Method for detecting DNA structure variation region of leukemia patient and application of method

Abstract

The invention discloses a method for detecting DNA genome structure variation of a leukemia patient. The variation comprises insertion, deletion, fusion, repetition and the like. A specific sequence on a DNA genome of a leukemia patient is complementarily hybridized by using a peptide nucleic acid molecule combined with fluorescence so as to be marked with a marker, then the whole DNA genome is dyed by using a non-specific fluorescence marker, and a plurality of nucleic acid molecules of the marked DNA can be used for analyzing the fluorescence of the specific sequence after being stretched by a nano-channel, so that the detection sensitivity of the leukemia patient is improved. Position information between the specific sequences can be determined, and after the position information and the position information of the specific sequence of the reference genome are compared and analyzed, the structural variation on the DNA genome of the leukemia patient can be analyzed. The method disclosed by the invention is simple to operate, short in time consumption, high in resolution, good in specificity, high in efficiency and good in stability.

Description

Technical Field

[0001] The invention belongs to the field of nanotechnology, and specifically relates to a method for detecting DNA structural variation regions of leukemia patients and an application thereof. Background Art

[0002] Leukemia is a common hematological malignancy, and its occurrence is related to multiple gene mutations. Some genes are originally transcription factors that regulate cell proliferation, differentiation and apoptosis. When genes change, they directly affect the downstream signal transduction pathways, leading to enhanced cell proliferation, blocked apoptosis, differentiation disorders, etc., leading to the occurrence of leukemia. Through genetic testing, gene mutations in leukemia patients can be detected, thereby helping doctors to more accurately judge the condition, formulate treatment plans and predict prognosis. To help leukemia patients and their families better understand the condition and treatment plan, patients and their families can understand the patient's gene mutations through genetic testing results, as well as the impact of these mutations on treatment plans and prognosis. This helps patients and their families better participate in treatment decisions and disease management.

[0003] Variation is the most important factor leading to genomic differences, which can be specifically divided into single base pair variations (SNVs / SNPs), small insertions or deletions (InDels≤50bp) and structural variations (Structure Variation, SVs>50bp). According to the different types of structural variations, structural variations can be further divided into insertions, deletions, duplications, inversions, translocations, copy number variations, etc. of DNA sequences. Structural variations can affect the stability of the genome, the expression regulation of related genes, and thus determine the phenotype of the species. According to statistics, each human genome has more than 20,000 structural variations, and there are more than 1,000 diseases that may be caused by genomic structural variations, such as ALS, schizophrenia and cancer. The impact of structural variations is greater than that of SNVs / SNPs or InDels. Multiple studies have shown that structural variations can better analyze population structure and provide us with further effective information for a deeper understanding of the domestication process of animals and plants.

[0004] Cytogenetic methods, such as karyotyping or fluorescence in situ hybridization (FISH), are commonly used to analyze structural variation in the prior art. However, these methods have the disadvantages of relatively low sensitivity and resolution when detecting structural variation at the medium to small sequence level, and are complex to operate, time-consuming, and costly. Since the 21st century, with the development of experimental techniques such as microarrays and bacterial artificial chromosomes, people have begun to use methods such as array comparative genomic hybridization (aCGH) to detect structural variation. This method has improved resolution, throughput, and sensitivity, but it is still laborious and costly.

[0005] Current second-generation sequencing methods, especially whole-genome sequencing, are widely used to detect structural variations. Although the accuracy is high, the read length is short and cannot span most of the longer SV regions. It can only be detected based on sequence assembly methods and cannot provide sufficient information. Although single-molecule sequencing third-generation sequencing technology (such as PacBio, nanopore sequencing) can measure sequences up to 100kp, its error rate is high (about 15%), the cost is high, and the operation is also complicated.

[0006] Therefore, a highly sensitive and wide-coverage detection method is needed to detect gene mutations in leukemia patients and to adjust treatment plans and prognosis in a timely manner based on the test results. Summary of the invention

[0007] The purpose of the present invention is to provide a method for detecting structural variation analysis of DNA genome in leukemia patients. The method provided by the present invention is not only simple to operate, short in time, high in resolution, good in specificity, high in efficiency, good in stability, but also can cover structural variation at medium to small sequence levels.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a method for detecting DNA structural variation regions in leukemia patients, comprising the following steps:

[0010] (1) extracting multiple nucleic acid molecules from DNA of leukemia patients;

[0011] (2) mixing the multiple nucleic acid molecules of DNA in step (1) with peptide nucleic acid molecules carrying a first marker to obtain a labeled product;

[0012] (3) mixing the labeled product in step (2) with the second label, performing a hybridization reaction, adding the reverse product to magnetic beads for binding, washing, and dissolving in an eluent to obtain a plurality of labeled DNA nucleic acid molecules;

[0013] (4) Passing the multiple nucleic acid molecules of the DNA labeled in step (3) through a nanochannel to linearize the molecules, detecting the fluorescent signals of the two markers on the DNA backbone and the specific sequence for optical imaging, and comparing and analyzing with the reference genome to obtain the structural variation results of the DNA.

[0014] In step (1), the first marker is a fluorescent marker;

[0015] In step (3), the second marker is a non-specific fluorescent marker.

[0016] Preferably, the reference genome is a DNA genome of a healthy person, which is a completely complete genome sequence and has the same sequence and position information as the DNA molecules of leukemia patients that have been processed and labeled in the same way.

[0017] Preferably, the hybridization reaction process in step (3) of the above method includes performing a hybridization reaction in a hybridization solution of a mixture of the labeled product and the second marker in step (2), wherein the hybridization solution includes 150 mM NaCl, 15 mM sodium citrate and 0.02% Tween-20, and the hybridization conditions are pH 7.0 and reaction at 30°C for 1 hour.

[0018] Preferably, the elution buffer is selected from Low TE buffer (10 mM Tris-HCl, 0.1 mM EDTA) or nuclease-free water.

[0019] Preferably, step (4) of the above method also includes assembling the optical imaging of multiple nucleic acid molecules of the labeled DNA to construct a first map of the genomic region of the multiple nucleic acid molecules of the leukemia patient's DNA; comparing the first map of the genomic region with the second map of the reference genome to determine the similarities and differences between the first map and the second map, and then determining the region of structural variation of the leukemia patient's DNA; the multiple nucleic acid molecules of the leukemia patient's DNA cover the entire length or a portion of the length of the detected genomic region.

[0020] Preferably, the peptide nucleic acid sequence has an average repetition frequency of at least 1 site / 100 kb in the genomic region, preferably an average repetition frequency of 5-35 sites / 100 kb, and more preferably an average repetition frequency of 12-17 sites / 100 kb.

[0021] More preferably, said second profile is generated from a reference gene.

[0022] Preferably, the plurality of nucleic acid molecules of the leukemia patient's DNA have a length of at least 150 kb, and the linearization step in step (4) above comprises stretching the nucleic acid molecules to 70%-100% of their total length.

[0023] Preferably, the plurality of nucleic acid molecules of the leukemia patient's DNA are contacted with the peptide nucleic acid molecules.

[0024] Preferably, the plurality of nucleic acid molecules of the leukemia patient's DNA are contacted with molecules of the second marker.

[0025] The present invention also includes the application of the above detection method in the diagnosis, prognosis evaluation and treatment guidance of blood tumor diseases

[0026] Beneficial effects of the present invention:

[0027] 1. Simple operation and short time consumption: The labeling process of the present invention only takes 1 hour, which saves 4-5 hours compared with the existing enzymatic incubation labeling method.

[0028] 2. High resolution and good specificity: Currently, mature karyotype analysis can only stain about 500 bands, so it can only be observed within a resolution range of 5Mbp. This method has about 500,000 marker signals in the whole genome, with a resolution of about 500bp, which is 10,000 times that of karyotype analysis. The peptide nucleic acid used in this method only hybridizes and complements the specific sequence of the genome, and does not require recognition by the enzyme domain, so it has good specificity.

[0029] 3. High efficiency and good stability: The fluorescent marker is directly modified on the peptide nucleic acid molecule, and the peptide nucleic acid directly hybridizes the complementary genome specific sequence, without the intermediate enzyme recognition link, and does not need to recognize the specific base on the specific sequence. Under the same action time, it is more efficient and has a higher success rate. In addition, the peptide nucleic acid molecule is not degraded by any currently known nuclease or protease, and the affinity between it and DNA is more stable than the DNA / DNA hybrid chain, and it can form a very stable PNA2 / DNA three-chain structure with the Hoogsteen bond. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0031] Figure 1 It is a schematic diagram of the process of the present invention.

[0032] Figure 2 This is a diagram of structural variations associated with blood diseases obtained from the analysis of Sample 1 in Example 1.

[0033] Figure 3 Schematic diagram of the location of the PML::RARα fusion variation obtained from the analysis of Sample 1 in Example 1.

[0034] Among them, Ref represents the reference genome, BMA001 represents the genome of sample 1, the black vertical lines on the strips represent the markers of specific sequences, and the purple area represents the location where the fusion occurs.

[0035] Figure 4This is a schematic diagram of the location of the 3.2 kb insertion variation on the NCF4 gene obtained from the analysis of Sample 1 in Example 1. Ref represents the reference genome, BMA001 represents the genome of Sample 1, the black vertical lines on the strips represent markers for specific sequences, and the shaded area represents the location where the insertion occurred.

[0036] Figure 5 This is a schematic diagram of the location of the 11 kb deletion mutation upstream of the BPGM gene obtained from the analysis of Sample 1 in Example 1. Ref represents the reference genome, BMA001 represents the genome of Sample 1, the black vertical lines on the strips represent markers for specific sequences, and the shaded area represents the location where the deletion occurs.

[0037] Figure 6 This is the result of qPCR to verify the PML::RARα fusion variant of sample 1 in Example 1.

[0038] Figure 7 This is a diagram of structural variations associated with blood diseases obtained from the analysis of Sample 2 in Example 2.

[0039] Figure 8 This is a schematic diagram of the location of the 6kb insertion mutation on intron5 of the PTPRD gene obtained from the analysis of sample 2. Ref represents the reference genome, BMA002 represents the genome of sample 2, and the black vertical lines on the strips represent markers for specific sequences.

[0040] Fig. 9 Schematic diagram of the location of the 9.5 kb insertion mutation on intron1 of the ARHGEF18 gene obtained from the analysis of sample 2. Ref represents the reference genome, BMA002 represents the genome of sample 2, the black vertical lines on the strips represent markers for specific sequences, and the shaded area represents the location where the insertion occurred.

[0041] Fig.10 Schematic diagram of the location of the ZNF480::ZNF528 fusion variant obtained from the analysis of sample 2.

[0042] Among them, Ref represents the reference genome, BMA002 represents the genome of sample 2, the black vertical lines on the strips represent markers of specific sequences, and the shaded area represents the location where the fusion occurs.

[0043] Fig.11 Schematic diagram of the location of the LAMA1::PTPRM mutation caused by the 497kb tandem duplication event analyzed for sample 2. Ref represents the reference genome, BMA002 represents the genome of sample 2, the black vertical lines on the strips represent markers for specific sequences, and the shaded area represents the location where the duplication occurred.

[0044] Fig.12This is the result of qPCR to verify the ZNF480::ZNF528 fusion mutation of sample 2 in Example 2. DETAILED DESCRIPTION

[0045] The present invention mixes multiple nucleic acid molecules of leukemia patient DNA with peptide nucleic acid molecules carrying a first marker, and the labeled product undergoes hybridization reaction with a second marker. The multiple nucleic acid molecules of leukemia patient DNA that have been labeled twice undergo DNA linearization through a nanochannel, and finally detect the fluorescence on the DNA skeleton and specific sequence for optical imaging, assemble the images of the multiple nucleic acid molecules of the leukemia patient DNA to construct a first map of the genomic region of the nucleic acid molecules of the leukemia patient DNA, and compare and analyze with the second map of the reference genome to obtain the structural variation results of the leukemia patient DNA.

[0046] The schematic diagram of the detection method of the present invention is as follows Figure 1 shown.

[0047] The present invention provides a method for detecting DNA structural variation regions in leukemia patients, which specifically comprises the following steps:

[0048] (1) extracting multiple nucleic acid molecules from DNA of leukemia patients;

[0049] (2) mixing the multiple nucleic acid molecules of DNA in step (1) with peptide nucleic acid molecules carrying a first marker to obtain a labeled product;

[0050] (3) mixing the labeled product in step (2) with the second label, performing a hybridization reaction, adding the product after the reaction to magnetic beads for binding, washing, and dissolving in an eluent to obtain a plurality of labeled DNA nucleic acid molecules;

[0051] (4) Passing the multiple nucleic acid molecules of the DNA labeled in step (3) through a nanochannel to linearize the molecules, detecting the fluorescent signals of the two markers on the DNA backbone and the specific sequence for optical imaging, and comparing and analyzing with the reference genome to obtain the structural variation results of the DNA.

[0052] Preferably, the multiple nucleic acid molecules of the leukemia patient DNA of the present invention are DNA, including double-stranded DNA and single-stranded DNA.

[0053] Preferably, the first marker of the present invention is a fluorescent marker modified on the peptide nucleic acid molecule, and labeling is achieved thereby; the second marker is a non-specific nucleic acid fluorescent marker selected from EB, SYBR Green I, SYBRGold, SYBR safe, GelStar, PI, DAPI, Hoechst and DRAQ5.

[0054] The multiple nucleic acid molecules of the leukemia patient's DNA of the present invention have a length of at least 150kb, wherein the linearization step includes stretching the nucleic acid molecules to 70%-100% of their total length; the multiple nucleic acid molecules cover the entire length or a partial length of the genomic region, and the second map is generated independently from the genomic sequence information or from a second nucleic acid sample.

[0055] The peptide nucleic acid molecule sequence in the genomic region of the nucleic acid molecule constructed by the present invention is selected from the peptide nucleic acid base sequence table for human genome marking in Table 1 of the present invention; at least one repeated specific sequence of the peptide nucleic acid sequence in the genomic region is a reverse complementary sequence of the peptide nucleic acid sequence, preferably with an average repetition frequency of 5-35 sites / 100kb, and more preferably with an average repetition frequency of 12-17 sites / 100kb.

[0056] The embodiments of the present invention involve the use of peptide nucleic acids (PNA) molecules to mark specific positions on nucleic acid molecules, and the sequence of the peptide nucleic acid molecules is reverse complementary to the specific sequence. Peptide nucleic acid is a nucleotide-like substance formed by replacing the pentose phosphodiester bond backbone in DNA with a polyamide bond composed of an electrically neutral peptide chain amide 2-aminoethylglycine. PNA also carries four bases, ATCG, and can recognize the corresponding base sequence according to the principle of base complementarity. PNA oligomers have good thermal stability, high binding affinity and the particularity of being independent of salt concentration. They are not degraded by any nuclease or protease currently known.

[0057] Due to the neutral backbone of PNA, there is no phosphate group in its molecule, so PNA chains are more likely to bind to DNA chains with negatively charged complementary sequences, and the formed complex molecules are more stable than DNA / DNA hybrid chains. When a PNA molecule invades the target DNA sequence, a DNA chain is displaced and rapidly and highly specifically binds to its complementary DNA through the principle of base complementary pairing. After that, the second PNA molecule forms a very stable PNA2 / DNA triple-strand structure with the PNA / DNA double helix through Hoogsteen bonds, leaving the unbound chain in a single-stranded state. As long as the PNA is modified with a fluorescent marker, the specific sequence on the DNA molecule can be stably labeled.

[0058] When these labeled nucleic acid molecules are linearly stretched in the nanochannel, the spatial distance between the fluorescent marker signals from the peptide nucleic acid can be measured consistently and quantitatively, thus generating a unique characteristic image reflecting the region. Based on a series of characteristic images, a map reflecting the structural information of the entire genome can be assembled.

[0059] In principle, as long as a specific sequence that meets the above conditions can be found, a peptide nucleic acid with fluorescent modification that is reverse complementary to the specific sequence can be designed. Based on the frequency of occurrence of the specific sequence in the genome, the following is a list of preferred peptide nucleic acid base sequences.

[0060] Table 1 Peptide nucleic acid base sequences used for human genome labeling

[0061]

[0062]

[0063] Embodiments of the present invention also relate to the analysis of a given labeled nucleic acid by comparison with a reference genome or a data set of a reference genome. A reference genome is a completely complete genome sequence. A reference genome can be a digitally stored data set of sequence information obtained by computer analysis that can be compared with the sample to be analyzed, or it can be sequence and position information that is processed and labeled in the same way as the DNA molecules of leukemia patients, or a DNA genome of a healthy person.

[0064] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0065] The ultra-high molecular weight extraction kit of the present invention was purchased from Bone Marrow Aspirate (BMA) DNA Isolation Kit v2, Bionano. The various instruments, equipment, raw materials or reagents used in the examples are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, and can also be prepared according to conventional methods well known to those skilled in the art.

[0066] Example 1

[0067] The peptide nucleic acid sequence (GAAGAGC) selected in Table 1 was labeled with a green fluorescent marker SYTO9 and synthesized for later use. Ultra High Molecular Weight Extraction Kit (Bone Marrow Aspirate (BMA) DNA Isolation Kit v2, Bionano) extract samples (bone marrow sample 1 from AML patient, 1.5x10 6 cells).

[0068] DNA was mixed with peptide nucleic acid molecules (70 nM) labeled with fluorescent markers and DAPI dye (40 μM) and reacted at 30°C for 1 hour in hybridization solution (150 mM NaCl, 15 mM sodium citrate, 0.02% Tween-20, pH 7.0). After hybridization, magnetic beads were added for binding, washed, and finally dissolved in the elution solution.

[0069] The labeled DNA molecules are linearized through the nanochannel of the Saphyr system (Bionano Genomics), and then the fluorescence on the DNA backbone and specific sequences is detected for optical imaging and comparative analysis with the reference genome.

[0070] The results are as follows Figure 2 As shown in Figure 1, the structural variations of the sample, such as insertions, deletions, and translocations, that are associated with blood diseases, are directly visualized. Further refined analysis revealed a PML::RARα fusion variation, as shown in Figure 1. Figure 3 The specific location information is shown in Table 2 below:

[0071] Table 2: Specific location information

[0072] type translocation RefcontigID1 17 RefcontigID2 15 RefStartPosition 40,348,183 RefEndPosition 74,011,987 Size(bp) -1 Confidence 1.00 Presentin% ofControl 0.0 VAF 0.39 OverlapGenes PML; RARA PutativeGeneFusion PML-RARA

[0073] A 3.2 kb insertion mutation in the NCF4 gene, the insertion position may involve exon 2 to 5, such as Figure 4 As shown in Table 3 below:

[0074] Table 3: NCF4 gene mutation information

[0075]

[0076]

[0077] A mutation with a deletion of 11 kb upstream of the BPGM gene may affect the promoter, such as Figure 5 As shown in Table 4 below:

[0078] Table 4: BPGM gene mutation information

[0079] type deletion RefcontigID1 7 RefcontigID2 7 RefStartPosition 134,620,893 RefEndPosition 134,641,476 Size(bp) 11,640 Confidence 0.99 Presentin% ofControl 0.0 VAF 0.49 NearestNon-OverlapGene BPGM

[0080] Verification Example 1:

[0081] In order to test the effect of the present invention, A fluorescence quantitative PCR (qPCR) experiment was designed. To detect the results of the fusion gene. According to the location of the fusion of the present invention, the fusion primers and probes are designed, and then the primers and probes of the internal reference gene ABL1 are designed. The same bone marrow blood sample 1 is used to extract its RNA nucleic acid, reverse transcribe it into cDNA, and then perform qPCR. The specific experimental steps are as follows:

[0082] 1. RNA extraction

[0083] (1) Add 5 volumes of 1X red blood cell lysis buffer to 1 volume of sample.

[0084] (2) Incubate on ice for 10-15 min. Vortex twice during incubation.

[0085] (3) Centrifuge at 4°C, 2100 rpm for 10 min and completely remove the supernatant.

[0086] (4) Add 1X red blood cell lysis buffer (twice the original volume of the sample) to the white blood cell pellet and resuspend the cells.

[0087] (5) Centrifuge at 2100 rpm at 4°C for 10 min and completely remove the supernatant.

[0088] (6) Add lysis buffer RLH (add β-mercaptoethanol before use) to the leukocyte pellet and vortex or use a pipette to mix.

[0089] (7) The solution was transferred to the filter column CS (the filter column CS was placed in the collection tube), centrifuged at 12000 rpm for 2 min, the filter column was discarded, and the filtrate was collected.

[0090] (8) Add 1 volume of 70% ethanol to the filtrate and mix well. Transfer the resulting solution and precipitate into adsorption column CR4 (the adsorption column is placed in a collection tube). Centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column CR4 back into the collection tube.

[0091] (9) Add 700 μl of deproteinized solution RW1H (with anhydrous ethanol added in advance) to the adsorption column CR4, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid in the collection tube.

[0092] (10) Add 500 μl of rinsing solution RW (with anhydrous ethanol added in advance) to the adsorption column CR4, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 30 sec, pour out the waste liquid in the collection tube, and put the adsorption column CR4 back into the collection tube. Repeat once.

[0093] (11) Centrifuge at 12000 rpm for 2 min and discard the waste liquid. Place the adsorption column CR4 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.

[0094] (12) Transfer the adsorption column CR4 into a new RNase-Free centrifuge tube, add 30 μl RNase-Free ddH2O, leave at room temperature for 2 min, and centrifuge at 12000 rpm for 2 min to obtain RNA solution.

[0095] 2. Reverse transcription of RNA into cDNA

[0096] (1) Remove the reaction buffer, random primer, and RNA and dissolve them on ice. After they are fully dissolved, vortex to mix and centrifuge briefly, then place on ice for later use.

[0097] (2) Gently flick Enzyme Mix to mix thoroughly and place on ice for later use.

[0098] (3) The RNA sample was diluted to 4 μl with nuclease-free water, first reacted at 70°C for 5 min to open the secondary structure, and then quickly placed on ice for at least 5 min.

[0099] (4) Add the following reagents to the RNA sample tube:

[0100] Table 5: RNA sample tube reagent components

[0101] Components Volume (μl) Previous RNA 4 ReactionBuffer,RandomPrimer 4 EnzymeMix 2 Total 10

[0102] (5) Use a pipette to gently pipette up and down to mix (5-10 times). After a brief centrifugation, place the tube in a PCR instrument and perform the following procedure:

[0103] Table 6: PCR instrument execution program

[0104] Temperature(℃) time 25 5min 42 60min 70 15min 4 Hold

[0105] 3. qPCR experiment

[0106] (1) Take out the primer mix and probe mix, place them on ice to dissolve, vortex and mix thoroughly, centrifuge briefly, and place them on ice for later use.

[0107] (2) TaqPro U+Multiple Probe qPCR Mix (Novozyme) was gently flicked to mix and placed on ice for later use.

[0108] (3) The reaction system was prepared according to the following table:

[0109] Table 7: Reaction system composition

[0110] Components Volume (μl) Previous cDNA 5 Primer Mix (6pM) 1 Probe Mix (4pM) 1 TaqProU+MultipleProbeqPCRMix 10 Nuclease-free water 3 Total 20

[0111] Among them, the primer sequences for detecting the fusion position are:

[0112] 5'-GGCACCCGCAAGACCAACA-3' and

[0113] 5'-GGGCACTATCTCTTCAGAACTGCT-3', the probe sequence is: 5'-FAM-CAACCCCAACCACCGCACCCCT-3'-TAMRA. The primer sequences for detecting the internal reference gene ABL1 are: 5'-CTCAGATCTCGTCAGCCATG-3' and 5'-CCCCTGTCATCAACCTGCTCA-3', and the probe sequence is: 5'-VIC-TCCCCTACCAGGCAGTTTCGG-3'-TAMRA.

[0114] (4) Use a pipette to gently pipette up and down to mix (5-10 times). After a brief centrifugation, seal the tube with a sealing film and place it in a PCR instrument to perform the following procedure. Collect fluorescence at the end of each round:

[0115] Table 8: PCR instrument execution program

[0116]

[0117] 4. Result analysis: The results are as follows Figure 6 As shown in the figure, it can be seen that sample 1 does have the PML::RARα fusion mutation, which is consistent with the results of the method of the present invention.

[0118] Embodiment 2:

[0119] The peptide nucleic acid sequence (GGATAC) molecule selected from Table 1 was labeled with a green fluorescent marker SYTO9 and synthesized for later use. The sample (bone marrow sample 2 of AML patient, 1.5×10 6 cells). DNA was mixed with peptide nucleic acid molecules (70nM) and DAPI dye (40μM) labeled with fluorescent markers, and reacted at 30°C for 1h in hybridization solution (150mM NaCl, 15mM sodium citrate, 0.02% Tween-20, pH 7.0). After hybridization, magnetic beads were added for binding, washed, and finally dissolved in the eluent. The labeled DNA molecules were linearized by the nanochannel of the Saphyr system (Bionano Genomics), and then the fluorescence on the DNA backbone and specific sequences was detected for optical imaging and comparative analysis with the reference genome.

[0120] The structural variations of the sample, such as insertions, deletions, and duplications, which are related to blood diseases, are directly visualized. Figure 7Further refined analysis revealed two insertion mutations, namely a 6 kb insertion mutation on intron5 of the PTPRD gene and a 9.5 kb insertion mutation on intron1 of the ARHGEF18 gene, as shown in Figure 8 , 9 As shown in Table 9 below:

[0121] Table 9: Variant site information

[0122]

[0123]

[0124] An 81Kb deletion caused the ZNF480::ZNF528 fusion mutation, the specific location information is as follows Fig.10 As shown in Table 6 below:

[0125] Table 10: Specific location information

[0126] type deletion RefcontigID1 19 RefcontigID2 19 RefStartPosition 52,320,340 RefEndPosition 52,410,477 Size(bp) 81,926 Confidence 0.99 Presentin% ofControl 0.0 VAF 0.95 OverlapGenes ZNF480; ZNF610; ZNF880; ZNF528-AS1 PutativeGeneFusion ZNF480-ZNF528

[0127] A 497 kb tandem duplication event caused the LAMA1::PTPRM mutation, such as Fig.11 As shown in Table 7 below:

[0128] Table 11: LAMA1::PTPRM mutation information

[0129] type Duplication_split RefcontigID1 18 RefcontigID2 18 RefStartPosition 7,079,454 RefEndPosition 7,577,072 Size(bp) 497,618 Confidence -1.0 Presentin% ofControl 0.0 VAF 1.00 OverlapGenes LAMA1; LRRC30; LOC112577592; PTPRM PutativeGeneFusion LAMA1-PTPRM

[0130] Verification Example 2:

[0131] In order to further test the detection effect of the present invention, another qPCR experiment was designed for the results of the fusion gene of sample 2. Primers and probes were designed according to the location of the fusion of sample 2 of the present invention. The primer sequences for detecting the fusion location were: 5'-TGCATCAAAGGTGTGAAC-3' and 5'-CTTACACCCAGAAATTTTCCT-3', respectively, and the probe sequence was: 5'-FAM-TTGTTTCCTGCATTACTTCCCA-3'-TAMRA. The primers, probe sequences and other steps for detecting the internal reference gene ABL1 were the same as those in Verification Example 1.

[0132] The results are as follows Fig.12 As shown in the figure, it can be seen that sample 2 does have the ZNF480-ZNF528 fusion mutation, which is consistent with the results of the method of the present invention.

[0133] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting DNA structural variation regions in leukemia patients, characterized in that: The following steps are involved: (1) extracting multiple nucleic acid molecules from DNA of leukemia patients; (2) mixing the multiple nucleic acid molecules of DNA in step (1) with peptide nucleic acid molecules carrying a first marker to obtain a labeled product; (3) mixing the labeled product in step (2) with the second label, performing a hybridization reaction, adding the reverse product to magnetic beads for binding, washing, and dissolving in an eluent to obtain a plurality of labeled DNA nucleic acid molecules; (4) passing the multiple nucleic acid molecules of the DNA labeled in step (3) through a nanochannel to linearize the molecules, detecting the fluorescent signals of the two markers on the DNA backbone and the specific sequence for optical imaging, and comparing and analyzing with the reference genome to obtain the structural variation results of the DNA; In step (1), the first marker is a fluorescent marker; The peptide nucleic acid molecule sequence in step (2) is reverse complementary to the specific sequence of multiple nucleic acid molecules of DNA; In step (3), the second marker is a non-specific fluorescent marker.

2. The method according to claim 1, characterized in that: The reference gene is a DNA genome of a healthy population, which is a completely complete genome sequence and has sequence and position information that is processed and labeled in the same way as the DNA molecules of leukemia patients.

3. The method according to claim 1, characterized in that: The hybridization reaction process in step (3) includes hybridizing the mixture of the labeled product in step (2) and the second marker in a hybridization solution, wherein the hybridization solution includes 150 mM NaCl, 15 mM sodium citrate and 0.02% Tween-20; the second marker is selected from EB, SYBR Green I, SYBRGold, SYBR safe, GelStar, PI, DAPI, Hoechst and DRAQ5.

4. The method according to claim 1, characterized in that: The step (4) also includes assembling the optical imaging of the multiple nucleic acid molecules of the labeled DNA to construct a first map of the genomic region of the multiple nucleic acid molecules of the leukemia patient's DNA; comparing the first map of the genomic region with a second map of the reference genome to determine the similarities and differences between the first map and the second map, and then determining the region of structural variation of the leukemia patient's DNA; the multiple nucleic acid molecules of the leukemia patient's DNA cover the entire length or a portion of the length of the detected genomic region.

5. The method according to any one of claims 1 to 4, characterized in that: The peptide nucleic acid sequence has an average repetition frequency of 5-35 sites / 100 kb in the genomic region.

6. The method according to claim 4, characterized in that: The second profile is generated from a reference gene.

7. The method according to any one of claims 1 to 4, characterized in that: The multiple nucleic acid molecules of the leukemia patient's DNA have a length of at least 150 kb, wherein the linearization step in step (4) of claim 1 includes stretching the multiple nucleic acid molecules of the labeled DNA to 70%-100% of their total length.

8. The method according to any one of claims 1 to 7, characterized in that: The plurality of nucleic acid molecules of the leukemia patient's DNA are contacted with the peptide nucleic acid molecules.

9. The method according to any one of claims 1 to 8, characterized in that: The plurality of nucleic acid molecules of the leukemia patient's DNA are contacted with molecules of the second marker.

10. Use of the method according to any one of claims 1 to 9 in the diagnosis, prognosis assessment and treatment guidance of blood tumor diseases.

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