Genetic analysis method

Abstract

This genetic analysis method comprises: isolating fixed maternal blood cells one by one; de-crosslinking DNA from protein in a proteinase-containing buffer for each of the isolated blood cells; extracting the de-crosslinked DNA; subjecting the extracted DNA to whole genome amplification; and amplifying a target sequence by using a plurality of specific markers from the amplified whole genome amplification product.

Description

Genetic analysis methods

[0001] The present disclosure relates to a method for genetic analysis of maternal blood cells.

[0002] Previously, attempts have been made to concentrate or isolate fetal cells from maternal whole blood for analysis, and there is a strong demand for minimally invasive genetic testing using fetal cells as an alternative to highly invasive amniocentesis and chorionic villus sampling.

[0003] Patent Document 1 discloses a screening test method for determining whether a prenatal fetus has an aneuploidy, such as Down syndrome, by analyzing cell-free DNA contained in maternal whole blood. Patent Document 2 discloses a method for obtaining chromosomal DNA from fetal cells. Patent Document 3 discloses a method for decrosslinking fixed tissue sections and obtaining nucleic acids. Patent Document 4 discloses genetic analysis of placenta-derived fetal trophoblast cells. Patent Document 5 discloses a method for preparing and detecting fetal cells from a sample derived from a pregnant woman using a magnetic substance-antibody conjugate. Patent Document 6 discloses a method for digesting and dissolving crosslinked proteins from a fixed biological sample, specifically protein-crosslinked nucleic acids, with a protease. Patent Document 7 discloses a process for separating anucleated red blood cells from a nucleated cell-enriched fraction derived from blood and a method for detecting fetal abnormalities using fetal mesenchymal stem cells. Patent Document 8 discloses a method for isolating fetal cells from a sample from a pregnant woman. Patent Document 9 discloses a method for isolating and detecting fetal membrane cells from maternal blood using a fetal membrane cell marker. Non-Patent Document 1 discloses a method for amplifying the whole genome from a single cell. Non-Patent Document 2 discloses a method for decrosslinking multiple immobilized cells.

[0004] Patent Document 1: EP 2473638 B1 Patent Document 2: WO 2018 / 123220 Patent Document 3: WO 2011 / 104027 Patent Document 4: WO 2020 / 245459 Patent Document 5: Japanese Patent Application Laid-Open No. 2023-156347 Patent Document 6: Japanese Patent Application No. 2023-521579 Patent Document 7: US 2022 / 0389384 A1 Patent Document 8: US 2023 / 0295683 A1 Patent Document 9: EP 4445135 A1

[0005] Unlicensed Document 1: QIAGEN REPLI-g Advanced DNA Single Cell Kit Handbook (https: / / www.qiagen.com / jp / resources / download.aspx?id=6e9c72bc-1959-4350-9527-df8e1e1258b9&lang=en) Non-licensed literature 2: U. Oba, K. Kohashi, Y. Sangatsuda, Y. Oda, K. Sonoda, S. Ohga, K. Yoshimoto, Y. Arai, S. Yachida, T. Shibata, T. Ito & F. Miura: An efficient procedure for the recovery of DNA from formalin-fixed paraffin-embedded tissue sections. Biol Methods Protoc. 2022 Jul 26;7(1):bpac014. doi: 10.1093 / biomethods / bpac014

[0006] In the case of the test using cell-free DNA disclosed in Patent Document 1, the test accuracy, particularly the positive predictive value, is low, so if the test result is positive, it is necessary to undergo highly invasive amniocentesis or chorionic villus sampling. Furthermore, Patent Document 2 describes a method for obtaining chromosomal DNA derived from fetal cells, but does not describe a specific genetic analysis method. Furthermore, Patent Document 3 describes a method for decrosslinking fixed tissue sections and obtaining nucleic acids, but does not describe a specific genetic analysis method. In addition, Patent Document 4 describes genetic analysis using placenta-derived fetal trophoblast cells, but this technology cannot be applied to the analysis of fetal nucleated red blood cells. Furthermore, placenta-derived fetal trophoblast cells exhibit so-called mosaicism, which differs from the actual genetic information of the fetus, posing problems for tests using placenta-derived fetal trophoblast cells.

[0007] The above-mentioned Non-Patent Document 1 discloses a method for amplifying the whole genome from a single cell, but when whole genome amplification is performed on a single cell immobilized by the same method, amplification is not performed or amplification is biased, making it impossible to obtain the target marker and making genetic analysis impossible. Furthermore, the above-mentioned Non-Patent Document 2 discloses a method for decrosslinking multiple immobilized cells, but when whole genome amplification is subsequently attempted on a single cell immobilized by the same method, amplification is not performed or amplification is biased, making it impossible to obtain the target marker and making genetic analysis impossible.

[0008] In order to avoid the above-mentioned mosaicism, it is extremely difficult to concentrate or isolate fetal nucleated red blood cells from maternal whole blood for genetic analysis. Furthermore, since it takes time from transportation to analysis after blood collection, fetal nucleated red blood cells are lost, fused, destroyed, or killed during the process, making subsequent analysis difficult and impractical. Furthermore, when cells are fixed in order to suppress such cell changes or deterioration over time, genome amplification is difficult in a single isolated cell, making genetic analysis, particularly next-generation sequencer (NGS) analysis, difficult.

[0009] Furthermore, when genome amplification was performed after decrosslinking of immobilized cells, amplification did not occur in a single isolated cell, or amplification was biased, making genetic analysis, particularly NGS analysis, difficult. In particular, analyses requiring quantitative analysis, such as aneuploidy analysis, were too inaccurate to be put to practical use. Furthermore, nucleated red blood cells exist as those of fetal and maternal origin, and antibodies also exhibit nonspecific adsorption, making it difficult to isolate only fetal cells using antibodies. Therefore, fetal cell identification is also necessary, but attempting to analyze multiple cells individually results in low throughput and high costs, making practical application difficult.

[0010] The present disclosure provides a genetic analysis method that reduces variation in genome amplification after decrosslinking each individual fixed and isolated cell to reduce changes or deterioration of cells over time, such as cell loss, fusion, destruction, or death.

[0011] A genetic analysis method according to a first aspect of the present disclosure includes isolating immobilized maternal blood cells one by one, decrosslinking proteins and DNA from each of the isolated blood cells using a proteinase-containing buffer, extracting the decrosslinked DNA, performing whole-genome amplification of the extracted DNA, and amplifying target sequences from the amplified whole-genome amplification product using multiple specific markers.

[0012] A genetic analysis method according to a second aspect of the present disclosure has the same configuration as that of the first aspect, but wherein the specific marker is a short tandem repeat (STR) marker.

[0013] A genetic analysis method according to a third aspect of the present disclosure has the same configuration as the first or second aspect, and furthermore, the STR marker corresponds to a target sequence of a gene belonging to at least one of chromosome 13, chromosome 18, chromosome 21, and a sex chromosome.

[0014] A genetic analysis method according to a fourth aspect of the present disclosure has the same configuration as any one of the first to third aspects, wherein the blood cells are fixed with a 0.01 to 6% by mass paraformaldehyde solution or a 0.001 to 0.2% by mass glutaraldehyde solution immediately after collection from the subject.

[0015] A genetic analysis method according to a fifth aspect of the present disclosure has the same configuration as any one of the first to fourth aspects, and further comprises heating at 70 to 100° C. during the decrosslinking.

[0016] A genetic analysis method according to a sixth aspect of the present disclosure has the same configuration as any one of the first to fifth aspects, wherein the proteinase is proteinase K.

[0017] A genetic analysis method according to a seventh aspect of the present disclosure includes, in addition to the configuration of any one of the first to sixth aspects, adding a 5 to 1,000 mM buffer to adjust the pH to 7 to 9 during the decrosslinking.

[0018] A genetic analysis method according to an eighth aspect of the present disclosure has the same configuration as the seventh aspect, wherein the buffer is a Tris-HCl buffer.

[0019] A genetic analysis method according to a ninth aspect of the present disclosure has the same configuration as any one of the first to eighth aspects, but further comprises amplifying the target sequence using a next-generation sequencer.

[0020] Since the aspects of the present disclosure are configured as described above, it is possible to provide a genetic analysis method that suppresses changes or deterioration of cells over time, such as cell loss, fusion, destruction, or death, and that suppresses variation in genome amplification after de-crosslinking each of the immobilized and isolated cells.

[0021] It is a flowchart showing an overview of the steps in an embodiment of the present disclosure. It is a schematic diagram showing the state of DNA and proteins in immobilized cells. It is a schematic diagram showing the state where proteins are decomposed from the state of FIG. 2-1. It is a schematic diagram showing the state where decrosslinking is performed from the state of FIG. 2-2. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 1. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 2. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 3. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 4. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 5. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 6. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 7. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 8. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 9. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 10. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 11. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 12. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 13. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 14. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 15. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 16. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 17. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 18. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 19. It is a graph showing the amplification result of the target sequence by the STR marker in Sample 20.

[0022] Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. In this disclosure, when a numerical range is indicated as "A to B," it means "not less than A and not more than B" unless otherwise specified.

[0023] 1 is a flowchart showing an outline of the steps in an embodiment of the present disclosure. First, prior to the step shown in S1, the immobilization and isolation of maternal blood cells will be described.

[0024] [Immobilization of maternal blood cells] Maternal blood cells specifically refer to blood cells, the majority of which are red blood cells, followed by white blood cells and platelets. Maternal blood cells also include fetal nucleated red blood cells, which are the subject of analysis in the present disclosure.

[0025] Nucleated red blood cells are erythroblasts that have lost the ability to divide. Red blood cells are generated by the differentiation and maturation of hematopoietic stem cells. During the process of differentiation and maturation, myeloid progenitor cells, erythroid-megakaryocytic progenitor cells, early erythroid progenitor cells (BFU-E), late erythroid progenitor cells (CFU-E), proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, normochromatic erythroblasts, reticulocytes, and red blood cells appear in this order from hematopoietic stem cells.

[0026] Erythroblasts include proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, and normochromatic erythroblasts. During the differentiation process of normochromatic erythroblasts into reticulocytes, the nucleus is lost from the blood cell. Normochromatic erythroblasts usually lose the ability to divide.

[0027] Nucleated red blood cells are normally found in the bone marrow, but very small amounts of nucleated red blood cells are found in the blood. Also, very small amounts of nucleated red blood cells of both maternal and fetal origin are found in maternal blood. In maternal blood, the number of nucleated red blood cells of fetal origin is usually lower than the number of nucleated red blood cells of maternal origin.

[0028] Although it is desirable to analyze maternal blood cells immediately after collection from the mother, in many cases several days pass between collection and analysis, and therefore preservation is necessary. Therefore, for preservation, maternal blood cells are subjected to fixation treatment. For fixation treatment, a fixative consisting of a paraformaldehyde solution or a glutaraldehyde solution is used.

[0029] In fixation treatment using a paraformaldehyde solution as a fixative, the paraformaldehyde concentration is 0.1 to 6% by mass, preferably 0.1 to 5% by mass, and more preferably 1 to 4% by mass, and the fixation time is 5 to 30 minutes, preferably 10 to 20 minutes, and more preferably 10 to 15 minutes. In fixation treatment using a glutaraldehyde solution as a fixative, the glutaraldehyde concentration is 0.001 to 0.2% by mass, preferably 0.001 to 0.1% by mass, and more preferably 0.01 to 0.08% by mass, and the fixation time is 5 to 30 minutes, preferably 10 to 20 minutes, and more preferably 10 to 15 minutes.

[0030] Immediately after collection from the mother, maternal blood cells are removed using the density gradient centrifugation method described above (see WO 2012 / 023298 A1) or a commercially available hemolysis reagent such as OptiLyse (Beckman Coulter) to concentrate fetal nucleated red blood cells and recover them as blood cell components. The recovered blood cells are dispersed in the fixative solution. After the fixation time has elapsed, the blood cell components recovered by a second centrifugation treatment are dispersed in an appropriate buffer and stored until analysis. Alternatively, blood may be collected from the mother using a blood collection tube containing a fixative solution, and red blood cells and other particles may be removed using the density gradient centrifugation method or a hemolysis reagent to concentrate fetal nucleated red blood cells and recover them as blood cell components.

[0031] The maternal blood cells fixed as described above are isolated one by one using a commercially available cell sorter (for example, BD or Beckman) prior to analysis.

[0032] [A. Decrosslinking of Cells] In fixed blood cells, as shown in Figure 2-1, DNA 10 is linked to protein 20 by crosslinks 30, making it difficult to separate DNA 10. Therefore, decomposition of proteins and solubilization of nucleic acids shown in S1 of Figure 1, and dissociation of crosslinks shown in S2 are carried out.

[0033] <A-1. Protein Degradation and Nucleic Acid Solubilization> First, the fixed and isolated blood cells are subjected to the step shown in S1 of Figure 1. That is, a buffer is added to each isolated cell fraction to adjust the pH to a predetermined level, and then proteinase is added and the mixture is heated to degrade the protein and solubilize the nucleic acid. As a result, the protein 20 shown in Figure 2-1 is degraded into amino acids 21 as shown in Figure 2-2. However, at this stage, peptide 22 is linked to DNA 10 via crosslink 30.

[0034] As the buffer (buffer solution), various buffers, including commercially available products, such as phosphate buffer or Tris-HCl buffer, can be used. The buffer concentration is 5 to 1,000 mM, preferably 20 to 800 mM, and more preferably 50 to 200 mM. When the buffer is a phosphate buffer, the buffer concentration is 5 to 400 mM, preferably 5 to 300 mM, and more preferably 5 to 200 mM. When the buffer is a Tris-HCl buffer, the buffer concentration is 100 to 1,000 mM, preferably 300 to 1,000 mM. The pH after adjustment with the buffer is 7 to 9, preferably 7.5 to 8.5, and more preferably 7.7 to 8.3. As the proteinase, various proteinases, including commercially available products, such as aspartic proteinase, metalloproteinase, serine proteinase, thiol proteinase, or proteinase K, can be used. The concentration of the proteinase is 0.001 to 200 mg / mL, preferably 0.01 to 10 mg / mL, and more preferably 0.05 to 5 mg / mL. When the proteinase is an aspartic proteinase, its concentration is 0.001 to 100 mg / mL, preferably 0.005 to 10 mg / mL, more preferably 0.005 to 1 mg / mL, and particularly preferably 0.01 to 0.05 mg / mL. When the proteinase is a serine proteinase, its concentration is 0.001 to 100 mg / mL, preferably 0.05 to 10 mg / mL, and more preferably 0.1 to 2 mg / mL. When the proteinase is a thiol proteinase, its concentration is 0.01 to 50 mg / mL, preferably 1 to 40 mg / mL, and more preferably 10 to 30 mg / mL. When the proteinase is proteinase K, its concentration is 0.01 to 200 mg / mL, preferably 1 to 150 mg / mL, more preferably 40 to 120 mg / mL, and particularly preferably 50 to 100 mg / mL. The temperature for the heat treatment is 40 to 70°C, preferably 50 to 60°C, and more preferably 54 to 58°C, and the heating time is 10 to 120 minutes, preferably 30 to 90 minutes, and more preferably 45 to 75 minutes. The buffer preferably contains a sugar.Examples of sugars include glucose, altrose, galactose, mannose, idose, fructose, sorbose, tagatose, lactose, sucrose, kojibiose, sophorose, nigerose, laminaribiose, maltose, cellobiose, isomaltose, gentiobiose, and trehalose, with trehalose being more preferred. The concentration of trehalose contained in the buffer is not particularly limited, but is preferably 0.001 to 1 mol / L, more preferably 0.01 to 0.4 mol / L, and particularly preferably 0.1 to 0.3 mol / L.

[0035] 1, an aminocarboxylic acid chelating agent such as EDTA, a phosphonic acid chelating agent such as HEDP, a chelating metal salt such as an EDTA metal salt, an anionic surfactant such as sodium dodecyl sulfate solution, or a nonionic surfactant such as a glycerin fatty acid ester or a polyoxyethylene alkyl ether may be used in combination. According to embodiments of the present disclosure, it has been shown that amplification of a target sequence can be performed without a chelating agent or a surfactant, and therefore EDTA or sodium dodecyl sulfate may not be included.

[0036] <A-2. Dissociation of Crosslinks> Next, in the step shown in S2 of Figure 1, the nucleic acid (DNA) solubilized by decomposing the protein 20 in step A-1 above is heat-treated to dissociate the crosslinks. The heat treatment temperature is 70 to 100°C, preferably 75 to 95°C, and more preferably 80 to 90°C, and the heating time is 10 to 120 minutes, preferably 30 to 90 minutes, and more preferably 45 to 75 minutes. This dissociates the crosslinks 30 between the DNA 10 and the peptide 22, as shown in Figure 2-3.

[0037] [B. Whole Genome Amplification] Subsequently, in the step shown in S3 of FIG. 1, a WGA (whole genome amplification) reaction mix is ​​added to the cell lysate solution obtained by dissociating the crosslinks in step A-2 above, and the whole genome is amplified, followed by heating to inactivate the polymerase. Although a commercially available whole genome amplification kit can be used as the WGA reaction mix, it is preferable to perform whole genome amplification using the WGA reaction mix used in the MDA method (multiple displacement amplification). Whole genome amplification can be performed by reaction at about 30 ° C. for about 18 hours, but may be adjusted or changed as appropriate depending on the type of whole genome amplification kit or the amount or bias of genome amplification. The polymerase can be inactivated by heat treatment at about 96°C for about 5 minutes, but this may be adjusted or changed as appropriate.

[0038] [C. Amplification of Target Sequences] <C-1. Tagging for Cell Distinction> Subsequently, in the step shown in S4 of Figure 1, a primer cocktail of short tandem repeat (STR) markers or single nucleotide polymorphism (SNP) markers, each of which has an index tag attached to it for distinguishing between individual cells, is added to a predetermined amount (e.g., 100 ng) of the amplification product obtained by whole genome amplification in step B above. The sequence of the index tag is not particularly limited as long as it can distinguish between individual cells.

[0039] <C-2. Amplification of Target Sequence> Next, in the step S5 of Figure 1, the amplification product to which the primer cocktail was added in step C-1 above is subjected to multi-PCR using DNA polymerase to amplify the target sequence. From the viewpoint of identifying fetal cells, the target sequence is preferably an STR. Furthermore, it is desirable that this STR be contained in at least one of chromosomes 13, 18, 21, and a sex chromosome.

[0040] <C-3. Combining the Cells into a Single Sample> Next, in the step shown in S6 of Figure 1, the amplification products for each cell obtained by multi-PCR in step C-2 above are combined and treated as a single sample. It is preferable to perform bead purification of this single sample and then perform quality evaluation using Qubit or the like.

[0041] <C-4. Library Preparation> Next, in the step shown in S7 of Figure 1, one sample obtained in step C-3 above is subjected to NGS adapter ligation, followed by bead purification. After purification, it is preferable to perform quality evaluation using qPCR or the like. The created library is combined by pooling equimolar amounts of libraries from multiple samples into one.

[0042] <C-5. Identification of fetal cells> Next, in the step shown in S8 of Figure 1, the library pool obtained in step C-4 above is subjected to NGS to decode the NGS sequence. Here, since the cells from which the Y chromosome-derived reads were obtained are male, they can be determined to be cells derived from the "child." On the other hand, in the case of a girl, since it is not possible to distinguish between mother and child based on gender, the cells are classified into two types based on differences in the appearance pattern of STR markers, and the STR marker appearance pattern is compared with that of cells known to be derived from the mother, or a minority group of cells is determined to be cells derived from the "child."

[0043] <C-6. Genetic Analysis> At the same time, in the step shown in S9 of Figure 1, the NGS sequence obtained in the step C-5 above is decoded, and the presence or absence of a genetic disease is determined based on the presence or absence of a gene sequence caused by a specific genetic disease. In addition, in the case of determining copy number variation, the aneuploidy of the chromosome of interest can be identified based on whether or not a trisomy-specific STR signal (triallelic pattern) is detected from the appearance pattern of STR markers on the chromosome of interest.

[0044] <Summary of Embodiments> As described above, in the genetic analysis method according to the embodiments of the present disclosure, immobilized maternal blood cells are isolated one by one, proteins and DNA of each of the isolated blood cells are de-crosslinked using a proteinase-containing buffer, the de-crosslinked DNA is extracted, the extracted DNA is subjected to whole genome amplification, and target sequences are amplified using a plurality of specific markers for the amplified whole genome amplification product.

[0045] In the genetic analysis method according to the embodiment of the present disclosure, the specific marker is preferably a short tandem repeat (STR) marker, and the STR marker preferably corresponds to a target sequence of a gene belonging to at least one of chromosomes 13, 18, 21, and a sex chromosome.

[0046] In the genetic analysis method according to an embodiment of the present disclosure, the blood cells are preferably fixed with a 0.01 to 6% by mass paraformaldehyde solution or a 0.001 to 0.2% by mass glutaraldehyde solution immediately after collection from the subject. Furthermore, during the decrosslinking, the cells are preferably heated at 70 to 100°C. Furthermore, the proteinase is preferably proteinase K. Furthermore, during the decrosslinking, a 5 to 1,000 mM buffer is preferably added to adjust the pH to 7 to 9. Furthermore, the buffer is preferably Tris-HCl buffer.

[0047] In the gene analysis method according to the embodiment of the present disclosure, the amplification of the target sequence is preferably carried out using a next-generation sequencer.

[0048] In this example, approximately 20 mL of peripheral blood was provided from several pregnant women aged 12 to 20 weeks under appropriate management at a hospital. The blood samples were subjected to density gradient centrifugation (see WO 2012 / 023298) to obtain a concentrate of fetal nucleated red blood cells. The blood cells were then fixed as described below, and the fixed blood cells were sorted using a cell sorter "FACSAria" manufactured by BD. ＴＭ Using "III", cells were isolated one by one and then decrosslinked as described below.

[0049] (1) Examples As examples, the following samples 1 to 15 were prepared.

[0050] (1-1) Sample 1: Immobilization was performed using a Streck blood collection tube for 30 minutes. The immobilized blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 5 mM phosphate buffer was added, and 0.01 mg / mL of aspartic proteinase was further added as a proteinase, followed by heating at 50°C for 90 minutes. For decrosslinking, heating was performed at 70°C for 60 minutes.

[0051] (1-2) Sample 2: Fixation was performed using 2% by mass paraformaldehyde for 20 minutes. The fixed blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 10 mM phosphate buffer was added, and 0.02 mg / mL of aspartic proteinase was also added as a proteinase, followed by heating at 50°C for 90 minutes. For decrosslinking, heating was performed at 80°C for 60 minutes.

[0052] (1-3) Sample 3: Fixation was performed using 3% by mass paraformaldehyde for 10 minutes. The fixed blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 100 mM phosphate buffer was added, and 0.1 mg / mL of serine proteinase was further added as a proteinase, followed by heating at 50°C for 90 minutes. For decrosslinking, heating was performed at 90°C for 30 minutes.

[0053] (1-4) Sample 4: Fixation was performed using 4% by mass paraformaldehyde for 10 minutes. The fixed blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 200 mM phosphate buffer was added, and 1 mg / mL of serine proteinase was further added as a proteinase, followed by heating at 50°C for 60 minutes. For decrosslinking, heating was performed at 100°C for 30 minutes.

[0054] (1-5) Sample 5: Fixation was performed using 5% by mass paraformaldehyde for 5 minutes. The fixed blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 300 mM Tris-HCl buffer was added, and 10 mg / mL thiol proteinase was further added as a proteinase, followed by heating at 60°C for 60 minutes. For decrosslinking, heating was performed at 70°C for 60 minutes.

[0055] (1-6) Sample 6: Fixation was performed using 5% by mass paraformaldehyde for 5 minutes. The fixed blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 500 mM Tris-HCl buffer was added, and 30 mg / mL thiol proteinase was further added as a proteinase, followed by heating at 60°C for 60 minutes. For decrosslinking, heating was performed at 80°C for 60 minutes.

[0056] (1-7) Sample 7: Fixation was performed using 5% by mass paraformaldehyde for 5 minutes. The fixed blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 800 mM Tris-HCl buffer was added, and 50 mg / mL proteinase K was further added as a proteinase, followed by heating at 60°C for 30 minutes. For decrosslinking, heating was performed at 90°C for 30 minutes.

[0057] (1-8) Sample 8: Fixation was performed using 6% by mass paraformaldehyde for 5 minutes. The fixed blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 1,000 mM Tris-HCl buffer was added, and 100 mg / mL proteinase K was further added as a proteinase, followed by heating at 60°C for 30 minutes. For decrosslinking, heating was performed at 100°C for 30 minutes.

[0058] (1-9) Sample 9: Immobilization was performed using 0.001% by mass glutaraldehyde for 30 minutes. The fixed blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 5 mM phosphate buffer was added, and 0.01 mg / mL of aspartic proteinase was added as a proteinase, followed by heating at 50°C for 90 minutes. For decrosslinking, heating was performed at 70°C for 60 minutes.

[0059] (1-10) Sample 10: Immobilization was performed using 0.01% by mass glutaraldehyde for 20 minutes. The immobilized blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 10 mM phosphate buffer was added, and 0.02 mg / mL of aspartic proteinase was added as a proteinase, followed by heating at 50°C for 90 minutes. For decrosslinking, heating was performed at 80°C for 60 minutes.

[0060] (1-11) Sample 11: Immobilization was performed using 0.01% by mass glutaraldehyde for 10 minutes. The immobilized blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 100 mM phosphate buffer was added, and 0.1 mg / mL of serine proteinase was added as a proteinase, followed by heating at 50°C for 90 minutes. For decrosslinking, heating was performed at 90°C for 30 minutes.

[0061] (1-12) Sample 12: Immobilization was performed using 0.01% by mass glutaraldehyde for 10 minutes. The immobilized blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 200 mM phosphate buffer was added, and 1 mg / mL of serine proteinase was added as a proteinase, followed by heating at 50°C for 60 minutes. For decrosslinking, heating was performed at 100°C for 30 minutes.

[0062] (1-13) Sample 13: Immobilization was performed using 0.05% by mass glutaraldehyde for 5 minutes. The fixed blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 500 mM Tris-HCl buffer was added, and 30 mg / mL thiol proteinase was added as a proteinase, followed by heating at 60°C for 60 minutes. For decrosslinking, heating was performed at 80°C for 60 minutes.

[0063] (1-14) Sample 14: Immobilization was performed using 0.1% by mass glutaraldehyde for 5 minutes. The fixed blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 800 mM Tris-HCl buffer was added, and 50 mg / mL proteinase K was further added as a proteinase, followed by heating at 60°C for 30 minutes. For decrosslinking, heating was performed at 90°C for 30 minutes.

[0064] (1-15) Sample 15: Immobilization was performed using 0.2% by mass glutaraldehyde for 5 minutes. The fixed blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 1,000 mM Tris-HCl buffer was added, and 100 mg / mL proteinase K was further added as a proteinase, followed by heating at 60°C for 30 minutes. For decrosslinking, heating was performed at 100°C for 30 minutes.

[0065] (2) Comparative Examples As comparative examples, the following samples 16 to 20 were prepared.

[0066] (2-1) Sample 16: Fixation was performed using 7% by mass paraformaldehyde for 3 minutes. The fixed blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 500 mM Tris-HCl buffer was added, and 30 mg / mL thiol proteinase was added as a proteinase, followed by heating at 60°C for 60 minutes. For decrosslinking, heating was performed at 80°C for 60 minutes.

[0067] (2-2) Sample 17: Fixation was performed using 5% by mass paraformaldehyde for 5 minutes. The fixed blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 1,500 mM Tris-HCl buffer was added, and 30 mg / mL aspartic proteinase was further added as a proteinase, followed by heating at 60°C for 60 minutes. For decrosslinking, heating was performed at 80°C for 60 minutes.

[0068] (2-3) Sample 18: Fixation was performed using 5% by mass paraformaldehyde for 5 minutes. The fixed blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 500 mM phosphate buffer was added, but no proteinase was added and no heating was performed. For decrosslinking, the cells were heated at 80°C for 60 minutes.

[0069] (2-4) Sample 19: Immobilization was performed using 0.01% by mass glutaraldehyde for 10 minutes. The immobilized blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 500 mM phosphate buffer was added, and 30 mg / mL of proteinase K was added as a proteinase, followed by heating at 60°C for 60 minutes. For decrosslinking, heating was performed at 60°C for 120 minutes.

[0070] (2-5) Sample 20: Immobilization was performed using a Streck blood collection tube for 10 minutes. The immobilized blood cells were then isolated one by one using a cell sorter. For proteolysis of the isolated blood cells, 500 mM phosphate buffer was added, and 30 mg / mL of serine proteinase was added as a proteinase, followed by heating at 60°C for 60 minutes. For decrosslinking, heating was performed at 100°C for 5 minutes.

[0071] (3) Whole genome amplification: For each of Samples 1 to 20, whole genome amplification was performed using the REPLI-g Advanced DNA Single Cell Kit (Qiagen) on the cell lysate obtained by dissociating the crosslinks. The yields of double-stranded DNA (dsDNA) obtained as a result are shown in Table 1 below.

[0072]

[0073] As shown in Table 1 above, for Samples 1 to 15, which are examples, 77.0 to 126.0 ng of dsDNA could be recovered from one cell. On the other hand, for Samples 16 to 20, which are comparative examples, the yield of dsDNA was lower than that of the examples, except for Sample 17.

[0074] (4) Amplification of target sequence A primer cocktail for STR markers was added to each of the whole genome amplification products of Samples 1 to 20. The amount of primer cocktail added was 22 μL per 100 ng of dsDNA. A total of 61 STR markers were used: 14 sets of markers for chromosome 13, 10 sets of markers for chromosome 18, 14 sets of markers for chromosome 21, and 23 sets of markers for sex chromosomes. Here, in the genetic analysis method of the present disclosure, "amplifying the target sequence" means that 6 or more of these 61 markers can be amplified, preferably 32 or more.

[0075] Multi-PCR was performed on the whole genome amplification products to which the primer cocktail had been added using Taq DNA Polymerase (Funakoshi). The PCR reaction consisted of 26 cycles of thermal denaturation at 95°C for 15 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 36 seconds. The amplification products obtained by multi-PCR were subjected to electrophoresis, and the results are shown in graphs by base pair number in Figures 3-1 to 3-15 (Examples) and Figures 4-1 to 4-5 (Comparative Examples).

[0076] The electrophoretic profiles of the Examples shown in Figures 3-1 to 3-15 show broad peaks ranging from low to high molecular weights, indicating that a relatively large number of STR markers have been added. In contrast, the electrophoretic profiles of the Comparative Examples shown in Figures 4-1 to 4-5 show fewer peaks than the Examples, indicating that the number of STR markers has not been increased sufficiently. The number of STR markers detected for each of the Examples and Comparative Examples is as shown in Table 2 below.

[0077]

[0078] As shown in Table 2 above, at least 32 of the 61 STR markers were detected in the amplification products of the example, which is the majority of the markers. More specifically, of the 15 samples, 40 or more markers were detected in 13 samples, and 50 or more markers were detected in 10 samples. Of these 10 samples in which 50 or more markers were detected, at least 11 of the 14 markers were detected for chromosome 13, at least 6 of the 10 markers were detected for chromosome 18, at least 10 of the 14 markers were detected for chromosome 21, and at least 17 of the 23 markers were detected for the sex chromosomes, suggesting that whole genome amplification had been performed generally evenly.

[0079] In addition, for sample 3, in which 49 markers were detected, 12 of the 14 markers were detected for chromosome 13, 6 of the 10 markers for chromosome 18, 13 of the 14 markers for chromosome 21, and 18 of the 23 markers for the sex chromosomes, suggesting that whole genome amplification comparable to that of samples with 50 or more detected markers was achieved. In addition, for sample 13, in which 47 markers were detected, 12 of the 14 markers were detected for chromosome 13, 6 of the 10 markers for chromosome 18, all 14 markers for chromosome 21, and 15 of the 23 markers for the sex chromosomes were detected, suggesting that whole genome amplification comparable to that of samples with 50 or more detected markers was achieved.

[0080] In addition, for sample 5, in which a total of 42 markers were detected, only 2 of the 10 markers were detected on chromosome 18, less than half. In addition, for sample 6, in which a total of 38 markers were detected, only 10 of the 23 markers were detected on the sex chromosomes, less than half. Furthermore, for sample 9, in which a total of 32 markers were detected, only 7 of the 14 markers were detected on chromosome 13, less than half; 5 of the 10 markers were detected on chromosome 18, less than half; and 11 of the 23 markers were detected on the sex chromosomes, less than half. Thus, for samples 5, 6, and 9, there was at least one chromosome in which the number of detected markers was less than half, suggesting that there was a region that was not sufficiently amplified.

[0081] In contrast, only a small amount of markers were detected in the amplification products of the comparative examples, or none at all. Here, for sample 16 of the comparative examples, the paraformaldehyde concentration as a fixative was 7% by mass, exceeding 6% by mass, which is thought to have caused excessive immobilization of cells, presumably resulting in insufficient protein degradation and nucleic acid solubilization. For sample 17, the amount of buffer added was 1,500 mM, exceeding 1,000 mM, presumably resulting in insufficient protein degradation. For sample 18, protein degradation was presumably insufficient because no proteinase was added or heating was performed. For sample 19, the decrosslinking temperatures exceeded 60°C and 70°C, and for sample 20, the decrosslinking times were less than 5 and 10 minutes, presumably resulting in insufficient decrosslinking in either case. From the above, it is presumed that the comparative examples did not achieve sufficient whole genome amplification for analysis.

[0082] From the above, it was inferred that the amplification products of the Examples could be used for testing and diagnosing abnormalities in each chromosome because multiple markers were detected for each chromosome. On the other hand, the amplification products of the Comparative Examples could not be used for testing and diagnosing abnormalities in each chromosome because there were chromosomes for which one or no markers were detected.

[0083] (5) Cell Tagging: When amplifying the target sequence as described in (4) above, it is desirable to amplify the target sequence with a different index tag attached to each primer for each sample. For example, by attaching index tag "1" to each primer for sample 1, index tag "2" to each primer for sample 2, and index tag "3" to each primer for sample 3, when the amplified products are analyzed together, it becomes possible to determine which target sequence was detected in each sample.

[0084] (6) Library Preparation: As described above, the amplification products of each sample using tagged primers are combined into one sample, and then NGS adapters are ligated using TruSeq DNA Library Prep Kits (Illumina). Then, bead purification is performed using magnetic beads for DNA purification (HighPrep PCR, Funakoshi). After purification, quality assessment is preferably performed using qPCR.

[0085] (7) Identification of fetal cells The resulting library pool can then be subjected to NGS using NextSeq 2000 (Illumina) to decode the NGS sequence. In the case of a boy, cells from which Y chromosome-derived reads are obtained are male and can be determined to be cells derived from the "child." In the case of a girl, since the mother and child cannot be distinguished based on gender, the cells can be classified into two types based on the difference in the appearance pattern of the STR marker, and the STR marker appearance pattern can be compared with that of cells known to be derived from the mother, or a minority group of cells can be determined to be cells derived from the "child."

Claims

1. A genetic analysis method comprising isolating fixed maternal blood cells one by one, decrosslinking the protein and DNA of each of the isolated blood cells using a proteinase-containing buffer, extracting the decrosslinked DNA, performing whole genome amplification of the extracted DNA, and amplifying target sequences from the amplified whole genome amplification product using multiple specific markers.

2. The genetic analysis method according to claim 1, wherein the specific marker is a short tandem repeat (STR) marker.

3. The method of genetic analysis according to claim 2, wherein the STR marker corresponds to a target sequence of a gene belonging to at least one of chromosomes 13, 18, 21 and a sex chromosome.

4. The genetic analysis method according to claim 1, wherein the blood cells are fixed in a 0.01 to 6% by mass paraformaldehyde solution or a 0.001 to 0.2% by mass glutaraldehyde solution immediately after collection from the subject.

5. The genetic analysis method according to claim 1, wherein the decrosslinking is carried out by heating at 70 to 100°C.

6. The method for gene analysis according to claim 1, wherein the proteinase is proteinase K.

7. The method for gene analysis according to claim 1, wherein during the decrosslinking, a 5 to 1,000 mM buffer is added to adjust the pH to 7 to 9.

8. The method for gene analysis according to claim 7, wherein the buffer is a Tris-HCl buffer.

9. The genetic analysis method according to claim 1, wherein the amplification of the target sequence is carried out using a next-generation sequencer.

10. The genetic analysis method according to claim 3, wherein the blood cells are fixed in a 0.01 to 6% by mass paraformaldehyde solution or a 0.001 to 0.2% by mass glutaraldehyde solution immediately after collection from the subject.

11. The genetic analysis method according to claim 10, wherein the decrosslinking is carried out by heating at 70 to 100°C.

12. The method for gene analysis according to claim 11, wherein the proteinase is proteinase K.

13. The genetic analysis method according to claim 12, wherein during the decrosslinking, a 5 to 1,000 mM buffer is added to adjust the pH to 7 to 9.

14. The method for gene analysis according to claim 13, wherein the buffer is a Tris-HCl buffer.

15. The genetic analysis method according to claim 14, wherein the amplification of the target sequence is carried out using a next-generation sequencer.

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