixture 2 was obtained; the mixture 2 was concentrated in a DNA vacuum concentrator until the liquid was evaporated to dryness, and 2×Hybridization Buffer and Hybridization Component A were added to obtain a mixture 3; the mixture 3 was mixed and centrifuged, and then heated at 95 ° C for 10 min, and added. In a 4000 human pathogenic target gene-specific probe library, a mixture 4 was obtained, and the mixture 4 was incubated at 47 ° C for 24 hours.

Preferably, the method of binding the magnetic beads to the DNA is: adding the hybridized sample to the pretreated magnetic beads, mixing and placing in a thermal cycler at 47 ° C for 45 minutes.

Preferably, the magnetic bead-DNA conjugate is eluted sequentially using Wash Buffer I, Stringent Wash Buffer, Wash Buffer I, Wash Buffer II, and Wash Buffer III.

Preferably, said steps S2-S5 and step S7 comprise a purification step.

Preferably, the purification step is: adding the corresponding reaction solution to the sample and mixing, incubating at room temperature for 10 minutes, and fully combining the DNA with the magnetic beads; placing the tube on the magnetic stand until the solution becomes clear, discarding the supernatant, and The tube will remain on the magnetic stand, add 80% alcohol, incubate for 30 seconds at room temperature, and take away the wine. Fine, keep the tube on the magnetic stand, add 80% alcohol, incubate for 30 seconds at room temperature, absorb alcohol; dry thoroughly at room temperature.

More preferably, when it is desired to separate the DNA from the magnetic beads, the tube is removed from the magnetic stand after purification, and ddH ₂ O or 10 mM Tris-HCl, pH 8.0, is added and incubated for 2 minutes at room temperature.

A kit for enriching 4000 human target genes, comprising a DNA end repair reagent, an A reagent, a linker reagent, an LM-PCR reagent, and a capture reagent.

Preferably, the capture reagent comprises 4000 human pathogenic target gene-specific probe libraries, capture magnetic beads and labeling sequences.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention selects 4000 targets from OMIM, HGMD, UniProt, HUGO, NCBI and Refseq databases and currently known human pathogenic genes, and combines them together by biotin probe capture technology. Gene sequence, parallel high-throughput sequencing, can simultaneously check multiple variant types of genes, and high detection sensitivity, which can make 4000 pathogenic genes become the main diagnostic test methods in clinical, which is beneficial to reduce costs, reduce missed diagnosis, and improve detection positive. The rate is conducive to its clinical promotion. The kit of the invention facilitates the enrichment of pathogenic genes, simplifies the detection operation and saves time.

DRAWINGS

Figure 1 is a flow chart of the present invention.

Fig. 2 is a fragmentation electrophoresis pattern in step S1.

Figure 3 is an electrophoresis pattern of the purified LM-PCR product in step S5.

Figure 4 is an electrophoresis pattern of the purified PCR product in step S7.

detailed description

The invention will be further described with reference to the drawings and specific embodiments. Unless otherwise stated, the reagents, equipment or methods used in the present invention are well known to those skilled in the art and will not be further described herein.

The formulations of the following reagents used in the present invention are as follows:

End Repair Master Mix (20 μl):

Water 8μl

10×KAPA End Repair Buffer 7μl

KAPA End Repair Enzyme Mix 5μl.

Add A reaction mixture (A-Tailing Master Mix, 50 μl):

Water 42μl

10×KAPA A-Tailing Buffer 5μl

KAPA A-Tailing Enzyme 3μl.

Ligation Master Mix (55 μl):

Water 30μl

5×KAPA Ligation Buffer 10μl

KAPA T4 DNA Ligase 5 μl.

Example 1

For the sake of simplicity, the present embodiment only uses six samples as an example to illustrate the method for enriching a human pathogenic target gene of the present invention. As shown in FIG. 1, the method includes the following steps:

S1, extracting total DNA and breaking it to obtain a DNA fragment

DNA was extracted from 300 μl of whole blood of each sample according to a conventional method, and 2 μl of the DNA sample was subjected to concentration determination on a NanoDrop. The measured DNA concentration requires an OD260/OD280 ratio between 1.8 and 2.0, and an OD260/OD230 ratio between 1.8 and 2.2. The above two ratios can determine the purity of the extracted DNA. If it is outside the above range, it can be considered that the purity of the extracted DNA does not meet the requirements and needs to be re-extracted or re-purified. Further diluted to a concentration of 25 ng/μl with DNA lysis buffer according to the determined concentration.

Total DNA was interrupted with a Q800R ultrasonic disruptor. Specific steps are as follows:

Add pure water to the cup probe cup and have no titanium probe. Transfer 40 μl of 25 ng/μl DNA into a 0.5 ml centrifuge tube, attach it to the sample holder, and gently bounce off the bubbles at the bottom. Place the sample holder into the cup probe and add pure water to the cup probe again until the level of pure water is flush with the DNA level in the tube.

Open the Q800R's circulating condensate system in advance and set the temperature at 3 °C. Set the interrupt program on the host: Pulse on20sec; Pulse off30sec; Time20min; Amplitude 40%. When the temperature drops to the set temperature of 3 °C, the interrupting program can be turned on. After the program is run, store the sample in the microtube at -20 °C. After all samples have been interrupted, turn off the circulating condensate system and shut down the engine.

2 μl of the fragmented DNA fragment was taken from each sample and detected by 1.5% agarose gel electrophoresis. Finally, the fragment length of each sample should be below 500 bp, and the peak value at 350 bp was acceptable. The results of agarose gel electrophoresis are shown in Figure 2. The first lane is a molecular size marker, and the second to seventh lanes are for DNA fragmentation of different samples, and the fragment size is about 500 bp or less. The subsequent step is to purify and collect a fragment having a peak of about 350 bp.

S2, end repair

50 μl of the DNA fragment was taken from each sample, and 20 μl of the end-recovery reaction mixture was added in a total volume of 70 μl. Incubate at 20 ° C for 30 min on a thermocycler (amplifier) and purify immediately after completion.

The specific method of purification was as follows: 120 μl of Agencourt ^R AMPure ^R XP reagent was added to each sample in a total volume of 190 μl. Use a pipette to repeatedly pipe up and down and mix well. Incubate for 10 minutes at room temperature to allow the DNA to bind well to the magnetic beads. Place the tube on the magnetic stand until the solution becomes clear and carefully discard the supernatant. Leave the tube on the magnetic stand and add 200 μl of 80% alcohol. Incubate for 30 seconds at room temperature and carefully remove the alcohol. Leave the tube on the magnetic stand and add 200 μl of 80% alcohol. Incubate at room temperature for more than 30 seconds, carefully remove the alcohol. This step should be as clean as possible, but care should be taken not to remove the magnetic beads from the bottom. Dry at room temperature. Take the tube out of the magnetic stand.

S3, plus A

To each tube containing the repair DNA-magnetic beads, 50 μl of the reaction mixture of the addition reaction mixture A was added in a total volume of 50 μl. The pipette is repeatedly sucked up and down, and the sample is thoroughly mixed. The thermocycler (amplifier) was incubated at 30 ° C for 30 min and purified immediately after completion.

The specific method of purification was as follows: 90 μl of PEG/NaclSPRI ^R Solution was added to each sample in a total volume of 140 μl. Use a pipette to repeatedly pipe up and down and mix well. Incubate for 10 minutes at room temperature to allow the DNA to bind well to the magnetic beads. Place the tube on the magnetic stand until the solution becomes clear and carefully discard the supernatant. Leave the tube on the magnetic stand and add 200 μl of 80% alcohol. Incubate for 30 seconds at room temperature and carefully remove the alcohol. Leave the tube on the magnetic stand and add 200 μl of 80% alcohol. Incubate at room temperature for more than 30 seconds, carefully remove the alcohol. This step should be as clean as possible, but care should be taken not to remove the magnetic beads from the bottom. Dry at room temperature. Take the officer out of the magnetic stand.

S4, add connector

Add the following reaction solution to each DNA-magnetic bead sample tube to which A is added:

Use a pipette to repeatedly pipe up and down, and mix the sample thoroughly. The thermocycler (amplifier) was incubated at 20 ° C for 15 min, and the subsequent purification step was performed immediately after the end. The specific method of purification is as follows:

50 μl of PEG/Nacl SPRI ^R Solution was added to each sample in a total volume of 100 μl. Use a pipette to repeatedly pipe up and down and mix well. Incubate for 10 minutes at room temperature to allow the DNA to bind well to the magnetic beads. Place the tube on the magnetic stand until the solution becomes clear and carefully discard the supernatant. Leave the tube on the magnetic stand and add 200 μl of 80% alcohol. Incubate for 30 seconds at room temperature and carefully remove the alcohol. Leave the tube on the magnetic stand and add 200 μl of 80% alcohol. Incubate at room temperature for more than 30 seconds, carefully remove the alcohol. This step should be as clean as possible, but care should be taken not to remove the magnetic beads from the bottom. Dry at room temperature. Take the tube out of the magnetic stand. 50 μl of 10 mM Tris-HCl (pH 8.0) was added and incubated for 2 minutes at room temperature to separate the DNA from the magnetic beads. Transfer all of the supernatant to a new tube and continue with the rest.

S5, Pre-capture LM-PCR

The reaction system and reaction conditions of Pre-capture LM-PCR were established according to the standards shown in Tables 1 and 2.

Table 1. Reaction system of Pre-capture LM-PCR

Reagent		Dosage
2×KAPA HiFiHotStartReadyMix	25μl

Pre-LM-PCR Oligos 1&2, 5μM	5μl
Adapter-ligated Sample Library	20μl
total capacity	50μl

Table 2. Reaction conditions of Pre-capture LM-PCR

Samples were stored at 4 ° C or -20 ° C for 72 hours or directly subjected to subsequent purification experiments. The specific method for purification of Pre-capture LM-PCR products is:

50 μl of Agencourt ^R AMPure ^R XP reagent was added to each sample in a total volume of 100 μl. Use a pipette to repeatedly pipe up and down and mix well. Incubate for 10 minutes at room temperature to allow the DNA to bind well to the magnetic beads. Place the tube on the magnetic stand until the solution becomes clear and carefully discard the supernatant. Leave the tube on the magnetic stand and add 200 μl of 80% alcohol. Incubate for 30 seconds at room temperature and carefully remove the alcohol. Leave the tube on the magnetic stand and add 200 μl of 80% alcohol. Incubate at room temperature for more than 30 seconds, carefully remove the alcohol. This step should be as clean as possible, but care should be taken not to remove the magnetic beads from the bottom. Dry at room temperature. Take the tube out of the magnetic stand. 52 μl of ddH ₂ O was added and incubated for 2 minutes at room temperature to separate the DNA from the magnetic beads. Place the tube back onto the magnetic stand until the solution becomes clear. Transfer 50 μl of the supernatant to a new tube and continue with the subsequent steps. The amplified DNA concentration and fragment size were determined by 1.5% agarose gel electrophoresis, Qubit and qPCR. The results of agarose gel electrophoresis are shown in Fig. 3. The first lane is a molecular size marker, and the eighth to the 26th lanes are for pre-LM-PCR of different samples, and the fragment size is about 400 bp. The results of the DNA concentration after amplification of the Qubit assay are shown in Table 3.

Table 3. Qubit determination of DNA concentration after amplification

S6, hybrid capture

(1) Reagent and instrument preparation

1 hybrid preparation

a) Turn on the PCR instrument and set the temperature to 95 ° C and maintain the stable temperature.

b) Take an appropriate amount of 4000 human pathogenicity target gene-specific probe library 4.5 μl and let it thaw at room temperature.

2 Take 6 samples as an example to illustrate the preparation of HE Universal Oligo and HE Index Oligo.

Table 4, HEUniversal Oligo and HE Index Oligo Table

composition	Quantity
SeqCap HE Universal Oligo	1500pmol (3μl of 500μM)
SeqCap HE Index 2 Oligo	250pmol (0.5μl of 500μM)

SeqCap HE Index 4 Oligo	250pmol (0.5μl of 500μM)
SeqCap HE Index 5 Oligo	250pmol (0.5μl of 500μM)
SeqCap HE Index 6 Oligo	250pmol (0.5μl of 500μM)
SeqCap HE Index 7 Oligo	250pmol (0.5μl of 500μM)
SeqCap HE Index 12 Oligo	250pmol (0.5μl of 500μM)
Final concentration	3,000 pmol (6 μl of 500 μM)

Preparation of 3 DNA sample mixture

According to the sample concentration determined by Nanodrop, 300 ng of each sample was mixed in a 1.5 ml tube to prepare a mixed library sample, and finally 1.5 μg of the mixed library sample was used for hybridization.

4 sequence capture and preparation of magnetic bead eluent

The 10× eluent (I, II, III and Stringent) and 2.5×Bead eluate in the NimbleGenSeqCap EZ Hybridization and Wash kit were diluted to 1× working solution to prepare an eluent for capturing the unit amount of working solution. The dosage is shown in Table 5.

Table 5, dilution table of eluent

Reagent dosage	+PCR grade water	Reaction volume (1X)
10×Stringent Wash Buffer(vial 4)–40μl	360μl	400μl
10×Wash Buffer I(vial 1)–30μl	270μl	300μl
10×Wash Buffer II(vial 2)–20μl	180μl	200μl
10×Wash Buffer III(vial 3)–20μl	180μl	200μl
2.5×Bead Wash Buffer(vial 7)–200μl	300μl	500μl

The working solution can be stored for 2 weeks at room temperature. The buffers were placed in a 47 ° C water bath for equilibration 2 hours prior to elution.

5 Capture Beads pretreatment

The captured magnetic beads were returned to room temperature 30 minutes before use. The beads were vortexed thoroughly for 15 seconds. Add 100 μl of magnetic beads to a 1.5 ml centrifuge tube per capture unit. Each centrifuge tube can add up to 6 capture units of magnetic beads. Place the tube on the magnetic stand. When the liquid becomes clear (this process does not exceed 5 minutes), carefully discard the supernatant (do not suck the magnetic beads at the bottom), and the residual liquid will be in the subsequent elution step. The step is removed.

Add 1x Bead Wash Buffer twice the volume of the beads to the tube of the magnetic stand. The tube was removed from the magnetic stand and vortexed for 10 seconds. Return the tube to the magnetic stand to bond the beads. Once the liquid becomes clear, the supernatant can be discarded. Repeat this step once.

Resuspend the beads with an equal volume of 1 x Bead Wash Buffer. 100 μl of resuspended magnetic beads were added to a new 0.2 ml centrifuge tube. Return the tube to the magnetic stand to bond the beads. Once the liquid becomes clear, the supernatant can be discarded. At this point, the capture magnetic beads are pre-treated and can bind the captured DNA. Immediately after this step, follow-up operations should be taken to avoid the water droplets from drying out. A small amount of magnetic bead eluent does not interfere with the capture of magnetic beads in combination with DNA.

(2) Hybridization

5 μl of 1 mg/ml COT DNA and 1.5 μg of the mixed library sample were added to a new 1.5 ml centrifuge tube to obtain a mixture 1. To the mixture 1, 3 μl of 500 μM eqCap HE Universal Oligo and 3 μl of 500 μM eq Cap HE Index Oligo (for example, 6 samples) were added to obtain a mixture 2 (sample library/COT DNA/SeqCap HE Universal Oligo-SeqCap HE Index Oligo mixture), and the mixture was obtained. The composition of 2 is shown in Table 6.

Table 6, composition of mixture 2

ingredient	composition
COT DNA	5μg
Mixed library sample	1.5μg≤50ul
500μMSeqCap HE Universal Oligo	3μl
500μMSeqCap HE Index Oligos	3μl
total capacity	Volume ≤57μl

The tube lid of the mixture 2 was opened, placed in a DNA vacuum concentrator at 60 ° C, and concentrated for 15 min. As the volume of the added liquid increases, the concentration time is appropriately extended until the liquid is evaporated to dryness.

To the evaporated mixture 2, 7.5 μl of 2×Hybridization Buffer (vial 5) and 3 μl of Hybridization Component A (vial 6) were added to obtain a mixture 3. The composition of the mixture 3 is shown in Table 7.

Table 7, composition of mixture 3

ingredient	composition
COT DNA	5μg
Mixed library sample	1.5μg≤50ul
500μMSeqCap HE Universal Oligo	3μl
500μMSeqCap HE Index Oligos	3μl
2×Hybridization Buffer(vial 5)	7.5μl
Hybridization Component A (vial 6)	3μl
Final volume	10.5 μl

Mixture 3 was mixed for 10 seconds and centrifuged at maximum speed for 10 seconds in the centrifugation mode of the concentrator. The mixture was placed on a 95 ° C heating module and heated for 10 mins to denature the DNA. Centrifuge at maximum speed for 10 seconds at room temperature. The denatured mixture was added to a 0.2 ml tube of 4.5 μl of 4000 human pathogenic target gene-specific probe library. Vortex for 3 seconds and centrifuge for 10 seconds. All of the above mixture was transferred to a 0.2 ml tube and the tube lid was capped to give a mixture 4. Mixture 4 was incubated on a thermocycler for 24 hours at 47 ° C (hot lid 57 ° C). The composition of the mixture 4 is shown in Table 8.

Table 8, composition of mixture 4

ingredient	composition
COT DNA	5μg
Mixed library sample	1.5μg
500μM HE Universal Oligo and 500μM HE Index Oligos	1,500pmol each
2×Hybridization Buffer(vial 5)	7.5μl
Hybridization Component A (vial 6)	3μl
4000 human pathogenic target gene-specific probe library	4.5μl
Final volume	15μl

(3) Capture the binding of magnetic beads to DNA

The hybridized sample is added to the pretreated capture magnetic beads. The mixture was thoroughly mixed 10 times with a pipette, placed in a thermocycler at 47 ° C (hot lid 57 ° C) for 45 minutes. Vortex the sample every 15 minutes Mix for 3 seconds to ensure that the beads are evenly suspended.

(4) Magnetic beads-DNA conjugate elution

To 15 μl of the magnetic bead-DNA conjugate, 100 μl of 1×Wash Buffer I at 47 ° C was added. Vortex for 10 seconds. The contents of the 0.2 ml tube were all transferred to a 0.5 ml tube by transient centrifugation.

Return the tube to the magnetic stand to bond the beads. Once the liquid becomes clear, the supernatant can be discarded. Remove the tube from the magnetic stand, add 200 μl of 1×Stringent Wash Buffer at 47°C, and pipette up and down 10 times to mix. This process is fast and constant at 47 ° C to ensure that the temperature does not drop. Incubate at 47 ° C for 5 minutes. Repeat this step once.

Return the tube to the magnetic stand to bond the beads. Once the liquid becomes clear, the supernatant can be discarded. Add 200 μl of room temperature 1×Wash Buffer I and vortex for 2 minutes. If there is liquid residue on the tube cover, gently tap the tube to collect the liquid on the tube cover before proceeding to the next step. Return the tube to the magnetic stand to bond the beads. Once the liquid becomes clear, the supernatant can be discarded. Add 200 μl of room temperature 1×Wash Buffer II and vortex for 1 minute. Return the tube to the magnetic stand to bond the beads. Once the liquid becomes clear, the supernatant can be discarded. Add 200 μl of room temperature 1×Wash Buffer III and vortex for 30 seconds. Return the tube to the magnetic stand to bond the beads. Once the liquid becomes clear, the supernatant can be discarded. The tube was removed from the magnetic rack, each tube sample bind to the capture beads-DNA plus 50μl ddH ₂ O. The magnetic beads captured samples were stored at -15 ° C to -25 ° C. The DNA is not used to separate the magnetic beads again, and the magnetic bead captured sample will be used as a template for the next Post-capture LM-PCR.

S7, Post-capture LM-PCR

The system and reaction conditions of the Post-capture LM-PCR reaction were established in accordance with the requirements of Tables 9 and 10.

Table 9. Post-capture LM-PCR reaction system

Reagent	Dosage
Bead-bound captured DNA	20μl
KAPA HiFiHotStartReadyMix	25μl
Post-captureLM-PCR Oligos 1&2 (5μM)	5μl
total capacity	50μl

Table 10, Post-capture LM-PCR reaction conditions

Samples were stored at 4 ° C or -20 ° C for 72 hours or directly subjected to subsequent purification experiments. The post-capture LM-PCR post purification method is:

90 μl of Agencourt ^R AMPure ^R XP reagent was added to each sample tube in a total volume of 140 μl, and repeatedly pipetted up and down with a pipette to thoroughly mix the solution. Incubate for 15 min at room temperature to allow sufficient binding of the DNA to the magnetic beads. Place the tube on a magnetic stand and let stand until the liquid becomes clear. Carefully discard the supernatant. Leave the tube on the magnetic stand, add 200μl of 80% alcohol, incubate for 30 seconds at room temperature, and carefully remove the alcohol. Leave the tube on the magnetic stand, add 200μl of 80% alcohol, incubate for 30 seconds at room temperature, carefully absorb the alcohol and dry at room temperature. The tube was removed from the magnetic stand, 52 μl of ddH ₂ O was added, and incubated for 2 min at room temperature to separate the DNA from the magnetic beads. Place the tube back into the magnetic stand until the solution becomes clear. Each sample was directly transferred to a 50 μl to 1.5 ml centrifuge tube and the subsequent operations were continued.

The amplified DNA concentration and fragment size were determined by 1.5% agarose gel electrophoresis, Nanodrop, Qubit and qPCR. The results of agarose gel electrophoresis are shown in Fig. 4. The first lane is a molecular size marker, and the second to fourth lanes are post-PCR for different samples, and the peak value is about 400 bp. The results of DNA concentration after amplification by Qubit and qPCR are shown in Table 11.

Table 11, Qubit and qPCR determination of DNA concentration after amplification

Sample number		Group 1	Group 2	Group 3
Qubit measurement value (ng/μl)	2.44	2.14	2.64
qPCR measurement (nM)	14.36	6.7	7.84

The above-mentioned embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that those skilled in the art can also make a decision without departing from the inventive concept. Dry deformation and improvement are all within the scope of protection of the present invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims (10)

1. A method for enriching 4000 human pathogenic target genes, comprising the steps of:

   S1, extracting total DNA and breaking it to obtain a DNA fragment;

   S2, end-repairing the DNA fragment to obtain a blunt-ended DNA fragment;

   S3, adding A to the 3' end of the blunt-ended DNA fragment to obtain a DNA fragment with A added;

   S4, adding a linker at the 3' end of the DNA fragment added with A to obtain a DNA fragment to which a linker is added;

   S5, performing Pre-Capture LM-PCR on the DNA fragment of the adaptor;

   S6, mixing a Pre-capture LM-PCR product of 4000 human pathogenic target genes to obtain a mixed library sample, hybridizing with a mixed library sample using a 4000 human pathogenic target gene-specific probe library, and capturing the DNA and eluting the magnetic beads;

   S7, DNA samples captured by magnetic beads were subjected to Post-capture LM-PCR.
2. The method according to claim 1, wherein the reaction system of Pre-Capture LM-PCR is as follows:

   
3. The method according to claim 2, wherein the reaction conditions of Pre-Capture LM-PCR are as follows: 98 ° C 45 sec; 98 ° C 15 sec, 60 ° C 30 sec, 72 ° C 30 sec, 9 cycles; 72 ° C 1 min; 4 ° C.
4. The method of claim 1 wherein the system of the Post-capture LM-PCR reaction is as follows:

   
5. The method according to claim 4, wherein the conditions of the Post-capture LM-PCR reaction are as follows: 98 ° C 45 sec; 98 ° C 15 sec, 60 ° C 30 sec, 72 ° C 30 sec, 14 cycles; 72 ° C 1 min; 4 ° C.
6. The method according to claim 1, wherein the step of hybridizing the 4000 human pathogenic target gene-specific probe library with the mixed library sample is as follows: adding COT DNA and a sample library to the centrifuge tube to obtain a mixture 1; SeqCap HE Universal Oligo and SeqCap HE Index Oligos were separately added to obtain a mixture 2; the mixture 2 was concentrated in a DNA vacuum concentrator until the liquid was evaporated to dryness, and 2×Hybridization Buffer and Hybridization Component A were added to obtain a mixture 3; After heating at 95 ° C for 10 min, 4000 human pathogenic target gene-specific probe libraries were added to obtain a mixture 4, and the mixture 4 was incubated at 47 ° C for 24 hours.
7. The method according to claim 1, wherein the magnetic beads are combined with the DNA by adding the hybridized sample to the pretreated magnetic beads, mixing them, and placing them in a thermocycler at 47 ° C for 45 minutes. .
8. The method of claim 1 wherein said steps S2-S5 and S7 comprise a purification step.
9. A kit for enriching 4000 human target genes, comprising a DNA end repair reagent, an A reagent, a linker reagent, an LM-PCR reagent, and a capture reagent.
10. The kit according to claim 9, wherein the capture reagent comprises 4000 human pathogenic target gene-specific probe library, capture magnetic beads, and labeling sequences.

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