Method for amplifying DNA from formalin-fixed sample

Abstract

To provide a method for amplifying the target DNA from a formalin-fixed sample.SOLUTION: The present invention provides a method for amplifying DNA, comprising the following steps of: (1) extracting DNA from a formalin-fixed sample, (2) removing fragmented DNA from a solution containing the extracted DNA, and (3) amplifying the target DNA in the solution derived from the step (2).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for amplifying a target DNA from a formalin-fixed sample, the method for amplifying DNA of the present invention is characterized by comprising a step of removing fragmented DNA from a solution containing DNA extracted from a formalin-fixed sample. The present invention also relates to a sequencing method, etc., comprising a step of analyzing the sequence of the DNA amplified by the amplification method. [Background technology]

[0002] In recent pathological diagnosis, formalin-fixed paraffin-embedded (FFPE) tissues are widely used as samples for genetic testing using, for example, PCR or next-generation sequencing (NGS), and the test results are used for the diagnosis and companion diagnosis of various diseases (Non-Patent Document 1). In addition, genetic research using such samples is useful for the search for new therapeutic drugs, etc. (Non-Patent Document 1).

[0003] However, it has been reported that the quality of DNA in FFPE samples that have been poorly fixed or overfixed with formalin is poor, making it difficult to perform genetic tests such as PCR tests (Non-Patent Documents 2 and 3). In addition, even if the FFPE sample is created within three years, if the DIN (DNA Integrity Number) value does not meet the test standard due to overfixation or other reasons, the test will have to be abandoned. Although FFPE is a method that can preserve samples semi-permanently, the quality of DNA deteriorates with long-term storage, and the success rate of NGS, etc. decreases for samples that are more than three years old.

[0004] For the reasons mentioned above, currently, for example, when NGS is performed under insurance coverage, it is limited to samples less than three years old. In addition, although the DNA in frozen samples is of high quality, not all hospitals are able to perform it due to the cost and difficulty of long-term storage. In forensic medicine examinations, tests are sometimes performed from samples stored in formalin for a long time, but there is a problem that genetic testing cannot be performed due to factors such as DNA fragmentation. For this reason, it has been considered that formalin-fixed samples (e.g., FFPE samples, especially FFPE samples over three years old) are not suitable for PCR or other procedures in the first place because of the advanced DNA fragmentation. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Yasushi Y et al., Pathology International. 2020;20 (12) 921-931 [Non-Patent Document 2] Wang F et al., J Mol Diagn. 2007; 9(4):441-451 [Non-Patent Document 3] Srinivasan M et al., Am J Pathol. 2002; 161(6):1961-1971 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, an objective of the present invention is to provide a method for amplifying a target DNA from a formalin-fixed sample, and a sequencing method, etc., which includes a step of analyzing the sequence of the DNA amplified by the amplification method.

[0007] The present inventors conceived that the reason why formalin-fixed samples (e.g., FFPE samples, particularly FFPE samples over 3 years old) are not suitable as samples for PCR, etc. is that formalin fixation leads to increased fragmentation of DNA, and the short DNA generated by the fragmentation inhibits the amplification of the target DNA. Therefore, it was thought that the problem was not that formalin-fixed samples (e.g., FFPE samples, particularly FFPE samples over 3 years old) themselves are not suitable as samples for PCR, etc., but that such fragmented DNA is mixed in the extract when DNA is extracted from the sample.

[0008] Therefore, the present inventors investigated whether the desired DNA could be amplified by removing fragmented DNA from the extract and amplifying the target DNA by PCR or the like. Surprisingly, the present inventors succeeded in amplifying the target DNA from a formalin-fixed sample (FFPE sample) by removing fragmented DNA. The present inventors also succeeded in amplifying the target DNA from a formalin-fixed sample (FFPE sample) that had been stored for more than three years by removing fragmented DNA. Furthermore, the present inventors also succeeded in detecting gene rearrangements of immunoglobulin H chain (IgH) by PCR, which could not be detected by conventional methods, by removing fragmented DNA. The present inventors further conducted research based on these findings and completed the present invention.

[0009] That is, the present invention is as follows. [1] A method for amplifying DNA, comprising: (1) extracting DNA from a formalin-fixed sample; (2) removing the fragmented DNA from the solution containing the extracted DNA; and (3) amplifying the target DNA in the solution obtained in step (2); A method comprising: [2] The method according to [1], wherein the removal of fragmented DNA in step (2) is carried out using a silica-based solid phase material. [3] The method according to [2], wherein the silica-based solid phase material is a silica membrane. [4] The method according to any one of [1] to [3], wherein the formalin-fixed sample has been fixed for more than three years. [5] The method according to any one of [1] to [4], wherein the formalin-fixed sample has been fixed for 10 years or more. [6] The method according to any one of [1] to [5], wherein the formalin-fixed sample is a thymus sample. [7] The method according to any one of [1] to [6], wherein the amplification of the target DNA in step (3) is carried out by PCR. [8] The method according to any one of [1] to [7], wherein the target DNA is an IgH gene. [9] A sequencing method comprising a step of analyzing the sequence of DNA amplified by the method according to any one of [1] to [8].

[10] The method described in [9], wherein sequencing is performed using a next-generation sequencer.

[11] A kit for amplifying DNA that contains a silica-based solid phase material.

[12] The method according to any one of [1] to [8], wherein the fragmented DNA is DNA of 100 bp or less.

[13] The method according to

[12] , wherein the fragmented DNA is DNA of 50 bp to 100 bp. Effect of the Invention

[0010] According to the present invention, the desired DNA in a formalin-fixed sample can be amplified, making it possible to carry out genetic testing using PCR, a next-generation sequencer, or the like. In particular, the present invention is excellent in that it can amplify the desired DNA in a formalin-fixed sample that has been stored for more than three years, which was previously considered untestable. Furthermore, according to the present invention, genetic analysis can be carried out on old samples that have been stored for more than three years, making it possible to search for and identify novel diagnostic markers and causative genes of diseases. Furthermore, according to the present invention, it may be possible to carry out genetic testing on specimens that have been preserved in formalin in forensic genetic testing. [Brief description of the drawings]

[0011] [Figure 1] Figure 1 shows the state of DNA from each sample (Sample 1 (prepared in 2022), Sample 2 (prepared in 2013), and Sample 3 (prepared in 2010)) after step (1) (before purification) and after step (2) (after purification) in the amplification method of the present invention. [Diagram 2] 2 shows the results of amplification of a housekeeping gene (GAPDH) using DNA derived from samples 1 to 3 after completion (purification) of step (2) in the amplification method of the present invention. In Fig. 2, P22-4322-4 in the circled number 2 represents sample 1, P13-11275-14 in the circled number 4 represents sample 2, and P10-240-4 in the circled number 5 represents sample 3. [Diagram 3]3 shows the results of amplification of the IgH gene using DNA from samples 1 to 3 after step (1) (before purification) (left) and after step (2) (after purification) (right) in the amplification method of the present invention. In FIG. 3, P22-4322-4 in the circled number 2 represents sample 1, P13-11275-14 in the circled number 4 represents sample 2, and P10-240-4 in the circled number 5 represents sample 3 (note that in FIG. 3, circled numbers 1 and 3 represent IgH genes amplified by the same method as samples 1 to 3, and relate to FFPE samples produced in 2017 and 2016, respectively, derived from the thymus of patients diagnosed with MALT lymphoma). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] 1. DNA Amplification Method of the Present Invention The present invention provides a method for amplifying a DNA of interest from a formalin-fixed sample. Specifically, the method comprises: (1) extracting DNA from a formalin-fixed sample; (2) removing the fragmented DNA from the solution containing the extracted DNA; and (3) amplifying the target DNA in the solution obtained in step (2); (hereinafter, may be referred to as "the DNA amplification method of the present invention").

[0013] In this specification, "formalin" refers to an aqueous solution containing formaldehyde, which typically further contains methanol. The concentration of formaldehyde in formalin is typically 35 to 38% (w / v) in the Japanese Pharmacopoeia. In this specification, for example, 20% formalin contains 1 / 5 of the formalin stock solution (e.g., Japanese Pharmacopoeia formalin) by volume, and the same applies to formalins of other percentages.

[0014] In this specification, a "formalin-fixed sample" refers to a sample prepared by immersing a (biological) tissue fragment or other sample in a solution (hereinafter sometimes referred to as a "fixative") obtained by diluting a formalin stock solution (e.g., Japanese Pharmacopoeia formalin) for a predetermined period of time or more. The dilution rate of the formalin stock solution is typically 10% to 20% (v / v), and dilution can be performed, for example, using distilled water or various buffer solutions. In this specification, a formalin stock solution diluted with various buffer solutions is referred to as buffered formalin (e.g., 10% neutral buffered formalin, etc.).

[0015] The time for which a sample such as a biological tissue piece is immersed (hereinafter, may be referred to as "fixation time") varies depending on the fixative, the size of the biological tissue piece, etc., and can be appropriately determined by a person skilled in the art. For example, when a 10% neutral buffered formalin solution is used as the fixative, the immersion time is 6 to 72 hours, preferably 24 to 48 hours.

[0016] The formalin-fixed sample used in the present invention may be embedded in paraffin by a method known per se. The formalin-fixed sample embedded in paraffin is called formalin-fixed paraffin-embedded (FFPE). Paraffin embedding may be performed, for example, by the following method. The formalin-fixed sample is dehydrated by sequentially immersing it in a series of ethanol solutions with concentrations of 50 to 100% (for example, overnight in 70% ethanol, 30 minutes x 5 times in 99.5% ethanol, and 30 minutes x 1 time in 100% ethanol), and after reaching 100% ethanol, it is then sequentially immersed in a series of mixed solutions of ethanol and xylene to replace the solvent with xylene. Thereafter, it is sequentially immersed in a series of xylene solutions of paraffin. After the xylene concentration of the paraffin is reduced, it is transferred to a constant temperature bath at 60°C where the paraffin melts. Finally, a method of permeating 100% paraffin into the tissue and then lowering the temperature of the paraffin to solidify it, etc., are included, but are not limited to these.

[0017] The origin of the formalin-fixed sample used in the present invention is not particularly limited, and may be either an animal or a plant. Examples of animals include, but are not particularly limited to, fish, amphibians, reptiles, birds, mammals, etc., and are preferably mammals. Examples of mammals include, but are not particularly limited to, rodents such as rats, mice, hamsters, and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, and sheep, carnivores such as dogs and cats, and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees, and are preferably humans.

[0018] In addition, the formalin-fixed sample used in the present invention may be either plant tissue or animal tissue. Examples of animal tissue include, but are not limited to, skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood (including umbilical cord blood), bone marrow, heart, eye, and brain. Examples of plant tissue include, but are not limited to, meristematic tissue and permanent tissue.

[0019] The formalin-fixed sample used in the present invention may be one immediately after fixation, or one or more years (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 years) since fixation. In one embodiment, the formalin-fixed sample used in the present invention is one that has been fixed for 3 years or more. In another embodiment, the formalin-fixed sample used in the present invention is one that has been fixed for 10 years or more. The form of the formalin-fixed sample used in the present invention is not particularly limited, and examples thereof include permanent specimens and slide specimens.

[0020] The extraction of DNA from the formalin-fixed sample in step (1) of the present invention can be carried out by a method known per se (e.g., Agnieszka K Sarnecka et al., Contemp Oncol (Pozn). 2019;23(1):52-58. doi: 10.5114 / wo.2019.83875. Epub 2019 Mar 26., Samantha J McDonough et al., PLoS One. 2019 Apr 11;14(4):e0211400. doi: 10.1371 / journal.pone.0211400. eCollection 2019., etc.). Specifically, for example, a DNA extraction method using phenol / chloroform, ethanol, sodium hydroxide, CTAB, etc. can be mentioned. In the case of samples embedded in paraffin, briefly, formalin-fixed paraffin-embedded (FFPE) samples are treated with a deparaffinizing agent such as xylene or d-limonene to dissolve and remove the paraffin, and the deparaffinizing agent is washed with ethanol or the like, after which DNA is solubilized by proteinase K digestion. Alternatively, DNA is directly solubilized from the FFPE sample using a surfactant. After that, proteins are denatured with a protein denaturant such as phenol or guanidine thiocyanate and removed from the aqueous phase, and the DNA remaining in the aqueous phase may be extracted by adsorption and separation with a resin or the like. DNA can also be extracted using commercially available DNA extraction kits (e.g., QIAamp DNA FFPE Tissue Kit (QIAGEN), NucleoSpin® DNA FFPE XS (Takara Bio), Maxwell® FFPE Plus DNA Kit (Promega), WaxFree TM This may also be performed using a Paraffin Sample DNA Extraction Kit (TrimGen Corporation, etc.).

[0021] In the present specification, the "fragmented DNA" in step (2) is not particularly limited as long as it is shorter than the target DNA amplified in step (3) (i.e., the amplification product), and examples thereof include DNA of 500 bp or less, DNA of 400 bp or less, DNA of 300 bp or less, DNA of 200 bp or less, and DNA of 100 bp or less. Further examples thereof include DNA of 500 bp to 10 bp, DNA of 400 bp to 20 bp, DNA of 300 bp to 30 bp, DNA of 200 bp to 40 bp, and DNA of 100 bp to 50 bp.

[0022] The method for removing fragmented DNA from the solution containing the extracted DNA in step (2) is not particularly limited as long as it is capable of removing the desired fragmented DNA. Examples of the method include a method using a silica-based solid phase material (e.g., silica membrane, silica monolith, etc.), a method using an ion exchange carrier or gel filtration carrier, a method using magnetic beads, etc., with a method using a silica-based solid phase material being preferred.

[0023] The silica-based solid phase material is preferably a solid phase material having a silica monolith or a porous silica membrane. From the viewpoint of facilitating the removal of fragmented DNA and other impurities, or the operation of washing and elution of DNA, the solid phase material is preferably in the form of a spin column, a pipette tube with a column, or a column cartridge that can be attached to a centrifuge tube or a pipette tube. Furthermore, from the viewpoint of preventing contamination and operation efficiency, it is more preferable that the solid phase material is in the form of a column cartridge that can be attached to a spin column or a centrifuge tube. Furthermore, considering that the amount of DNA to be recovered is relatively small, the solid phase material is preferably in the form of a miniprep column or the like that can purify a small amount of DNA. For example, commercially available columns for DNA purification or concentration can be used as the solid phase material, and examples thereof include NucleoSpin (registered trademark) gDNA Clean-up XS (Machrei-Nagel), Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega), and minElute PCR Purification Kit (QIAGEN).

[0024] The washing solution used for removing or washing fragmented DNA and other impurities from a silica-based solid phase material can typically be a solution (e.g., an aqueous solution, etc.) containing at least one of a water-soluble organic solvent and a water-soluble salt. The water-soluble organic solvent contained in the washing solution can typically be a water-soluble alcohol. Examples of the water-soluble alcohol include methanol, ethanol, propanol, and butanol. The propanol may be either isopropanol or n-propanol, and the butanol may be either linear or branched. These water-soluble alcohols can also be used in combination. The amount of the water-soluble organic solvent contained in the washing solution may be an amount that can hold the DNA on the solid phase material and remove or wash fragmented DNA and other impurities, and can be appropriately determined by those skilled in the art, but typically includes, for example, 30 to 100% (v / v), 35 to 50% (v / v), etc. On the other hand, examples of the water-soluble salt contained in the washing solution include, for example, a halide salt, more specifically, a chloride salt. Examples of the water-soluble salt include monovalent or divalent cationic salts, specifically, for example, alkali metal salts, alkaline earth metal salts, and more specifically, sodium salts, potassium salts, etc. When the water-soluble salt is contained in the cleaning solution, the concentration is typically, for example, 10 mmol / l or more. On the other hand, the upper limit of the concentration is not particularly limited as long as it is within a range that does not impair the solubility of impurities, and is typically, for example, 1 mol / l.

[0025] The release of DNA from the solid phase material can typically be performed by passing an elution solution through the solid phase material to elute the DNA. As the elution solution used for eluting DNA, distilled water or Tris-EDTA (TE) buffer solution may be used. The washing solution and elution solution may be a washing solution and elution solution attached to a commercially available column for DNA purification or concentration, according to the manufacturer's instruction manual. The washing and elution of the solid phase material can be performed by a procedure known per se. Typically, for example, when the solid phase material is attached to a spin column, a solution containing DNA is passed through the column, a washing solution is added to the column to which the DNA is adsorbed, and the column is centrifuged to wash away the fragmented DNA and other impurities together with the washing solution. Subsequently, an elution solution is added to the column, and the column is centrifuged to elute the DNA from the column.

[0026] The method for amplifying the target DNA in step (3) is not particularly limited as long as it can amplify the target DNA, and can be carried out using polymerase chain reaction (PCR), ligase chain reaction, multiple annealing and looping-based amplification cycles (MALBAC), multiple displacement amplification (MDA), strand displacement amplification (SDA), rolling circle amplification (RCA), cross priming amplification, loop-mediated isothermal amplification (LAMP), isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN), etc. These methods are known to those skilled in the art and can be carried out by ordinary techniques. In one embodiment, the amplification of the target DNA in step (3) of the method of the present invention is carried out by PCR.

[0027] In the DNA amplification method of the present invention, the DNA (gene) to be amplified is not particularly limited, but may be, for example, a gene that is the subject of a genetic test for a specific disease. More specifically, for example, immune-related gene rearrangement (rearrangement) tests for malignant lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, etc. may be mentioned, and examples of the genes to be tested include IgH (immunoglobulin H chain) gene, IgK (immunoglobulin L chain κ) gene, IgL (immunoglobulin L chain λ) gene, TRB (T cell receptor β chain) gene, TRG (T cell receptor γ chain) gene, TRD (T cell receptor δ chain) gene, etc. In addition, for example, breast cancer HER2 protein (IHC) 4B5 test, epidermal growth factor receptor (EGFR) gene mutation test in lung cancer patients, detection of tuberculosis DNA, AmoyDx (registered trademark) lung cancer multi-gene PCR panel test (Riken Genesis Co., Ltd.), HRMA test, my Choic (registered trademark) diagnostic system test (Myriad Genetics, Inc.), KRAS G12C mutation analysis, EZH2 gene mutation analysis, melanoma BRAF mutation analysis, MSI test, MEBGEN RASKET TM These include genes that are the subject of testing using the -B kit (Medical and Biological Laboratories Co., Ltd.).

[0028] After step (2) or step (3) of the present invention, the quality of DNA may be confirmed. As an index of DNA quality, an index known per se may be used, for example, ΔCt value, DIN (DNA Integrity Number) value, etc. In the case of the DIN value, typically, 2.0 or more is preferable.

[0029] 2. Sequencing Method of the Present Invention The present invention provides a sequencing method, comprising the step of analyzing the sequence of the DNA amplified by the above-mentioned DNA amplification method of the present invention.The sequencing method may be performed by conventional techniques such as the Sanger method, or may be performed by massively parallel sequencing.The massively parallel sequencing includes, for example, next-generation sequencing (NGS).

[0030] Next-generation sequencing is a method for obtaining sequence information using a next-generation sequencer, and is characterized by the ability to perform a huge number of sequencing reactions simultaneously in parallel compared to the Sanger method (e.g., Rick Kamps et al., Int. J. Mol. Sci., 2017, 18(2), p.308 and Int. Neurourol. J., 2016, 20(Suppl. 2), S76-83). Various systems for next-generation sequencing are available, and include, but are not limited to, Roche's Genome Sequencer (GS) FLX System, Illumina's HiSeq, or Genome Analyzer (GA), Life technologies' Support Oligonucleotide Ligation Detection (SOLiD) system, Polonator's G.007 system, and Helicos BioSciences' HeliScope Gene Sequencing system.

[0031] Although not limited thereto, the general steps of next-generation sequencing are shown below. In next-generation sequencing, first, sample preparation is performed. In this step, DNA to be analyzed is enzymatically or mechanically fragmented according to the read length of the next-generation sequencer. In many cases, an adapter sequence required for the next sequencing step is then added. In addition, in order to analyze a specific gene region, the specific gene region may be enriched by PCR or the like, or a region having a specific sequence may be concentrated using a probe or the like. Enrichment of the gene region can be performed, for example, by an amplification step of 4 to 12 cycles, and concentration using a probe can be performed by utilizing a label attached to the probe (e.g., biotin, etc.).

[0032] Sequencing is then performed. Details of this process vary depending on the type of next-generation sequencer, but typically the DNA is linked to the substrate via an adapter sequence, and the sequencing reaction is performed using the adapter sequence as a priming site. For details of the sequencing reaction, see, for example, Rick Kamps et al., Int. J. Mol. Sci., 2017, 18(2), p.308 and Int. Neurourol. J., 2016, 20(Suppl. 2), S76-83. Finally, data output is performed. In this process, a collection of sequence information (reads) obtained by the sequencing reaction is obtained.

[0033] For matters other than those mentioned above in the sequencing method of the present invention, the contents described above in "1. DNA amplification method of the present invention" are all incorporated herein by reference.

[0034] 3. Amplification kit for DNA of the present invention The present invention provides a DNA amplification kit including a silica-based solid phase material. The silica-based solid phase material included in the DNA amplification kit of the present invention is not particularly limited as long as it contributes to removing or washing the fragmented DNA and other impurities as described above, and examples thereof include silica monoliths and silica membranes. The form of the solid phase material is also not particularly limited as long as it contributes to removing or washing the fragmented DNA and other impurities as described above, and examples thereof include spin columns, pipette tubes with columns, or column cartridges that can be attached to centrifuge tubes or pipette tubes. In addition, considering that the amount of DNA to be recovered is relatively small, the solid phase material may be in a form capable of purifying a small amount of DNA, such as a miniprep column.

[0035] The DNA amplification kit of the present invention may contain, in addition to the silica-based solid phase material, a washing solution used to remove or wash fragmented DNA and other impurities from the silica-based solid phase material, and an elution solution used to release (elute) DNA from the solid phase material. Typical examples of the washing solution include a solution (e.g., an aqueous solution, etc.) containing at least one water-soluble organic solvent and a water-soluble salt. Examples of the elution solution include distilled water and Tris-EDTA (TE) buffer. Furthermore, the DNA amplification kit of the present invention may contain, for example, an instruction manual. For matters other than those described above in the DNA amplification kit of the present invention, the contents described in "1. DNA amplification method of the present invention" above are all incorporated herein by reference.

[0036] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these in any way. EXAMPLES

[0037] Example 1: gDNA purification from FFPE samples <FFPEサンプル> The FFPE samples used were derived from the thymus of patients diagnosed by a pathologist as having MALT lymphoma: Sample 1 (prepared in 2022), Sample 2 (prepared in 2013), and Sample 3 (prepared in 2010). Sample 1 was fixed in 10% neutral buffered formalin (fixation time: 24 to 48 hours), while Samples 2 and 3 were fixed in 20% formalin (fixation time: 24 hours and one week).

[0038] <Obtaining pre-purified DNA> Promega's ReliaPrep TM DNA was extracted using the FFPE gDNA Miniprep System according to the makers protocol. The final extraction volume was 30 μl. After extraction, the concentration and 260 / 280 were measured using a Thermo Fisher NanoDorp.

[0039] <Obtaining purified DNA> The DNA obtained as described above in "<Obtaining pre-purified DNA>" was purified using NucleoSpin (registered trademark) gDNA Clean-up XS (Machrey-Nagel) (silica membrane method) according to the makers protocol.

[0040] The state of DNA for each sample before and after purification is shown in Table 1 and Figure 1.

[0041] [Table 1]

[0042] As a result of DNA purification, as shown in Table 1, the DNA concentration decreased before and after purification. This suggests that the purification removed low molecular weight DNA fragments, lowering the DNA concentration. In addition, using Agilent's Tape Station, it was confirmed that the DIN value of DNA increased before and after purification (Figure 1). From these results, it can be seen that low molecular weight DNA was removed by purification (Figure 1). Therefore, it was found that it is possible to reduce low molecular weight DNA by purification.

[0043] Example 2: PCR study before and after purification In normal lymph nodes, lymphocytes proliferate polyclonally, but in lymphomas and lymphocytic leukemias (e.g., MALT lymphomas, etc.), they proliferate monoclonally. In polyclonal lymphocyte populations, Ig / TCR genes are diverse, but in monoclonal lymphocyte populations, they are identical. Therefore, for example, if the IgH gene is amplified by PCR and the amplified genes are separated according to size by agarose gel electrophoresis, etc., in normal tissues where polyclonal proliferation occurs, the amplified genes spread throughout the gel and are detected as a smear. On the other hand, in lymphomas and lymphocytic leukemias (e.g., MALT lymphomas, etc.) where monoclonal proliferation occurs, the amplified genes are the same length, so they move to the same position in the gel, and amplification of a single gene is detected.

[0044] First, the housekeeping gene (GAPDH) was amplified by PCR using the purified DNA from Samples 1 to 3, and good amplification results were obtained for all DNAs (Figure 2). In addition, when the DNA from sample 3 in 2010 and sample 2 in 2013 before purification was used to perform PCR for IgH rearrangement and confirm DNA amplification by electrophoresis, the amplification could not be confirmed in the first place. On the other hand, when the DNA from the samples in 2010 and 2013 purified in Example 1 was used to perform PCR for IgH rearrangement and confirm DNA amplification by electrophoresis, the amplification could be confirmed as amplification of a single gene (Figure 3). In addition, for sample 1 in 2022, the desired IgH gene could be amplified more reliably by performing purification in Example 1 (Figure 3). As a result, by using the DNA amplification method of the present invention, it is possible to detect, for example, lymphoma and lymphocytic leukemia (e.g., MALT lymphoma, etc.) (Figure 3).

[0045] From the above, it was found that the amplification method of the present invention enables the amplification of DNA (desired gene) using DNA extracted from a formalin-fixed sample. Furthermore, it was demonstrated that, in a genetic test required for diagnosing lymphoma / lymphocytic leukemia (e.g., MALT lymphoma, etc.), the amplification method of the present invention makes it possible to perform the test using DNA extracted from a formalin-fixed sample, which was previously difficult to perform, particularly a formalin-fixed sample that has been stored for a long period of time (e.g., 3 years or more, particularly 10 years or more). [Industrial Applicability]

[0046] The present invention is useful because it can amplify desired DNA in a formalin-fixed sample, making it possible to perform genetic testing using PCR or a next-generation sequencer. In particular, the present invention is useful because it can amplify desired DNA in a formalin-fixed sample that has been stored for more than three years, which was previously considered untestable. In addition, the present invention is useful because it can perform genetic analysis on old samples that have been stored for more than three years, making it possible to search for and identify new diagnostic markers and causative genes of diseases. Furthermore, the present invention is useful because it can perform genetic testing on specimens that have been preserved in formalin in forensic genetic testing.

Claims

1. A method for amplifying DNA, comprising: (1) extracting DNA from a formalin-fixed sample; (2) removing the fragmented DNA from the solution containing the extracted DNA; and (3) amplifying the target DNA in the solution obtained in step (2); A method comprising:

2. 2. The method of claim 1, wherein the removal of fragmented DNA in step (2) is carried out using a silica-based solid phase material.

3. The method of claim 2 , wherein the silica-based solid phase material is a silica membrane.

4. The method according to any one of claims 1 to 3, wherein the formalin-fixed sample is fixed more than three years ago.

5. The method according to any one of claims 1 to 4, wherein the formalin-fixed sample has been fixed for more than 10 years.

6. The method according to any one of claims 1 to 5, wherein the formalin-fixed sample is a thymus sample.

7. The method according to any one of claims 1 to 6, wherein the amplification of the target DNA in step (3) is carried out by PCR.

8. The method according to any one of claims 1 to 7, wherein the target DNA is an IgH gene.

9. A sequencing method comprising a step of analyzing the sequence of DNA amplified by the method according to any one of claims 1 to 8.

10. The method of claim 9, wherein the sequencing is performed using a next-generation sequencer.

11. A kit for amplifying DNA comprising a silica-based solid phase material.

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