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WO2017193833A1 - Procédé et trousse comprenant 4 000 gènes cibles pathogènes humains - Google Patents

Procédé et trousse comprenant 4 000 gènes cibles pathogènes humains Download PDF

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Publication number
WO2017193833A1
WO2017193833A1 PCT/CN2017/082496 CN2017082496W WO2017193833A1 WO 2017193833 A1 WO2017193833 A1 WO 2017193833A1 CN 2017082496 W CN2017082496 W CN 2017082496W WO 2017193833 A1 WO2017193833 A1 WO 2017193833A1
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dna
capture
pcr
reagent
human pathogenic
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Chinese (zh)
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张巍
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Guangzhou Jiajian Medical Testing Co Ltd
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Guangzhou Jiajian Medical Testing Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads

Definitions

  • the invention relates to the field of gene detection, in particular to a method and a kit for enriching 4000 human pathogenic target genes.
  • 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
  • the protein coding sequence is less than 1.5% in the human genome.
  • regions of unknown function including many repeats, transposons, or pseudogenes.
  • genes and regulatory sequences there are still many regions of unknown function, including many repeats, transposons, or pseudogenes.
  • genes 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.
  • 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.
  • 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.
  • Illumina's Hiseq platform life technologies' PGM platform
  • Roche's 454 platform Roche's 454 platform.
  • High-throughput sequencing technology is extremely important in research and clinical applications due to its ultra-high sequencing capabilities.
  • 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.
  • 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.
  • a method for enriching 4000 human pathogenic target genes comprising the following steps:
  • the reaction system of Pre-capture LM-PCR is as follows:
  • 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.
  • the system of the Post-capture LM-PCR reaction is as follows:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • said steps S2-S5 and step S7 comprise a purification step.
  • 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.
  • 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.
  • the capture reagent comprises 4000 human pathogenic target gene-specific probe libraries, capture magnetic beads and labeling sequences.
  • 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.
  • 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.
  • reaction mixture (A-Tailing Master Mix, 50 ⁇ l):
  • 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:
  • DNA was extracted from 300 ⁇ l of whole blood of each sample according to a conventional method, and 2 ⁇ l 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/ ⁇ l with DNA lysis buffer according to the determined concentration.
  • Total DNA was interrupted with a Q800R ultrasonic disruptor. Specific steps are as follows:
  • the first lane is a molecular size marker
  • 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.
  • the specific method of purification was as follows: 120 ⁇ l of Agencourt R AMPure R XP reagent was added to each sample in a total volume of 190 ⁇ l. 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 ⁇ l 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 ⁇ l 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.
  • the specific method of purification was as follows: 90 ⁇ l of PEG/NaclSPRI R Solution was added to each sample in a total volume of 140 ⁇ l. 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 ⁇ l 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 ⁇ l 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.
  • thermocycler amplifier
  • 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.
  • SeqCap HE Index 4 Oligo 250pmol (0.5 ⁇ l of 500 ⁇ M) SeqCap HE Index 5 Oligo 250pmol (0.5 ⁇ l of 500 ⁇ M) SeqCap HE Index 6 Oligo 250pmol (0.5 ⁇ l of 500 ⁇ M) SeqCap HE Index 7 Oligo 250pmol (0.5 ⁇ l of 500 ⁇ M) SeqCap HE Index 12 Oligo 250pmol (0.5 ⁇ l of 500 ⁇ M) Final concentration 3,000 pmol (6 ⁇ l of 500 ⁇ M)
  • 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.
  • 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.
  • the captured magnetic beads were returned to room temperature 30 minutes before use. The beads were vortexed thoroughly for 15 seconds. Add 100 ⁇ l 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.
  • 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.
  • 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 ⁇ l 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.
  • 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.
  • each tube sample binds to the capture beads-DNA plus 50 ⁇ l 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.
  • the tube was removed from the magnetic stand, 52 ⁇ l 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 ⁇ l 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.

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Abstract

La présente invention décrit un procédé et une trousse comprenant 4 000 gènes cibles pathogènes humains. Le procédé comprend les étapes suivantes : fragmentation d'un ADN pour obtenir un fragment d'ADN, exécution d'une réparation d'extrémité, exécution de la queue A, addition d'un lieur, puis exécution de la LM-PCR pré-capture ; mélange des 4 000 gènes cibles pathogènes humains avec un produit de LM-PCR pré-capture pour obtenir un échantillon de bibliothèque mixte, mélange et hybridation d'une bibliothèque de sonde spécifique des 4 000 gènes cibles pathogènes humains à l'échantillon de bibliothèque mixte, puis exécution d'une capture sur billes magnétiques et lavage d'un ADN ; et exécution de LM-PCR post-capture. La trousse comprend un réactif de réparation d'extrémité d'ADN, un réactif de queue A, un réactif d'addition de lieur, un réactif de LM-PCR, et un réactif de capture. Les 4 000 gènes pathogènes humains sont sélectionnés comme cibles et combinés dans l'invention. Une technologie de capture de sonde biotine est ensuite utilisée pour exécuter l'amplification en une fois et le séquençage à haut débit simultanément, permettant ainsi le criblage simultané de multiple formes mutantes des gènes. Le procédé de test présente un niveau élevé de sensibilité, et peut utiliser 4 000 gènes pathogènes comme outil de diagnostic clinique principal. Le procédé peut réduire le coût, réduire la probabilité de mauvais diagnostic, améliorer le taux de test de vrai positif, et peut faire l'objet d'une promotion dans les environnements cliniques.
PCT/CN2017/082496 2016-05-10 2017-04-28 Procédé et trousse comprenant 4 000 gènes cibles pathogènes humains Ceased WO2017193833A1 (fr)

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CN113388676A (zh) * 2021-06-16 2021-09-14 安吉康尔（深圳）科技有限公司 一种用于检测结节性硬化症基因突变的探针集及其试剂盒
CN114045342A (zh) * 2021-12-01 2022-02-15 大连晶泰生物技术有限公司 一种游离DNA(cfDNA)甲基化突变的检测方法及试剂盒
CN114058681A (zh) * 2021-12-01 2022-02-18 大连晶泰生物技术有限公司 一种基于目标区域捕获的甲基化突变检测方法及试剂盒
CN115491337A (zh) * 2022-11-16 2022-12-20 南京北极光质检技术服务有限公司 聚多巴胺四氧化三铁磁珠捕获致病菌的方法及其在快检的应用
CN119913295A (zh) * 2025-02-20 2025-05-02 北京微未来科技有限公司 一种利用探针杂交捕获技术获得呼吸道多病原全基因组的方法、探针组及试剂盒

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CN112195521A (zh) * 2020-09-11 2021-01-08 翌圣生物科技（上海）有限公司 一种基于转座酶的dna/rna共建库方法、试剂盒及应用
CN113388676A (zh) * 2021-06-16 2021-09-14 安吉康尔（深圳）科技有限公司 一种用于检测结节性硬化症基因突变的探针集及其试剂盒
CN113388676B (zh) * 2021-06-16 2023-07-25 深圳雅济科技有限公司 一种用于检测结节性硬化症基因突变的探针集及其试剂盒
CN114045342A (zh) * 2021-12-01 2022-02-15 大连晶泰生物技术有限公司 一种游离DNA(cfDNA)甲基化突变的检测方法及试剂盒
CN114058681A (zh) * 2021-12-01 2022-02-18 大连晶泰生物技术有限公司 一种基于目标区域捕获的甲基化突变检测方法及试剂盒
CN115491337A (zh) * 2022-11-16 2022-12-20 南京北极光质检技术服务有限公司 聚多巴胺四氧化三铁磁珠捕获致病菌的方法及其在快检的应用
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