DNA size fractionation using ultrafiltration filters

Abstract

To provide methods and means for isolating or recovering short-stranded DNA, such as cell-free DNA (cfDNA), from blood samples. [Solution] A method for isolating or recovering short-chain DNA of 100 to 250 bp from a DNA sample, comprising the steps of introducing the DNA sample into a column equipped with an ultrafiltration filter, centrifuging the column, and recovering the filtrate containing the short-chain DNA.

Description

[Technical Field]

[0001] The present invention relates to a method and kit for isolating or recovering short-stranded DNA. Specifically, the present invention relates to a technique for removing contaminating genomic DNA during the isolation or recovery of cell-free DNA (cfDNA) from a blood sample. [Background technology]

[0002] Genetic testing to detect small amounts of cancer-derived DNA in blood uses next-generation sequencing (NGS) and digital polymerase chain reaction (dPCR). However, when using these highly sensitive detection technologies to detect cancer-derived DNA in blood, contamination of the collected cfDNA samples with genomic DNA from white blood cells is problematic. Genomic DNA contamination can occur for a variety of reasons, including blood sample storage conditions, the type of blood collection device used, and the time elapsed between blood collection and cfDNA extraction. It has been reported that this contaminating genomic DNA essentially exhibits wild-type activity, hindering the detection of low-frequency mutations and the calculation of mutation frequencies (Non-Patent Document 1). Because blood contains a large number of white blood cells, developing a technique to remove contaminating genomic DNA is more effective than developing a technique to prevent genomic DNA contamination in establishing accurate liquid biopsy testing.

[0003] While cfDNA present free in blood is fragmented to chain lengths of approximately 120 to 220 bp, the contaminating genomic DNA is characterized by its large chain lengths of several kbp, and a size-selective DNA recovery technique that takes advantage of this difference in DNA fragment size has been reported (Patent Document 1). This technique is characterized by concentrating DNA fragments shorter than a cutoff value by binding DNA molecules to carboxylated magnetic beads capable of adsorbing DNA in a solution containing a polymer, typically polyethylene glycol.

[0004] Meanwhile, in the life sciences field, ultrafiltration membranes (filters) are used as a fractionation technique that takes advantage of differences in molecular weight. Ultrafiltration membranes are filtration membranes that can be used to fractionate or concentrate molecules between approximately 1 and 1,000 kDa (Daltons). The performance of ultrafiltration membranes is expressed in terms of molecular weight cutoff. Molecules larger than this cutoff are retained on the membrane, while salts and water pass through the membrane, making them useful for concentrating large molecules such as proteins (Non-Patent Document 1). Pall Corporation's application notes (Non-Patent Document 1) state in an example of fractionation that they are "effective for applications such as removing unincorporated labels and purifying PCR products after amplification reactions," demonstrating that the target molecule is a large molecular weight substance and removing small molecular weight substances. An example of PCR product concentration using an ultrafiltration column is shown in Figure 1. The target sample is placed on the ultrafiltration filter column in a centrifuge tube and centrifuged (spin). After centrifugation, the remaining solution is collected from the top of the filter (Recover DNA fragments), yielding a sample with a concentrated target solute (the filtrate is discarded). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] US2022 / 0259637A1 [Patent Document 2] US2021 / 0009990A1 [Patent Document 3] WO2021 / 148393A [Non-patent literature]

[0006] [Non-Patent Document 1] (10) NanoSep Centrifugal Filtration Device Application Protocol Collection: 21 / 03 / 08 First Edition [Non-patent document 2] Boom, R. et al., Journal of Clinical Microbiology (1990) 28:495-503 [Non-patent document 3] Hawkins, TL et al., Nucleic Acids Research (1994) 22:4543-4544 Summary of the Invention [Problem to be solved by the invention]

[0007] Commonly used cfDNA extraction reagents use the Boom method (Non-Patent Document 2), which adsorbs nucleic acids onto a silica surface in the presence of chaotropic salts for nucleic acid adsorption purification. However, this technique lacks the size fractionation capability to distinguish DNA fragments. Techniques for selecting DNA of specific sizes by modifying the Boom method have also been reported (Patent Documents 2 and 3), but this is achieved by adjusting the concentration of the reagents used, which requires multiple processing steps.

[0008] On the other hand, an example of a technology for fractionating DNA by chain length is the Beckman Coulter product (Ampure), which utilizes the SPRI method (Non-Patent Document 3), which takes advantage of the chain-length dependence of DNA adsorption to silica surfaces in the presence of polyethylene glycol. While this product is widely used in NGS pretreatment kits and is well-known, it is not currently used as a cfDNA extraction reagent. While the technique of fractionating DNA fragments by chain length using a polymer mixture solution is similar to the technique described in Patent Document 1, DNA fragment fractionation techniques using a polymer mixture solution have not yet been widely adopted in cfDNA extraction techniques. These techniques select DNA size by mixing a sample solution and a polymer solution at the desired ratio, but the general difficulty of accurately dispensing the highly viscous polymer solution and the fact that the technique differs from the Boom method, a standard DNA fragment adsorption purification method, are thought to be hindering their widespread use. [Means for solving the problem]

[0009] As a result of investigating solutions to the above problems, the inventors discovered that short-chain DNA such as cfDNA can be fractionated by a size fractionation process using ultrafiltration, and that short-chain DNA can be efficiently fractionated by adding chaotropic salts to DNA samples. Furthermore, based on the current cfDNA extraction method, the Boom method, they developed a cfDNA fractionation technology that more efficiently removes contaminating genomic DNA by adding a size fractionation process using ultrafiltration prior to the Boom method.

[0010] Thus, the present invention encompasses, for example, the following aspects: In one aspect, the present invention provides a method for isolating or recovering short-stranded DNA of 100 to 250 bp from a DNA sample, comprising: introducing the DNA sample into a column equipped with an ultrafiltration filter and centrifuging it; A step of recovering the filtrate containing short-chain DNA The present invention provides a method comprising:

[0011] In one embodiment, the method of the present invention comprises: dissolving the DNA sample in a chaotropic salt solution; a step of contacting the filtrate containing the short-chain DNA with a silica carrier and adsorbing the short-chain DNA onto the silica carrier Further includes:

[0012] In another aspect, the present invention provides a kit for use in a method for isolating or recovering short-chain DNA of 100 to 250 bp from a DNA sample, comprising: a column equipped with an ultrafiltration filter, The kit further includes the method comprising introducing the DNA sample into a column equipped with the ultrafiltration filter, centrifuging the column, and then recovering the filtrate.

[0013] In one embodiment, the kit further comprises a column comprising a silica support, The method includes introducing the DNA sample dissolved in a chaotropic salt solution into a column equipped with the ultrafiltration filter, centrifuging the column, and then adsorbing the DNA contained in the filtrate onto the silica carrier. [Effects of the Invention]

[0014] The present invention enables simple and efficient removal of contaminating genomic DNA during isolation or recovery of cfDNA from a sample, thereby contributing to improved mutation detection rates and accurate calculation of mutant gene ratios in genetic testing using liquid biopsies that utilize cfDNA. [Brief explanation of the drawings]

[0015] [Figure 1] This is a diagram illustrating the conventional method of using an ultrafiltration filter column. The DNA fragments to be recovered remain in the column, and the filtrate is discarded. [Figure 2] Electrophoresis photograph (A) shows the analytical results of the filtrate from the ultrafiltration filter after purification on a silica column, and a table (B) shows the DNA concentration. The results are shown for a DNA sample solution prepared in an aqueous solution. [Figure 3] Electrophoresis photograph (A) shows the analytical results of the ultrafiltration filtrate after purification on a silica column, and a table (B) shows the DNA concentration. The results are shown for a DNA sample solution prepared with a chaotropic salt solution. [Figure 4] 1 is a photograph showing an example of a fractionation and purification device in which an ultrafiltration filter unit and a silica filter unit are combined, with the ultrafiltration filter unit being placed above the silica filter unit. [Figure 5] Electrophoresis photograph (A) showing the analysis results after treatment with the combined fractionation and purification device, and a table (B) showing the DNA concentrations. [Figure 6] (A) shows an electrophoresis photograph showing the analysis results after treatment with the combined fractionation and purification device, and (B) shows a table showing the DNA concentration. The effect of changing the volume of sample solution was verified. The centrifugal temperature during fractionation and purification was set to 25°C. [Figure 7](A) shows an electrophoresis photograph showing the analysis results after treatment with the combined fractionation and purification device, and (B) shows a table showing the DNA concentration. The effect of changing the volume of sample solution was verified. The centrifugation temperature during fractionation and purification was set at 4°C. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention relates to methods and kits for isolating or recovering short-stranded DNA (e.g., cfDNA) from a DNA sample such as blood.

[0017] In the present invention, the short-stranded DNA to be isolated or recovered is DNA with a length of 100 to 250 bp, including, for example, cell-free DNA (cfDNA). cfDNA refers to fragmented DNA of approximately 120 to 220 bp derived from broken cells and released into the blood, and cfDNA derived from cancer cells is particularly called ctDNA (circulating tumor DNA). Here, the 120 to 220 bp of cfDNA corresponds to a molecular weight of 79,200 to 145,200 Da, or approximately 80 to 145 kDa. In one embodiment, the short-stranded DNA to be isolated or recovered in the present invention is not primer DNA or probe DNA.

[0018] The DNA sample is not particularly limited as long as it contains short-stranded DNA to be isolated or recovered, and examples thereof include blood samples, plasma samples, lymph, saliva, spinal fluid, semen, feces, sputum, milk, etc. In one embodiment, the DNA sample comprises a blood sample or a plasma sample. The origin of the DNA sample is also not particularly limited, and a DNA sample derived from any living organism, such as a vertebrate (e.g., mammal, bird, reptile, fish, amphibian, etc.), can be used. In a preferred embodiment, the DNA sample is derived from a mammal, particularly a human, or a laboratory animal (e.g., a mouse, rat, monkey, etc.). The DNA sample may be treated with, for example, a DNA protecting agent, a DNase inhibitor, and / or an RNase, to selectively isolate or recover DNA. Additionally, a detergent (e.g., a non-ionic detergent such as Triton X-100 (octylphenol ethoxylate), Tween® 20 (polyoxyethylene sorbitan monolaurate), or Tween® 80 (polyoxyethylene sorbitan oleate)) may be added to the DNA sample to release the cfDNA encapsulated in the lipid vesicles.

[0019] As used herein, "isolation" and "recovery" refer to the partial or complete separation and removal of short-chain DNA contained in a DNA sample from non-DNA contaminants and longer-chain DNA (e.g., genomic DNA), and the removal of contaminating long-chain DNA. Isolation or recovery is performed so that at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% of the isolated or recovered DNA is short-chain DNA. Furthermore, at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% of the genomic DNA contaminating the DNA sample is removed.

[0020] In one aspect, the present invention provides a method for isolating or recovering short-stranded DNA of 100 to 250 bp from a DNA sample, comprising: introducing the DNA sample into a column equipped with an ultrafiltration filter and centrifuging it; A step of recovering the filtrate containing short-chain DNA The present invention relates to a method comprising:

[0021] In one embodiment, the DNA sample is in an aqueous solution.

[0022] In one embodiment, the method may further include dissolving the DNA sample in a chaotropic salt solution, which improves the recovery rate of short-chain DNA.

[0023] The chaotropic salt is not particularly limited as long as it is a known chaotropic salt, and examples thereof include guanidine salts such as guanidine isothiocyanate, guanidine thiocyanate, guanidine sulfate, and guanidine hydrochloride, urea, sodium iodide, potassium iodide, sodium bromide, potassium bromide, calcium bromide, ammonium bromide, sodium perchlorate, sodium cyanate, potassium cyanate, sodium thiocyanate, sodium perchlorate, sodium trichloroacetate, and sodium trifluoroacetate, and preferably guanidine salts. These chaotropic salts may be used alone or in combination.

[0024] Those skilled in the art can easily determine the optimal concentration of the chaotropic salt. The concentration of the chaotropic salt, as the final concentration when mixed with the DNA sample, may be, for example, 0.1% by weight or more, 0.5% by weight or more, 1% by weight or more, 2% by weight or more, 5% by weight or more, or 10% by weight or more, or may be, for example, 90% by weight or less, 80% by weight or less, 70% by weight or less, 60% by weight or less, 50% by weight or less, or 40% by weight or less, for example, 0.1% by weight to 90% by weight, 0.5% by weight to 80% by weight, 1% by weight to 70% by weight, 2% by weight to 60% by weight, 5% by weight to 50% by weight, or 10% by weight to 40% by weight.

[0025] Ultrafiltration filters are known in the art and are made from materials such as cellulose acetate, aromatic polyamide, polyvinyl alcohol, polysulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, etc. Columns equipped with ultrafiltration filters are also known in the art and are commercially available in a variety of forms.

[0026] The molecular weight cutoff of the ultrafiltration filter can be determined by considering the centrifugal speed and temperature to be used so that short-chain DNA can pass through. For example, if the DNA sample is an aqueous solution, an ultrafiltration filter with a molecular weight cutoff of 100 kDa is used at a centrifugal speed of 2,000 x g to 8,000 x g. If the DNA sample is a chaotropic salt solution, an ultrafiltration filter with a molecular weight cutoff of 10 kDa is used at a centrifugal speed of 8,000 x g to 14,000 x g, and / or an ultrafiltration filter with a molecular weight cutoff of 30 kDa is used at a centrifugal speed of 2,000 x g to 8,000 x g.

[0027] In the method of the present invention, a DNA sample is introduced into a column equipped with an ultrafiltration filter and centrifuged. The amount of DNA sample solution introduced can be appropriately determined depending on the volume of the column and the centrifugation conditions (speed and temperature). Centrifugation can be performed at an appropriate speed and temperature using a centrifuge known in the art.

[0028] The recovery amount and degree of purification (proportion of other DNA contamination) of short-chain DNA vary by changing the molecular weight cutoff of the ultrafiltration filter, the volume of DNA sample solution, the centrifugation speed, and the centrifugation temperature (Example 4). Therefore, in one embodiment, in the method of the present invention, the recovery rate of short-chain DNA is adjusted using at least two variables selected from the group consisting of the molecular weight cutoff of the ultrafiltration filter, the volume of DNA sample solution, the centrifugation speed, and the centrifugation temperature. In another embodiment, the degree of purification of short-chain DNA is adjusted using at least two variables selected from the group consisting of the molecular weight cutoff of the ultrafiltration filter, the volume of DNA sample solution, the centrifugation speed, and the centrifugation temperature.

[0029] After centrifugation, the filtrate contains the target short-chain DNA, and by collecting the filtrate, the short-chain DNA can be isolated or collected simply and efficiently.

[0030] The method of the present invention may involve using a DNA sample mixed with a chaotropic salt solution used in the Boom method as a target sample, and applying ultrafiltration as a size fractionation technique prior to the Boom method purification step. The Boom method is a method for isolating nucleic acids from biological samples by utilizing the property of nucleic acids to adsorb to silica in the presence of a chaotropic substance.

[0031] When using the boom method, the method of the present invention comprises: dissolving the DNA sample in a chaotropic salt solution; a step of contacting the filtrate containing the short-chain DNA with a silica carrier and adsorbing the short-chain DNA onto the silica carrier; It may further include:

[0032] For example, in one embodiment, the method of the present invention comprises: dissolving the DNA sample in a chaotropic salt solution; introducing the chaotropic salt solution of the DNA sample into a column equipped with an ultrafiltration filter and centrifuging the column; A step of contacting the filtrate containing short-chain DNA with a silica carrier to adsorb the short-chain DNA onto the silica carrier. Includes.

[0033] As described above, the chaotropic salt is not particularly limited, and examples thereof include those that promote binding between DNA and the silica carrier (DNA adsorption carrier), such as guanidine salts such as guanidine isothiocyanate, guanidine thiocyanate, guanidine sulfate, and guanidine hydrochloride; urea; sodium iodide; potassium iodide; sodium bromide; potassium bromide; calcium bromide; ammonium bromide; sodium perchlorate; sodium cyanate; potassium cyanate; sodium thiocyanate; sodium perchlorate; sodium trichloroacetate; and sodium trifluoroacetate, with guanidine salts being preferred. These chaotropic salts may be used alone or in combination. The concentration of the chaotropic salt is also as described above, and should be sufficient to bind DNA to the silica carrier; the optimal value can be easily determined by one skilled in the art.

[0034] The DNA sample is dissolved in a chaotropic salt solution, introduced into a column equipped with an ultrafiltration filter, and centrifuged. The centrifugation through the ultrafiltration filter can be carried out in the same manner as described above.

[0035] The filtrate containing the short-chain DNA is then contacted with a silica carrier to adsorb the short-chain DNA to the silica carrier. Silica carriers are known to be DNA adsorbing carriers, and their shape can be any shape, such as a column, powder, fiber, bead, membrane, or porous, as long as they are capable of adsorbing DNA. In one embodiment, the silica carrier is a silica filter. The silica carrier may be magnetic or modified.

[0036] In one embodiment, the method of the present invention further comprises a step of eluting the short-chain DNA adsorbed from the silica carrier. The short-chain DNA adsorbed to the silica carrier can be isolated or recovered by eluting the DNA with an eluent. Such an eluent is not particularly limited as long as it elutes the DNA from the silica carrier. Examples of such an eluent include water and buffers (e.g., Tris-HCl buffer), and those skilled in the art can select an appropriate eluent.

[0037] Before elution from the silica carrier, the silica carrier may be washed with a washing solution. The washing solution is not limited as long as it can wash away chaotropic salts remaining on the silica carrier to which DNA has been adsorbed and contaminants derived from the DNA sample. Examples of the washing solution include organic solvents in which DNA does not dissolve, water-soluble polymer solutions, and sugar solutions. Examples of solvents contained in the washing solution include ethanol, isopropanol, and acetone, and those skilled in the art can easily determine the optimal concentrations of these solvents. After washing, the silica carrier may be centrifuged, heat-treated, and air-dried to remove the washing solution.

[0038] In this way, by eluting DNA from the silica carrier, short-chain DNA can be isolated or recovered simply and efficiently.

[0039] The method according to the present invention can be carried out more easily and simply by using a kit including at least a column equipped with an ultrafiltration filter. That is, in a further aspect, the present invention provides a kit for use in a method for isolating or recovering short-chain DNA of 100 to 250 bp from a DNA sample, comprising: a column equipped with an ultrafiltration filter, The method relates to a kit in which the DNA sample is introduced into a column equipped with the ultrafiltration filter, followed by centrifugation, and then collecting the filtrate.

[0040] When using the kit of the present invention, the DNA sample may be dissolved in a chaotropic salt solution, and therefore the kit of the present invention may further comprise a chaotropic salt solution.

[0041] In another embodiment, the kit further comprises a column comprising a silica support, The method includes introducing the DNA sample dissolved in a chaotropic salt solution into a column equipped with the ultrafiltration filter, centrifuging the column, and then adsorbing the DNA contained in the filtrate onto the silica carrier.

[0042] The column equipped with an ultrafiltration filter and the column equipped with a silica carrier are as described above, and a person skilled in the art can prepare a column with a suitable configuration. In one embodiment, a column equipped with an ultrafiltration filter and a column equipped with a silica carrier are integrated. Preferably, a column equipped with an ultrafiltration filter at the top and a column equipped with a silica carrier at the bottom are integrated to form a column (for example, the configuration shown in Figure 4). With this configuration, the filtrate containing short-chain DNA that has passed through the ultrafiltration filter is introduced directly into the column equipped with the silica carrier, and the short-chain DNA is adsorbed to the silica carrier.

[0043] In addition to a column equipped with an ultrafiltration filter and a column equipped with a silica carrier, the kit of the present invention may also include a chaotropic salt solution, an elution solution for eluting DNA from the silica carrier, a washing solution, etc. The kit of the present invention may also include instructions describing the conditions for centrifugation in ultrafiltration (such as the molecular weight cutoff of the ultrafiltration filter, the amount of DNA sample, the centrifugation speed, and the centrifugation temperature). Providing the kit in this manner enables the isolation or recovery of short-chain DNA to be performed more simply and efficiently.

[0044] As used herein, elements referred to in the singular are intended to include the plural unless the context clearly indicates otherwise.

[0045] The present invention should not be construed as being limited to the description of the embodiments shown in this specification. Those skilled in the art will readily understand that the specific configuration can be modified within the scope of the idea or intent of the present invention.

[0046] In order to facilitate understanding of the invention, the position, size, shape, range, etc. of each component shown in the drawings etc. may not represent the actual position, size, shape, range, etc. Therefore, the present invention is not necessarily limited to the position, size, shape, range, etc. disclosed in the drawings etc.

[0047] The present invention will be specifically described below by way of examples, but these examples are provided merely to illustrate the present invention and are not intended to limit or restrict the scope of the invention disclosed in this application. [Example]

[0048] [Example 1] Since the molecular weight of DNA is approximately 660 per bp, the molecular weight of the cfDNA to be recovered is calculated by multiplying this value by the cfDNA chain length of 120-220 bp, resulting in a molecular weight of 79,200-145,200. This study aimed to compare products with fractionation performance close to the molecular weight of cfDNA. Pall's Nanosep centrifugal filter device, which can be centrifuged in a rotor for 1.5 mL tubes, was used as the ultrafiltration filter. Three molecular weight cutoff performances were selected: 10 kDa, 30 kDa, and 100 kDa.

[0049] The DNA sample was a 172-bp short-chain DNA as a model length for cfDNA, and a mixture of 3-kbp and 10-kbp DNA fragments as model samples for genomic DNA. Aqueous solutions were prepared using 20 ng of each of the three DNA samples as solutes and 60 μL of distilled water as solvent.

[0050] A 60 μL aqueous solution sample was placed on an ultrafiltration filter as the DNA solution and centrifuged for 10 minutes using an angle rotor. Three centrifugal force settings were used: 2,000 x g, 8,000 x g, and 14,000 x g. The filtrate obtained by centrifugation was analyzed after purification of the recovered sample. In this study, DNA was purified and recovered using the NucleoSpin Gel and PCR Clean-up (TaKaRa) purification kit, which uses the boom method with a silica filter column. The eluate volume during recovery was standardized to 20 μL. DNA fragment size and concentration were measured using a 4200 TapeStation System (Agilent, hereafter referred to as TapeStation) and the Genomic DNA ScreenTape System (Agilent) as reagents.

[0051] Aqueous solutions of cfDNA dissolved in distilled water were treated with a 10-minute centrifugation time, varying the molecular weight cutoff and centrifugal force. The resulting filtrate was purified and 1 μL of the filtrate was evaluated by electrophoresis (Figure 2). Figure 2A shows the electrophoretic image, and Figure 2B shows the concentration of each DNA fragment measured in the recovered solution. In the control sample (PC) without fractionation, each of the three DNA fragments of different chain lengths was clearly observed. Using the 100 kDa column with the largest pore size, three DNA fragments, including a 10 kb fragment, were identified in the recovered sample at 8,000 xg and 14,000 xg, demonstrating the feasibility of recovering DNA fragments from the ultrafiltration filter filtrate. Furthermore, a small amount of a 172 bp DNA fragment was detected at 2,000 xg using the 100 kDa column. These results suggest that selective recovery of cfDNA can be achieved by adjusting the processing conditions.

[0052] [Example 2] Considering that fractionation using an ultrafiltration column would be performed as a pretreatment for DNA purification using the Boom method, we attempted fractionation of a chaotropic salt solution sample containing chaotropic salts.

[0053] The following is a test in which the DNA sample solution was replaced with a chaotropic salt solution. The conditions and steps were the same as in Example 1, except that 60 μL of distilled water, which was the solvent in Example 1, was replaced with 60 μL of chaotropic salt solution. The chaotropic salt solution used in this test was prepared by diluting the binding buffer of NucleoSpin Gel and PCR Clean-up (TaKaRa) to the concentration specified in the manual.

[0054] The results of the fractionation experiment using the chaotropic salt solution sample are shown in Figure 3. Figure 3A shows the electrophoretic image, and Figure 3B shows the concentration of each DNA fragment measured in the recovery solution. Under these solution conditions, DNA fragment recovery was observed with all three ultrafiltration filters (10 kDa, 30 kDa, and 100 kDa), confirming that the addition of chaotropic salt affects filtration performance. The amount of recovered DNA tended to increase with increasing column molecular weight cutoff, i.e., larger pore diameter (Figure 3B). Furthermore, for the same fractionation performance, increasing the centrifugal force resulted in increased recovery. The concentrations of the three recovered DNA fragments demonstrated that cfDNA fractionation and recovery were possible using a 10 kDa or 30 kDa column at settings of 2,000 x g and 8,000 x g.

[0055] [Example 3] Figure 4 shows a composite column constructed for simultaneous processing of two columns: size fractionation using an ultrafiltration column and purification using a silica column based on the Boom method. The lower column is a silica column (silica filter unit 102) with the function of adsorbing and recovering nucleic acids. An ultrafiltration column (ultrafiltration filter unit 101) with size fractionation function is installed above it. This configuration aims to allow a chaotropic salt solution sample added to the top of the ultrafiltration column to be adsorbed to the silica column after size fractionation in a single centrifugation process. In this example, a 10 kDa column and a 30 kDa column were used for comparison. The silica column used was the silica column included with NucleoSpin Gel and PCR Clean-up (TaKaRa). The manual for this reagent kit specifies a centrifugal force setting of 11,000 xg during nucleic acid adsorption processing, but the tests were performed at settings of 2,000 xg and 8,000 xg, the size fractionation settings examined in Examples 1 and 2.

[0056] The results of analyzing the purified and recovered DNA samples are shown in Figure 5. Panel A of Figure 5 is an electrophoretic image, and Panel B of Figure 5 shows the concentration of each DNA fragment measured in the recovered solution. In Figure 5, PC represents the untreated DNA sample for both fractionation and purification, while "Sil" represents the results of purification using a silica column at each centrifugal force setting without a fractionation column. Fractionation of the 172-bp cfDNA fragment was confirmed in the 8,000xg treatment of the 10-kDa column (10-kDa + Sil.) and the 2,000xg treatment of the 30-kDa column (30-kDa + Sil.) of the combined column, while 3-kbp and 10-kbp fragments were below the detection limit. In the 8,000xg treatment using the 30kDa column (30kDa + Sil.), a small amount of 3kbp fragment was detected in addition to the 172bp fragment. However, assuming that the 3kbp and 10kbp fragments recovered using the silica column (Sil.) constituted contaminating genomic DNA, the residual genomic DNA percentage was calculated as (remaining genomic DNA = 0.17ng / μL) / (contaminating genomic DNA = 14.1 + 9.84ng / μL) × 100 = 0.71%, indicating that 99% of the mixed genomic DNA was removed.

[0057] [Example 4] A test was conducted to verify the effect of adjusting the amount of sample solution. Using 10 ng of each of the three types of DNA samples shown above as solutes, sample solutions were prepared with the composition shown in Table 1 below. The amount of DNA sample added and the concentration of chaotropic salt were the same; only the amount of solution was changed. The sample solution processing and analysis process were the same as in Example 3, but in this test, the centrifuge temperature was set to 25°C or 4°C and the centrifuge was set to 8000 x g.

[0058] [Table 1]

[0059] The results are shown in Figure 6 (25°C) and Figure 7 (4°C). In Figures 6 and 7, A is the electrophoretic image, and B is the concentration of each DNA fragment measured in the recovery solution. In the figure, C is the result of purification using a silica column without size fractionation. This test showed that the same solution volume and temperature during centrifugation are factors that affect DNA fractionation performance or cfDNA recovery rate. It was shown that adjusting these variables depending on the goal, such as aiming for a high recovery rate or high degree of purification, is effective.

[0060] The publications and patent documents cited in this specification are incorporated herein by reference in their entirety. [Explanation of symbols]

[0061] 101 Ultrafiltration filter unit 102 Silica filter unit

Claims

1. A method for isolating or recovering short-chain DNA of 100 to 250 bp from a DNA sample, comprising: introducing the DNA sample into a column equipped with an ultrafiltration filter and centrifuging it; A step of recovering the filtrate containing short-chain DNA A method comprising:

2. dissolving the DNA sample in a chaotropic salt solution The method of claim 1 further comprising:

3. dissolving the DNA sample in a chaotropic salt solution; a step of contacting the filtrate containing the short-chain DNA with a silica carrier and adsorbing the short-chain DNA onto the silica carrier The method of claim 1 further comprising:

4. a step of eluting the short-chain DNA adsorbed from the silica carrier; The method of claim 3 further comprising:

5. The method of claim 3, wherein the silica support is a silica filter.

6. 2. The method of claim 1, wherein the short-stranded DNA is cell-free DNA (cfDNA).

7. 10. The method of claim 1, wherein the DNA sample comprises a blood sample or a plasma sample.

8. 4. The method of claim 2 or 3, wherein the chaotropic salt comprises a guanidine salt.

9. 2. The method according to claim 1, wherein the recovery rate of the short-chain DNA is adjusted using at least two variables selected from the group consisting of the molecular weight cutoff of the ultrafiltration filter, the amount of solution of the DNA sample, the centrifugation speed, and the centrifugation temperature.

10. 2. The method according to claim 1, wherein the degree of purification of the short-chain DNA is adjusted using at least two variables selected from the group consisting of the molecular weight cutoff of the ultrafiltration filter, the amount of solution of the DNA sample, the centrifugation speed, and the centrifugation temperature.

11. A kit for use in a method for isolating or recovering short-chain DNA of 100 to 250 bp from a DNA sample, comprising: a column equipped with an ultrafiltration filter, The method of the kit comprises introducing the DNA sample into a column equipped with the ultrafiltration filter, centrifuging the column, and then recovering the filtrate.

12. The kit of claim 11 , further comprising a chaotropic salt solution.

13. Further comprising a column with a silica support, The kit according to claim 11, wherein the method comprises introducing the DNA sample dissolved in a chaotropic salt solution into a column equipped with the ultrafiltration filter, centrifuging the column, and then adsorbing the DNA contained in the filtrate onto the silica carrier.

14. The kit according to claim 13, wherein a column equipped with the ultrafiltration filter and a column equipped with the silica carrier are integrated together.

15. 14. The kit of claim 13, further comprising a chaotropic salt solution and / or an elution solution for eluting DNA from the silica support.

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