WO2025205105A1 - Genetic analysis method - Google Patents

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

This disclosure relates to a method for genetic analysis of maternal blood cells.

Efforts have been made to concentrate or isolate fetal cells from maternal whole blood and analyze them, 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.

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-antibody conjugate. Patent Document 6 discloses a method for dissolving fixed biological samples, specifically protein-crosslinked nucleic acids, by digesting crosslinked proteins 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 separating 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.

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: JP 2023-156347 A Patent Document 6: JP 2023-521579 A Patent Document 7: US 2022/0389384 A1
Patent Document 8: US 2023/0295683 A1
Patent Document 9: EP 4445135 A1

Non-Patent 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-patent document 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

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 will be necessary to undergo highly invasive amniocentesis or chorionic villus sampling. Furthermore, Patent Document 2 describes a method for obtaining chromosomal DNA 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. Additionally, 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 mosaicism, which differs from the actual genetic information of the fetus, posing problems for tests using placenta-derived fetal trophoblast cells.

Non-Patent Document 1 above discloses a method for amplifying the whole genome from a single cell, but when whole genome amplification is performed on a single cell immobilized using the same method, amplification either does not occur or is biased, making it impossible to obtain the desired marker and making genetic analysis impossible. Furthermore, Non-Patent Document 2 above discloses a method for de-crosslinking multiple immobilized cells, but when whole genome amplification is subsequently attempted on a single cell immobilized using the same method, amplification either does not occur or is biased, making it impossible to obtain the desired marker and making genetic analysis impossible.

In order to avoid the aforementioned mosaicism, it is extremely difficult to concentrate or isolate fetal nucleated red blood cells from maternal whole blood for genetic analysis. Furthermore, because time is required from blood collection through transportation to analysis, fetal nucleated red blood cells are lost, fused, destroyed, or killed during this process, making subsequent analysis difficult and impractical. Furthermore, if cells are fixed in order to prevent such cellular changes or deterioration over time, genome amplification becomes difficult in a single isolated cell, making genetic analysis, particularly analysis using a next-generation sequencer (NGS), difficult.

Furthermore, when genome amplification was performed after decrosslinking fixed cells, amplification either did not occur in the isolated single cell or 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 both of fetal and maternal origin, and antibodies are subject to nonspecific adsorption, making it difficult to isolate only fetal cells using antibodies. Therefore, it is necessary to identify fetal cells, but attempting to analyze multiple cells individually results in low throughput and high costs, making it difficult to put into practical use.

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

The genetic analysis method of the first aspect of the present disclosure involves isolating fixed maternal blood cells one by one, de-crosslinking the protein and DNA of each of the isolated blood cells using a proteinase-containing buffer, extracting the de-crosslinked DNA, performing whole-genome amplification of the extracted DNA, and amplifying target sequences from the amplified whole-genome amplification product using multiple specific markers.

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

A genetic analysis method according to a third aspect of the present disclosure has the same configuration as the first or second aspect, but 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.

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, except that 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.

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

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, except that the proteinase is proteinase K.

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

The genetic analysis method of the eighth aspect of the present disclosure has the same configuration as the seventh aspect, except that the buffer is a Tris-HCl buffer.

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 in addition, the amplification of the target sequence is carried out using a next-generation sequencer.

As described above, the aspects of the present disclosure are configured to provide a genetic analysis method that suppresses changes or deterioration of cells over time, such as cell loss, fusion, destruction, or death, and reduces variation in genome amplification after decrosslinking each immobilized and isolated cell.

1 is a flowchart outlining the steps in an embodiment of the present disclosure. FIG. 1 is a schematic diagram showing the state of DNA and proteins in immobilized cells. FIG. 2-1 is a schematic diagram showing the state in which the protein is decomposed. FIG. 2-3 is a schematic diagram showing a state where the cross-linking has been removed from the state shown in FIG. 2-2. 1 is a graph showing the results of amplification of a target sequence using an STR marker in Sample 1. 1 is a graph showing the results of amplification of a target sequence using an STR marker in sample 2. 1 is a graph showing the results of amplification of a target sequence using an STR marker in Sample 3. 1 is a graph showing the results of amplification of a target sequence using an STR marker in Sample 4. 1 is a graph showing the results of amplification of a target sequence using an STR marker in sample 5. 1 is a graph showing the results of amplification of a target sequence using an STR marker in sample 6. 10 is a graph showing the results of amplification of a target sequence using an STR marker in sample 7. 10 is a graph showing the results of amplification of a target sequence using an STR marker in sample 8. 10 is a graph showing the results of amplification of a target sequence using an STR marker in sample 9. 1 is a graph showing the results of amplification of a target sequence using an STR marker in sample 10. 1 is a graph showing the results of amplification of a target sequence using an STR marker in sample 11. 1 is a graph showing the results of amplification of a target sequence using an STR marker in sample 12. 1 is a graph showing the results of amplification of a target sequence using an STR marker in sample 13. 1 is a graph showing the results of amplification of a target sequence using an STR marker in sample 14. 1 is a graph showing the results of amplification of a target sequence using an STR marker in sample 15. 1 is a graph showing the results of amplification of a target sequence using an STR marker in sample 16. 1 is a graph showing the results of amplification of a target sequence using an STR marker in sample 17. 1 is a graph showing the results of amplification of a target sequence using an STR marker in sample 18. 1 is a graph showing the results of amplification of a target sequence using an STR marker in sample 19. 1 is a graph showing the results of amplification of a target sequence using an STR marker in sample 20.

Embodiments of the present disclosure will be described below with reference to the drawings. Note that in this disclosure, when a numerical range is indicated as "A to B," it means "greater than or equal to A and less than or equal to B," unless otherwise specified.

Figure 1 is a flowchart showing an overview of the steps in an embodiment of the present disclosure. First, prior to step S1, we will explain the fixation and isolation of maternal blood cells.

[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.

Nucleated red blood cells are erythroblasts that have lost the ability to divide. Red blood cells are produced by the differentiation and maturation of hematopoietic stem cells. During the process of differentiation and maturation, the following cells emerge from hematopoietic stem cells, in order: myeloid progenitor cells, erythroid-megakaryocyte 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.

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

Nucleated red blood cells are normally found in the bone marrow, but very small amounts of nucleated red blood cells can be found in the blood. Also, very small amounts of nucleated red blood cells of both maternal and fetal origin can be 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.

It is desirable to analyze maternal blood cells immediately after collection from the mother, but in many cases several days will pass between collection and analysis, so they must be stored. Therefore, for preservation, maternal blood cells are fixed. A fixative consisting of a paraformaldehyde solution or a glutaraldehyde solution is used for fixation.

For fixation using a paraformaldehyde solution as the 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. For fixation using a glutaraldehyde solution as the 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.

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 hemolyzing 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 described above. After the fixation time has elapsed, the recovered blood cell components are dispersed in an appropriate buffer and stored until analysis. Alternatively, blood may be collected from the mother using a blood collection tube containing fixative in advance, and red blood cells and other particles may be removed using the density gradient centrifugation method or a hemolyzing reagent described above to concentrate fetal nucleated red blood cells and recover them as blood cell components.

Prior to analysis, the maternal blood cells fixed as described above are isolated one by one using a commercially available cell sorter (e.g., BD or Beckman).

A. Cell Decrosslinking
In the 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, the decomposition of proteins and solubilization of nucleic acids shown in S1 of Figure 1, and the dissociation of crosslinks shown in S2 are carried out.

<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 cells are heated to decompose the proteins and solubilize the nucleic acids. As a result, the protein 20 shown in Figure 2-1 is decomposed into amino acids 21 as shown in Figure 2-2. However, at this stage, the peptide 22 is linked to the DNA 10 via a crosslink 30.

As a 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 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 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 a proteinase, various proteinases, including commercially available products, such as aspartic proteinase, metalloproteinase, serine proteinase, thiol proteinase, and 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 heat treatment temperature is 40 to 70°C, preferably 50 to 60°C, 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.

In the step shown in S1 of Figure 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 polyoxyethylene alkyl ether may also be used in combination. According to an embodiment of the present disclosure, it has been shown that amplification of a target sequence can be performed without a chelating agent or surfactant, and therefore EDTA or sodium dodecyl sulfate may not be included.

<A-2. Dissociation of Crosslinks>
Next, in the step 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.

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. A commercially available whole genome amplification kit can be used for the WGA reaction mix, but 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.

C. Amplification of Target Sequences
<C-1. Tagging to distinguish cells>
Next, in the step 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. The sequence of the index tag is not particularly limited as long as it can distinguish between individual cells.

<C-2. Amplification of target sequence>
Next, in step S5 of Figure 1, the amplification product to which the primer cocktail was added in step C-1 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.

<C-3. Combine all cells into one specimen>
Next, in the step S6 of Fig. 1, the amplification products obtained for each cell by multi-PCR in step C-2 are combined and treated as a single sample. It is preferable to purify this single sample with beads and then evaluate its quality using Qubit or the like.

<C-4. Library Adjustment>
Next, in step 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 assessment using qPCR or the like. The created library is combined by pooling equimolar amounts of libraries from multiple samples into one.

<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 females, 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."

<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 causing the 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 the STR markers on the chromosome of interest.

<Summary of the embodiment>
As described above, in the genetic analysis method according to an embodiment 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.

In the genetic analysis method according to an embodiment of the present disclosure, the specific marker is preferably a short tandem repeat (STR) marker. Furthermore, it is preferable that 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.

In the genetic analysis method according to an embodiment of the present disclosure, it is preferable that 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. Furthermore, it is preferable that the cells are heated at 70 to 100°C during the de-crosslinking. Furthermore, it is preferable that the proteinase is proteinase K. Furthermore, it is preferable that the pH is adjusted to 7 to 9 by adding a 5 to 1,000 mM buffer during the de-crosslinking. Furthermore, it is preferable that the buffer is Tris-HCl buffer.

In the genetic analysis method according to an embodiment of the present disclosure, amplification of the target sequence is preferably performed using a next-generation sequencer.

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. This blood sample was subjected to density gradient centrifugation (see WO 2012/023298) to obtain a concentrate of fetal nucleated red blood cells, after which the blood cells were fixed as described below. The fixed blood cells were isolated one by one using a BD cell sorter "FACSAria ^™ III," and then decrosslinked as described below.

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

(1-1) Sample 1
Immobilization was performed using a Streck blood collection tube 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 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.

(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 added as a proteinase, followed by heating at 50°C for 90 minutes. For decrosslinking, heating was performed at 80°C for 60 minutes.

(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 added as a proteinase, followed by heating at 50°C for 90 minutes. For decrosslinking, heating was performed at 90°C for 30 minutes.

(1-4) Sample 4
The 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, the cells were heated at 100°C for 30 minutes.

(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 added as a proteinase, followed by heating at 60°C for 60 minutes. For decrosslinking, heating was performed at 70°C for 60 minutes.

(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 added as a proteinase, followed by heating at 60°C for 60 minutes. For decrosslinking, heating was performed at 80°C for 60 minutes.

(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.

(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.

(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 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.

(1-10) Sample 10
Immobilization was performed using 0.01% by mass glutaraldehyde 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 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.

(1-11) Sample 11
Immobilization was performed using 0.01% by mass glutaraldehyde 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 added as a proteinase, followed by heating at 50°C for 90 minutes. For decrosslinking, heating was performed at 90°C for 30 minutes.

(1-12) Sample 12
Immobilization was performed using 0.01% by mass glutaraldehyde 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 added as a proteinase, followed by heating at 50°C for 60 minutes. For decrosslinking, heating was performed at 100°C for 30 minutes.

(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.

(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.

(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.

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

(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.

(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 added as a proteinase, followed by heating at 60°C for 60 minutes. For decrosslinking, heating was performed at 80°C for 60 minutes.

(2-3) Sample 18
The 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.

(2-4) Sample 19
Immobilization was performed using 0.01% by mass glutaraldehyde for 10 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, and 30 mg/mL 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.

(2-5) Sample 20
Immobilization was performed using a Streck blood collection tube for 10 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, 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.

(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.

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 dsDNA yield was lower than that of the examples, except for Sample 17.

(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.

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 graphed by base pair number in Figures 3-1 to 3-15 (Example) and Figures 4-1 to 4-5 (Comparative Example).

The electrophoresis 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 electrophoresis profiles of the Comparative Examples shown in Figures 4-1 to 4-5 show fewer peaks than the Examples, indicating that the STR markers have not been sufficiently added. The number of STR markers detected for each of the Examples and Comparative Examples is shown in Table 2 below.

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 more than half of the markers. More specifically, of the 15 samples, 40 or more markers were detected in 13 samples, and 50 or more markers in 10 samples. Of these 10 samples in which 50 or more markers were detected, at least 11 of the 14 markers were detected on chromosome 13, at least 6 of the 10 markers were detected on chromosome 18, at least 10 of the 14 markers on chromosome 21, and at least 17 of the 23 markers on the sex chromosomes, suggesting that whole genome amplification had occurred fairly evenly.

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

In sample 5, where a total of 42 markers were detected, only 2 of the 10 markers were detected on chromosome 18, less than half. In sample 6, where a total of 38 markers were detected, only 10 of the 23 markers were detected on the sex chromosomes, less than half. Furthermore, in sample 9, where 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 on the sex chromosomes, less than half. Thus, in samples 5, 6, and 9, there was at least one chromosome where less than half were detected, suggesting that there was an area that was not sufficiently amplified.

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

From the above, it can be inferred that the amplification products of the Examples all have multiple markers detected for each chromosome, and therefore can be used for testing and diagnosis when an abnormality is present in each chromosome. On the other hand, the amplification products of the Comparative Examples all have chromosomes for which one or no markers were detected, and therefore cannot be used for testing and diagnosis when an abnormality is present in each chromosome.

(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.

(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.

(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 (15)

Isolating the fixed maternal blood cells one by one;
Decrosslinking the proteins and DNA of each of the isolated blood cells with a proteinase-containing buffer;
Extracting the decrosslinked DNA;
The extracted DNA is subjected to whole genome amplification;
A genetic analysis method in which target sequences are amplified using multiple specific markers from an amplified whole genome amplification product. The genetic analysis method of claim 1, wherein the specific marker is a short tandem repeat (STR) marker. The genetic analysis method described in 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. The genetic analysis method of 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. The genetic analysis method according to claim 1, wherein the decrosslinking is performed by heating at 70 to 100°C. The genetic analysis method according to claim 1, wherein the proteinase is proteinase K. The genetic analysis method 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. The genetic analysis method according to claim 7, wherein the buffer is Tris-HCl buffer. The genetic analysis method of claim 1, wherein the amplification of the target sequence is performed using a next-generation sequencer. The genetic analysis method described in 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. The genetic analysis method described in claim 10, wherein the decrosslinking is performed by heating at 70 to 100°C. The genetic analysis method according to claim 11, wherein the proteinase is proteinase K. 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. The genetic analysis method according to claim 13, wherein the buffer is Tris-HCl buffer. The genetic analysis method described in claim 14, wherein the amplification of the target sequence is performed using a next-generation sequencer.