Methods and kits for DNA isolation

Abstract

The present invention relates to improved methods and systems for isolating cell free DNA (cfDNA) present in a liquid biological sample.SOLUTION: A method for isolating cell-free DNA from a liquid biological sample comprising the steps of a) providing a liquid biological sample, b) adding to said sample a solid-phase capable of binding DNA, a binding buffer comprising a detergent and a chaotropic agent, and 2-propanol to form a binding mixture thereof, c) washing the solid-phase to remove unbound material, and d) eluting the bound cell-free DNA, wherein the majority of the eluted DNA is below 400bp.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to improved methods and systems for isolating cell-free DNA (cfDNA) present in liquid biological samples. More particularly, the present invention relates to methods and systems for isolating cfDNA from plasma by size selection to facilitate the enrichment and recovery of small fragment cfDNA while minimizing the recovery of larger, high molecular weight genomic DNA (gDNA) fragments. [Background technology]

[0002] Fragmented cfDNA molecules were first discovered in the human circulatory system by Mandel and Metais in 1948. cfDNA, also known as circulating free DNA or circulating cell-free DNA, is DNA fragments released into the bloodstream by cells. Several mechanisms for the release of cfDNA molecules into the blood have been proposed, including necrosis, apoptosis, phagocytosis, active cellular secretion, exosome release, pyroptosis, mitotic cell death, and autophagy, resulting in the presence of cfDNA populations with diverse physical properties in the circulation. In healthy individuals, cfDNA fragments vary between 100 and 250 bp, with the most common size being 166 bp, which corresponds to a nucleosome complex of DNA molecules bound to a histone core. The term cfDNA can be used to refer to various forms of fragmented DNA circulating freely in the bloodstream, such as cell-free fetal DNA (cffDNA), circulating tumor DNA (ctDNA), or circulating cell-free mitochondrial DNA (ccf mtDNA).

[0003] The clinical importance of cfDNA was recognized when researchers observed differences in cfDNA characteristics between healthy and diseased individuals. Many studies have demonstrated that cancer patients generally have higher levels of cfDNA compared to healthy controls. Elevated cfDNA levels in cancer patients are thought to be caused by excessive DNA release by apoptotic and necrotic cells, and / or the accumulation of cfDNA due to chronic inflammation and excessive cell death. In healthy individuals, cfDNA levels are primarily low, but may temporarily increase after strenuous exercise. It has also been demonstrated that cfDNA fragments derived from tumor cells are shorter than those derived from non-malignant cells. Similarly, cfDNA of fetal origin contains a high proportion of DNA smaller than 150 bp. Increased proportions of smaller fragments have also been reported in donor-derived fractions in autoimmune diseases and after transplantation. Therefore, size selection of smaller cfDNA fragments could be used to increase the amount of target cfDNA fragments (i.e., tumor-derived cfDNA for cancer diagnosis or fetal cfDNA for non-invasive prenatal testing). cfDNA in cancer patients contains unique genetic and epigenetic alterations characteristic of the tumors from which they originate. Therefore, genetic analysis and molecular profiling of cfDNA hold promise for clinical applications in cancer detection, prognosis, staging, monitoring, and treatment selection. Two FDA-approved applications for cfDNA assays in routine clinical practice—the cobas EGFR Mutation Test v2 for lung cancer patients and Epi proColon, a colorectal cancer screening test based on the methylation status of the SEPT9 promoter—have successfully demonstrated cfDNA as a biomarker for cancer management. Fetal cfDNA present in maternal blood has also been successfully used to detect fetal abnormalities. cfDNA analysis has also shown potential clinical uses in organ transplantation, autoimmune diseases, and sepsis, where the cfDNA fraction is enriched in smaller DNA molecules.

[0004] In the blood of cancer patients, cfDNA originates from multiple sources, including cancer cells as well as cells from the tumor microenvironment and other non-cancerous cells from various parts of the body. DNA from cancer cells is most notably released by apoptosis, necrosis, and active secretion. Apoptosis systematically cleaves chromosomal DNA into multiples of 160–180 bp in length, resulting in the presence of mononucleosomes and polynucleosomes outside the cell. The majority of cfDNA generated by apoptosis is 167 bp in size (147 bp of DNA wrapped around the nucleosome and approximately 20 bp of linker DNA connecting the two nucleosome cores).

[0005] Solid tumor biopsies are expensive and invasive, making them less than ideal for elderly or very young patients. On the other hand, cfDNA analysis as a disease biomarker can be performed using noninvasive liquid biopsies, utilizing patient liquid biological samples such as plasma, urine, or serum. The amount of ctDNA in the total cfDNA pool can vary greatly depending on the patient, cancer type, and stage of cancer, ranging from 0.01% to 90% in advanced metastases. It is widely agreed that ctDNA is more highly fragmented and has shorter fragment sizes (<150 bp) than cfDNA derived from healthy cells. Both the low abundance and short fragment size of ctDNA pose significant challenges for cfDNA isolation and further analysis. Furthermore, intratumor genetic heterogeneity poses another challenge in clinical oncology, and identification of minor subclonal populations is essential for detecting emerging chemotherapy resistance, detecting minimal residual disease, and noninvasively monitoring disease progression. Detection limits are adversely affected by the presence of contaminating high-molecular-weight gDNA, which may be present in plasma from lysed blood cells. Therefore, it is important to select a cfDNA extraction method that not only achieves a high cfDNA yield but also enables efficient recovery of shorter cfDNA fragments and negatively selects for high-molecular-weight DNA. Detection of rare, low-level resistance mutations is more likely when tumor-derived cfDNA is abundant in the sample and background gDNA from blood cells is minimized. Therefore, cfDNA is typically purified from white blood cell (WBC)-free plasma or serum to prevent gDNA contamination caused by WBC lysis. Contaminating gDNA can dilute tumor cfDNA and hinder the detection of rare variants. Because increased cfDNA fragmentation has been widely reported in fetal cfDNA, donor cfDNA after organ transplantation, and autoimmune diseases, size-selection-based cfDNA extraction offers clear advantages beyond cancer diagnosis. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide an improved size-selective method for isolating cfDNA from a liquid biological sample, such as plasma. [Means for solving the problem]

[0007] The distinct advantage of this method is that it can efficiently isolate the major cfDNA fraction together with fragments that are smaller and more highly degraded than the high-molecular-weight gDNA that is recognized as a contaminant.This size-dependent DNA binding can specifically enrich the extracted cfDNA in the fraction of interest, for example, tumor-derived cfDNA in cancer, or fetal cfDNA in prenatal testing for suspected aneuploidy.This makes the method of the present invention highly suitable for liquid biopsy-based diagnosis.

[0008] Another advantage of this method is that the required volume of input plasma sample is very small, ranging from 0.5 ml to 4 ml.

[0009] Another advantage of this method is that it is rapid, and the cfDNA isolation procedure can be completed in less than 2 h to generate high-quality cfDNA suitable for downstream applications.

[0010] According to one embodiment of the present invention, a method for isolating cell-free DNA from a liquid biological sample comprises the steps of: a) providing a liquid biological sample; b) adding to said sample - a solid phase capable of binding DNA, - a binding buffer containing a detergent and a chaotropic agent, and - adding 2-propanol to form a combined mixture thereof; c) washing the solid phase to remove unbound material; and d) eluting the bound cell-free DNA, wherein the majority of the eluted DNA is less than 400 bp.

[0011] According to another aspect of the present invention, a method for size-selectively isolating cell-free DNA from a liquid biological sample comprises the steps of: a) providing a liquid biological sample; b) adding to said sample - an aqueous suspension of silica-coated magnetic microbeads capable of binding DNA; - a binding buffer containing guanidine thiocyanate and a non-ionic detergent such as Triton X-100, and - adding 2-propanol to form a conjugation mixture therebetween, the conjugation mixture comprising about 20-30% w / v of a non-ionic detergent such as Triton X-100, about 1.5-2.5 M guanidine thiocyanate, and about 15-25% v / v of 2-propanol; c) incubating the binding mixture at room temperature for about 10-30 minutes to promote binding of cell-free DNA to the magnetic microbeads; d) washing the magnetic microbeads with one or more washing buffers comprising ethanol; e) adding an elution buffer to the washed magnetic beads of step d) to release the cell-free DNA bound to the magnetic microbeads in solution; and f) Optionally, analyzing or quantifying the cell-free DNA obtained in step e).

[0012] According to another aspect of the present invention, a binding buffer composition is formed using guanidine thiocyanate and Triton X-100 for size-selective binding of cell-free DNA present in plasma to silica-coated magnetic microbeads in an aqueous suspension of 20-200 mg / ml, and the binding buffer is intended to be contacted with 2-propanol, plasma, and magnetic microbeads to form a binding mixture containing about 1.5-2.5 M guanidine thiocyanate, about 20-30% w / v Triton X-100, about 15-25% v / v 2-propanol, and about 25-40% v / v plasma.

[0013] According to another aspect of the present invention, a kit is described that includes silica-coated microbeads capable of binding 50-400 bp DNA from a biological sample in the presence of guanidine thiocyanate, Triton X-100, and 2-propanol.

[0014] Further advantages and benefits of the present invention will become readily apparent to those skilled in the art from the following detailed description.

[0015] The invention will now be described in more detail with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 illustrates the general methodology used to extract cfDNA from whole blood according to the methods of the present invention. [Figure 2a] FIG. 1 shows a bioanalyzer plot showing the fragment size dependent recovery of DNA fragments using the method of the present invention. [Figure 2b] FIG. 1 shows the percent recovery of low and high molecular weight DNA fragments selected using the method of the present invention. [Figure 3] FIG. 10 shows bioanalyzer plots for preparations 10A-10D showing the effect of varying the ratio of 2-propanol in the binding mixture. [Figure 4] FIG. 10 shows bioanalyzer plots for preparations 10E-10H showing the effect of varying the ratio of 2-propanol in the binding mixture. [Figure 5] FIG. 10 shows bioanalyzer plots of Preparation Nos. 13A repeat and 13G repeat showing the effect of varying the ratio of 2-propanol in the binding mixture. [Figure 6] FIG. 10 shows a bioanalyzer plot showing the effect of varying the ratio of 2-propanol in the binding mixture. [Figure 7] FIG. 7 shows an enlarged view of a portion of FIG. 6 relating to low molecular weight DNA fragments. [Figure 8]FIG. 7 shows another enlarged view of a portion of FIG. 6 relating to high molecular weight DNA fragments. [Figure 9] FIG. 1 shows a bioanalyzer plot showing the effect of varying the percentage of Triton X-100 (8.8% and 11.1%) in the binding mixture. [Figure 10] FIG. 1 shows a bioanalyzer plot showing the effect of varying the percentage of Triton X-100 in the binding mixture (8.8% and 4.5%). [Figure 11] FIG. 1 shows electropherograms to demonstrate the influence of plasma components on size selection. [Figure 12] FIG. 1 shows bioanalyzer plots demonstrating the scalability of the cfDNA isolation method of the present invention to varying plasma input amounts. [Figure 13] FIG. 1 shows a bioanalyzer plot showing the cfDNA recovery profile using plasma collected in standard EDTA tubes. [Figure 14] FIG. 1 shows the advantage of size selection of the method of the present invention in cancer mutation detection over commercially available kits that do not size select. DETAILED DESCRIPTION OF THE INVENTION

[0017] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, definitions of certain terms used throughout the specification and claims are provided below. Illustrative examples of certain terms herein should be considered as non-limiting examples.

[0018] The terms "comprising" or "comprises" have their conventional meaning throughout this application, meaning that an agent or composition must have the recited essential functions or ingredients, but others may also be present. The term "comprising" includes "consisting essentially of" as a preferred subset, which means that the composition has the recited ingredients, with no other functions or ingredients being present.

[0019] This written description uses examples to disclose the invention, including the best mode, and also enables any person skilled in the art to practice the invention, including making and using any devices or systems, and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements that differ insubstantially from the literal language of the claims.

[0020] List of abbreviations cfDNA: cell-free DNA cffDNA: cell-free fetal DNA ccf mtDNA: circulating cell-free mitochondrial DNA PBS: phosphate buffered saline GuSCN: Guanidine thiocyanate bp: base pair EDTA: Ethylenediaminetetraacetic acid NGS: Next Generation Sequencing SDS: sodium dodecyl sulfate gDNA: genomic DNA WBC: white blood cell PCR: polymerase chain reaction ddPCR: Digital Droplet PCR

[0021] List of equipment used in the examples 1. A centrifuge that can accommodate 15 mL centrifuge tubes and 1.5 mL microcentrifuge tubes 2. A standard laboratory shaker / mixer, e.g., an Eppendorf™ Thermomixer, to accommodate 15 mL centrifuge tubes and 1.5 mL microcentrifuge tubes. 3. Incubator 4. Vortex mixer 5. 15 mL centrifuge tubes and 1.5 mL microcentrifuge tubes 6. cfDNA stabilizing tubes for blood collection 7. Pipette tips with aerosol barriers 8. Magnetic racks suitable for 15 mL centrifuge tubes and 1.5 mL microcentrifuge tubes, e.g., MagRack 6 and MagRack Maxi (GE Healthcare) 9. Bioanalyzer 2100 (Agilent)

[0022] All tubes and pipette tips used were of DNase-free grade. Good clinical practice for drug safety was followed to prevent sample contamination.

[0023] Detailed Description The cfDNA isolation method of the present invention allows for rapid extraction and purification of cfDNA from small volumes of liquid biological samples, such as plasma, ranging from 0.5 ml to 4 ml, and provides high-resolution cfDNA size selection. This method is specifically designed to select short fragments of cfDNA (50 bp to 400 bp) from longer, high-molecular-weight contaminating gDNA. The isolation procedure of the present invention can be completed in less than two hours and produces high-quality cfDNA suitable for downstream applications such as PCR, digital droplet PCR (ddPCR), genotyping, and next-generation sequencing (NGS).

[0024] Figure 1 illustrates the general methodology used to extract cfDNA from whole blood according to the method of the present invention. To ensure the highest quality and quantity of cfDNA, blood samples are typically collected in cfDNA-stabilizing tubes, such as Streck cfDNA collection tubes. Streck cfDNA collection tubes are blood collection devices containing stabilizing reagents that stabilize nucleated blood cells and prevent the release of cellular DNA into the plasma, thereby preserving cfDNA in blood samples at room temperature for up to 14 days. As will be understood by those skilled in the art, EDTA tubes or heparin tubes can be used as alternatives. After blood collection, the collection tubes are stored at room temperature until further processing to obtain plasma. The collection tubes are centrifuged at a low speed of 1600 x g for 10 minutes at 20°C to separate the plasma from intact blood cells. The upper plasma fraction (approximately 4-5 mL per 10 mL of blood) is aspirated into a new tube without disturbing the buffy coat layer, which is located between the plasma and sedimented red blood cell layer. The tube is then centrifuged again at 16,000 x g for 10 minutes at 20°C to remove cellular debris and other contaminants, yielding clear plasma. The clear plasma fraction is aspirated into a new tube, leaving the cellular debris behind. As those skilled in the art know, to maximize plasma volume and ensure sample homogeneity, it is generally recommended to pool the individual aspirates and then realiquot them into convenient units for cfDNA isolation, e.g., 2.0 mL units. The plasma can then be processed immediately for cfDNA isolation or aliquoted and stored at -20°C / -80°C until needed. Purified cfDNA may be stored short-term at 2-8°C if it is to be used directly for analysis and / or downstream molecular biology applications. For long-term storage, -20°C or -80°C is recommended.

[0025] The predominant type of cfDNA found in plasma is derived from the nuclear genome and has a fragment size corresponding to a single nucleosome. These macromolecular complexes must be dissociated to release the cfDNA and facilitate its binding to the DNA-binding solid phase. In one embodiment of the present invention, the solid phase was preferably silica-coated magnetic microbeads, in which the silica bead surface directly participates in DNA binding via surface silane (Si-OH) groups. Some cfDNA is also believed to be encapsulated in lipid vesicles and must be released prior to the binding step. Release of cfDNA from these various macromolecular complexes and lipid vesicles is achieved by using a combination of a chaotropic agent and a detergent. The chaotropic agent disrupts nucleosomal units to release the cfDNA, while the detergent solubilizes and denatures proteins to help release non-covalently bound cfDNA. When blood is collected into Streck cfDNA collection tubes, proteinase K treatment is additionally required to reverse the effects of the Streck cfDNA stabilization chemistry by removing crosslinks that would otherwise prevent efficient recovery of cfDNA during the isolation method. As will be understood by those skilled in the art, when using other blood collection tubes, proteinase K treatment may not be necessary. Next, the denatured contaminants are removed by washing the silica beads with a washing buffer and then air-drying the silica beads. Next, the purified cfDNA is eluted from the silica beads using an elution buffer. In a preferred embodiment of the present invention, SeraSil-Mag 700 beads manufactured by GE Healthcare Life Sciences are used to bind the released cfDNA, GuSCN is used as a chaotropic agent, and 20% SDS (sodium dodecyl sulfate) is used as a detergent. As will be understood by those skilled in the art, any other DNA-binding solid phase can be used instead of silica beads. For example, the solid phase can be beads, particles, sheets, and membranes with inherent or additional DNA-binding capabilities.

[0026] Because the levels of cfDNA encountered in plasma are very low, isolation methods require significant reductions in volume to generate cfDNA at concentrations sufficient for analysis. Efficient binding, gentle washing, and minimal elution of cfDNA from plasma are key to providing purified cfDNA suitable for downstream applications. Because the levels of cfDNA in the final extract are typically low, UV absorbance-based analysis is generally not recommended. Instead, cfDNA concentration is assessed using qPCR or fluorescence-based methods such as Qubit™ (Invitrogen™). The Qubit™ dsDNA HS Assay Kit is compatible with a fluorometer or fluorescent plate reader and can accurately estimate total DNA concentrations down to 10 pg / µL. To assess the quality and yield of cfDNA in addition to the presence of gDNA, an Agilent 2100 Bioanalyzer system can be used with the Agilent High Sensitivity DNA Analysis Kit.

[0027] Method for purifying cfDNA from 1.0 to 4.0 mL of plasma [Example]

[0028] Whole blood samples collected in Streck cfDNA collection tubes were processed to separate plasma as described above. The following steps of the cfDNA isolation method were then performed to obtain purified cfDNA from the plasma sample. Each step was performed using three different input plasma volumes: 1 ml, 2 ml, and 4 ml of plasma sample.

[0029] Process 1.Dissolution This step is performed to release cfDNA from the macromolecular complex and reverse the Streck DNA stabilization chemistry. Proteinase K (20 mg / mL) solution and plasma sample were added to a 15 mL Streck cfDNA collection tube and briefly vortexed to mix. Next, 20% sodium dodecyl sulfate (SDS) was added to the tube. Either the proteinase K or the plasma can be added to the tube first. However, to prevent enzyme inactivation, the 20% SDS should not come into direct contact with the proteinase K solution. The tube was pulse-vortexed 2-3 times and the contents thoroughly mixed by vortexing for 15 seconds. The tube was then incubated at approximately 55-65°C for approximately 20-30 minutes. Table 1 below shows the various plasma input volumes used and the corresponding amounts of proteinase K and 20% SDS.

[0030] [Table 1]

[0031] Step 2: Preparation of the Binding Mixture The magnetic microbeads (Sera-Sil Mag 700, GE Healthcare Life Sciences) were thoroughly resuspended by vortexing before dispensing. The binding mixture was prepared by combining the plasma from step 1, binding buffer, aqueous suspension of magnetic beads, and 2-propanol. In this example, the conjugation reagent was first prepared by combining the binding buffer, aqueous suspension of magnetic beads, and 2-propanol. This premixed conjugation reagent was then added to the plasma-containing tube from step 1 and thoroughly mixed by pulse vortexing to form the binding mixture. Those skilled in the art will appreciate that the binding mixture can also be prepared by adding the binding buffer, magnetic beads, and 2-propanol one by one to the plasma-containing tube, followed by thorough pulse vortexing, rather than using a premixed conjugation reagent. In one embodiment of the present invention, the microbeads and binding buffer are added to the plasma sample before adding the 2-propanol.

[0032] The relative amounts of each component in the binding mixture are important to maximize cfDNA recovery and minimize gDNA binding. Table 2 below shows the amounts of premixed conjugation reagent used for three different input plasma volumes to form the binding mixture. Table 3 shows the amounts of the individual components of the conjugation reagent as shown in Table 2.

[0033] [Table 2]

[0034] [Table 3]

[0035] The binding buffer is usually composed of a detergent and a chaotropic agent. In this example, Triton X-100 was used as the detergent and GuSCN was used as the chaotropic agent. Triton X-100 is a non-ionic surfactant, e.g., a hydrophilic polyethylene oxide chain and an aromatic hydrocarbon lipophilic or hydrophobic group (C 14 H 22 O(CHO)n, where n=9-10). As will be appreciated by those skilled in the art, other detergents and chaotropic agents can be used with similar effects. Some examples of alternative detergents are Triton X-114, Nonidet P-40, and Igepal CA-630. One example of an alternative chaotropic agent is sodium perchlorate.

[0036] The tube containing the binding mixture was then incubated in a thermomixer (25°C, 1400 rpm) for 10 min, spun briefly, and placed on a magnetic rack for at least 5 min. The beads containing the bound cfDNA were collected on a magnet to form a bead pellet, and the clear supernatant containing the denatured proteins / lipids was carefully aspirated and discarded.

[0037] Step 3: Moving the beads The tube was removed from the magnetic rack, and 400 μL of Wash Buffer 1 was added directly to the bead pellet in the tube. Wash Buffer 1 consisted of 50% ethanol and 50% of a solution containing approximately 2.0 M GuSCN and approximately 22% w / v of a non-ionic surfactant, such as Triton X-100. The beads were completely resuspended by pulse-vortexing and brief spinning. The bead suspension was pipetted up and down, and the contents of the tube were transferred to a 1.5 mL microtube. Due to the viscosity of the liquid, the contents of the tip were slowly expelled to ensure complete transfer of the bead suspension. A second portion of Wash Buffer 1 (400 μL) was added to the tube. The tube was vortexed and briefly spun, and the contents were transferred to the same 1.5 mL microtube. The microtube was then placed on a magnetic rack for 1 minute to allow the beads to collect on the magnet, after which the supernatant was discarded.

[0038] Step 4: Washing the beads The microtube was removed from the magnetic rack, and 700 μL of Wash Buffer 1 was added to the microtube. The microtube was incubated in a thermomixer at 25°C / 1400 rpm for 1 minute, vortexed, and then briefly spun. The microtube was then placed on the magnetic rack for 1 minute, and the supernatant was then discarded. The microtube was removed from the magnetic rack, and 700 μL of Wash Buffer 2 was added to the microtube. Wash Buffer 2 consisted of 80% ethanol and 20% of a solution containing approximately 10 mM Tris-HCl, approximately 1.0 mM EDTA, and approximately 0.5% w / v of a polysorbate-type nonionic surfactant, such as TWEEN®-20. As one skilled in the art will appreciate, alternative nonionic surfactants with similar effects can also be used. Some examples are Tween®-80 or Tween®-60. Alternatively, the surfactant could be omitted altogether. The microtubes were incubated in a thermomixer at 25°C / 1400 rpm for 1 minute, vortexed, and briefly spun. The microtubes were then placed on a magnetic rack for 1 minute before the supernatant was discarded. Another wash was performed using Wash Buffer 2.

[0039] Step 5: Air drying The microtube was briefly spun to collect residual Wash Buffer 2 at the bottom of the microtube. The microtube was placed on a magnetic rack for 1 minute to collect the beads on the magnet. Using a small pipette tip, the clear residual supernatant was carefully removed from the bottom of the microtube. The bead pellet was then allowed to air dry for 5 minutes while still on the magnetic rack.

[0040] Step 6: Elution The microtube was removed from the magnetic rack. Elution buffer was added to the microtube and mixed thoroughly by vortexing to ensure complete resuspension of the bead pellet. The elution buffer contained approximately 10 mM Tris-HCl and approximately 0.5 mM EDTA, and the pH was adjusted to 8.0. The microtube was incubated in a thermomixer at 25°C / 1400 rpm for 3 minutes and briefly spun to collect the bead suspension at the bottom of the tube. The tube was placed on the magnetic rack for 1 minute to collect the beads on the magnet. Once the beads were collected on the magnet, the supernatant containing the isolated cfDNA was carefully transferred to a new microtube. Table 4 below shows the amount of elution buffer used for three different amounts of input plasma.

[0041] [Table 4]

[0042] Method for purifying cfDNA from 500 μL (0.5 ml) of plasma [Example]

[0043] Whole blood samples were processed to separate plasma as described above. The following steps of the cfDNA isolation method were then performed to obtain purified cfDNA from 0.5 ml of plasma sample.

[0044] Step 1: Melting Ten microliters of proteinase K (20 mg / mL) and 0.5 mL of plasma were added to a 2 mL microcentrifuge tube and mixed by brief vortexing. Next, 25 μL of 20% SDS was added to the tube. Either the proteinase K or the plasma could be added to the tube first. However, to prevent enzyme inactivation, the 20% SDS should not come into direct contact with the proteinase K solution. The tube was pulse-vortexed 2-3 times and the contents were thoroughly mixed by vortexing for 15 seconds. The tube was then incubated at approximately 55-65°C for approximately 20-30 minutes.

[0045] Step 2: Preparation of the Binding Mixture Magnetic microbeads (Sera-Sil Mag 700, GE Healthcare Life Sciences) were thoroughly resuspended by vortexing before dispensing. The conjugate reagent was prepared by combining the following three components and mixing thoroughly by pulse vortexing:

[0046] 1. 0.725 mL of binding buffer x (number of samples to be processed + 10%) 2. 0.35 mL of 2-propanol x (number of samples to be processed + 10%) 3. Magnetic bead suspension 3.75 μL (number of samples + 10%)

[0047] The binding mixture was prepared by adding 1.05 ml of freshly prepared conjugation reagent to the plasma-containing tube from step 1 and thoroughly mixing the contents by pulse-vortexing. As described in Example 1 above, the binding buffer, magnetic bead suspension, and 2-propanol could be added one at a time to the plasma-containing tube from step 1 instead of using a premixed conjugation reagent.

[0048] The tubes were then incubated in a thermomixer at 25°C / 1400 rpm for 10 minutes. The tubes were then briefly spun and placed on a magnetic rack for at least 5 minutes. The beads containing the bound cfDNA were collected by the magnet, and the clear supernatant was carefully aspirated and discarded.

[0049] Step 3: Washing the beads The tubes were removed from the magnetic rack and 700 μL of wash buffer was added to the tubes. Multiple washes were performed using wash buffers 1 and 2 as shown in Table 5 below.

[0050] [Table 5]

[0051] Wash buffers 1 and 2 used were as described in Example 1 above. The tubes were incubated in a thermomixer at 25°C / 1400 rpm for 1 minute, then vortexed and spun briefly. The tubes were then placed on a magnetic rack for 1 minute before the supernatant was discarded.

[0052] Step 4: Air drying The tube was briefly spun to collect two drops of residual wash buffer at the bottom of the tube. The tube was then placed on a magnetic rack for 1 minute to allow the beads to be attracted to the magnet. Using a small pipette tip, the residual clear supernatant was carefully removed from the bottom of the microtube, and the bead pellet was allowed to air-dry for 5 minutes while still on the magnetic rack.

[0053] Step 5: Elution The tube was removed from the magnetic rack. 15 μL of elution buffer was added to the tube, and the contents of the tube were mixed well by vortexing to ensure complete resuspension of the bead pellet. The elution buffer used was the same as described in Example 1 above. The tube was incubated in a thermomixer at 25°C / 1400 rpm for 3 minutes, followed by a brief spin to collect the bead suspension at the bottom of the tube. The tube was placed on the magnetic rack for 1 minute to collect the beads on the magnet. Once the beads had collected on the magnet, the supernatant containing the isolated cfDNA was carefully transferred to a new microcentrifuge tube.

[0054] Recovery vs. fragment size The method of the present invention is designed to maximize the recovery of small cfDNA fragments, such as those reported to be present in the plasma of patients with advanced cancer, representing a fraction rich in tumor-derived DNA. At the same time, the design of this method significantly reduces the co-purification of potentially high-molecular-weight gDNA from lysed blood cells. This synergistic effect of increasing the recovery of small fragments and reducing the recovery of large fragments is demonstrated in Examples 3 and 4 below, and is shown in Figures 2a and 2b, respectively. [Example]

[0055] Two ml of plasma was obtained from blood collected from two healthy human subjects in streck cfDNA collection tubes. Both plasma samples were spiked with a 50-bp DNA ladder at a concentration of 10 ng / mL of plasma, and each plasma sample was processed according to the method of the present invention to extract DNA. Next, 1 μl of DNA isolated from each sample was run on a high-sensitivity DNA chip in a Bioanalyzer 2100. The results are shown in Figure 2a, which shows a Bioanalyzer plot showing the size-dependent recovery of the 50-bp DNA ladder fragments used to spike the plasma. As shown in Figure 2a, two independent DNA extracts are shown by the blue and red lines, respectively. The reference ladder equivalent to the spiked input is shown by the green line. As can be seen from the plot, the 50-bp DNA ladder fragments corresponding to the major cfDNA peak (i.e., between approximately 100 bp and 300 bp) and fragments of 100 bp or less were efficiently recovered in both spiked samples. The relative recovery of 50 bp DNA ladder fragments shows minimal recovery at 2.5 kb. This example demonstrates that the cfDNA isolation method of the present invention significantly reduces the co-purification of high molecular weight gDNA contaminants from lysed blood cells that may be present. [Example]

[0056] Plasma was obtained from blood collected from two healthy human subjects in streck cfDNA collection tubes. Both plasma samples were spiked with a 50-bp DNA ladder at a concentration of 10 ng / mL of plasma, and each plasma sample was processed according to the method of the present invention to extract cfDNA. Based on eight independent experiments, the percent recovery of the 50-bp DNA ladder spiked into selected fragments of 50 bp, 100 bp, and 2.5 kbp was measured. Figure 2b shows a plot of the measurements, with error bars representing standard deviation. Figure 2b shows a recovery profile with high percent recovery of low molecular weight fragments, i.e., 50 bp and 100 bp, and low percent recovery of the high molecular weight fragment of 2.5 kb.

[0057] Synergistic effect: increased recovery of small cfDNA fragments combined with reduced recovery of larger, high molecular weight DNA The present inventors surprisingly discovered that by manipulating the relative ratios of Triton X-100, 2-propanol, and GuSCN in the binding mixture, it is possible to obtain a desired DNA fragment recovery profile from plasma samples. Increasing the ratio of both Triton X-100 and 2-propanol in the binding mixture was found to improve the recovery of short fragment-sized cfDNA and reduce the recovery of contaminating gDNA. Increasing the amount of guanidinium ions above a certain level was also found to increase the binding of high molecular weight fragments. As previously mentioned, the binding mixture is a combination of binding buffer, 2-propanol, an aqueous suspension of magnetic beads, and plasma.

[0058] Effect of varying the proportion of 2-propanol in the binding mixture [Example]

[0059] In this experiment, the percentage of 2-propanol in the binding mixture was varied to determine its effect on the cfDNA recovery profile. The various combinations tested are summarized in Table 6 below. The percentage of Triton X-100 was fixed at approximately 8.8% in the binding mixture. For preparations 10A-10D, the GuSCN in the binding mixture was fixed at 2M. For preparations 10E-10H, the GuSCN in the binding mixture was fixed at 2.4M.

[0060] [Table 6]

[0061] The extracted cfDNA was run on a Bioanalyzer 2100 to confirm the recovery profile. Figure 3 shows the Bioanalyzer plots for preparations 10A-10D. As shown in the plots, increasing the ratio of 2-propanol in the binding mixture increased the recovery of smaller DNA fragments. Figure 4 shows the Bioanalyzer plots for preparations 10E-10H with the same results. It was also observed that increasing the ratio of GuSCN in the binding mixture increased gDNA binding. [Example]

[0062] In this experiment, the effect of increasing 2-propanol from 22% to 25.2% in the binding mixture was tested while GuSCN in the binding mixture was fixed at 2 M and Triton X-100 at 8.8%.

[0063] This is summarized in Table 7 below.

[0064] [Table 7]

[0065] The extracted cfDNA was electrophoresed on a Bioanalyzer 2100 to confirm the recovery profile. Figure 5 shows the Bioanalyzer plots for preparations 13A repeat and 13G repeat. As shown in the plots, increasing the 2-propanol concentration from 22% to 25.2% resulted in a decrease in the binding of high molecular weight DNA. [Example]

[0066] In this experiment, the effects of 17.5%, 19%, 20.6%, and 22.2% 2-propanol in the binding mixture were tested, with GuSCN fixed at 2M and Triton X-100 fixed at 11.1%, as summarized in Table 8 below.

[0067] [Table 8]

[0068] The extracted cfDNA was run on a Bioanalyzer 2100 to confirm the recovery profile. Figure 6 shows a bioanalyzer plot demonstrating the effect of 2-propanol concentrations of 22.2% (A2), 20.6% (B2), 19% (C2), and 17.5% (D2) in the binding mixture on the recovery profile of the extracted cfDNA. As shown in the plot, increasing the 2-propanol concentration in the binding mixture from 17.5% to 22.2% decreased the binding of high-molecular-weight DNA and increased the binding of small-sized DNA. Figure 7 shows an expanded view of a portion of Figure 6 for low-molecular-weight DNA fragments. As shown in Figure 7, decreasing the 2-propanol concentration in the binding mixture from 22.2% to 20.6% decreased the 50-bp fragment recovery by at least 50%. Figure 8 shows another expanded view of a portion of Figure 6 for high-molecular-weight DNA fragments. As shown in Figure 8, a dramatic increase in recovery of the 2.5 kb fragment was observed when the proportion of 2-propanol in the ligation mixture was reduced from 22.2% to 19%.

[0069] Effect of varying the ratio of Triton X-100 in the binding mixture [Example]

[0070] In this experiment, the percentage of Triton X-100 in the binding mixture was varied to determine the effect on the cfDNA recovery profile. The various combinations tested are summarized in Table 9 below. The percentage of Triton X-100 in the binding mixture was tested at 8.8% and 11.1%, while the 2-propanol in the binding mixture was fixed at 22% and the GuSCN at 2M.

[0071] [Table 9]

[0072] The cfDNA extracts were run on a Bioanalyzer 2100 to determine the effect of the Triton X-100 ratio in the binding mixture on the cfDNA recovery profile. Figure 9 shows the Bioanalyzer plots for a fixed 2-propanol concentration of approximately 22%. As can be seen in Figure 9, increasing Triton X-100 from 8.8 to 11.1% reduced the binding of high molecular weight DNA and increased the recovery of small DNA fragments. [Example]

[0073] In this experiment, the 2-propanol in the binding mixture was fixed at approximately 25.2% and the GuSCN was fixed at 2M, while the Triton X-100 ratio in the binding mixture was tested at 8.8% and 4.5%. The various combinations tested are summarized in Table 10 below. The resulting cfDNA extracts were run on a Bioanalyzer 2100 to determine the effect on the cfDNA recovery profile. Figure 10 shows a Bioanalyzer plot in which increasing Triton X-100 increases the recovery of smaller sized DNA while simultaneously decreasing the recovery of larger sized DNA.

[0074] [Table 10]

[0075] Effect of plasma components in the binding mixture The inventors noted that plasma was required in the binding mixture to achieve the desired cfDNA recovery profile, as described in Example 10 below. [Example]

[0076] In this experiment, GuSCN was fixed at 2M, Triton X-100 was fixed at 11.1%, and 2-propanol was fixed at 22.2% in the binding mixture, as shown in Table 11 below. Size selection of cfDNA was tested in the absence of plasma. This was performed by replacing plasma once with NaCl and once with PBS. A 50-bp DNA ladder was spiked into each of the samples containing plasma, NaCl, and PBS to monitor size selection and DNA recovery.

[0077] [Table 11]

[0078] The extracted DNA was electrophoresed using a bioanalyzer to confirm the effect of plasma on size selection. Figure 11 shows an electropherogram that confirms that size selection is lost in the absence of plasma components.

[0079] Scalability of cfDNA isolation methods [Example]

[0080] A 50 bp DNA ladder was spiked into plasma obtained from blood collected in a Streck cfDNA collection tube at a concentration of 10 ng / ml of plasma. The spiked plasma was then processed according to the method of the present invention. In this experiment, four different plasma input volumes (0.5 ml, 1 ml, 2 ml, and 4 ml) were used to demonstrate the scalability of the isolation method. As shown in Table 12 below, the elution volume was scaled to match the input plasma volume to ensure equivalent DNA concentrations in the extract.

[0081] [Table 12]

[0082] 1 μl of each extract was run on a high-sensitivity DNA chip in a Bioanalyzer 2100. Figure 12 shows a Bioanalyzer 2100 plot showing the results achieved with various plasma input volumes (0.5 ml, 1 ml, and 4 ml) compared to a standard 2 ml input. As can be inferred from the overlapping lines in the plot, the method of the present invention can be used with various sample input volumes. Effective purification of cfDNA from plasma input volumes of 0.5 ml to 4 ml has been demonstrated.

[0083] Expected results from plasma collected in standard EDTA blood collection tubes The method of the present invention works best for extracting cfDNA from plasma collected in Streck cfDNA blood collection tube.However, as mentioned above, it is also possible to efficiently extract cfDNA from plasma collected in standard EDTA tube.However, in these cases, the recovery of smaller fragments may be lower than the level expected in Streck cfDNA blood collection tube. [Example]

[0084] Two milliliters of plasma collected in a standard EDTA tube was spiked with a 50-bp DNA ladder (10 ng / mL of plasma) and processed using the cfDNA isolation method of the present invention. One microliter of the extract was run on a high-sensitivity DNA chip along with the 50-bp DNA ladder input. Figure 13 shows a Bioanalyzer 2100 plot showing the recovery of cfDNA lines and 50-bp DNA ladder fragments from plasma collected in a standard EDTA tube (two independent extracts shown as blue and red lines, respectively, and ladder input shown as green).

[0085] The benefits of size selection for cancer mutation detection The method of the present invention allows for highly efficient extraction of cfDNA and minimal carryover of gDNA. This unique feature provides a distinct advantage in liquid biopsy-based applications, allowing for the detection of mutations present at very low levels that would normally be missed using standard isolation methods. This is illustrated in Example 13 below. [Example]

[0086] Three cancer patients were given 1 ml of plasma collected in standard EDTA blood collection tubes from a commercial source, and cfDNA was isolated using the method of the present invention and a standard commercially available kit without size selection. One microliter of the isolated cfDNA was run on a high-sensitivity DNA chip, and the results are shown in Figure 14. As shown in Figure 14, it is noteworthy that in patients 4 and 2, there is a significant presence of high-molecular-weight DNA (gDNA), as indicated by the green circles. The remaining eluate was concentrated and subjected to library preparation using the Target Selector™ NGS Lung Panel. The results, shown in Table 13 below, demonstrate that in patients 4 and 2, the size-selection-based extraction method detected significantly higher cancer-associated mutation frequencies than the standard isolation method. Importantly, in patient 3, the mutation frequency was below the detection level of the assay when extracted using the standard isolation method.

[0087] [Table 13]

[0088] It is understood that the present invention is not limited by the above-described embodiments and examples, and modifications within the scope of the appended claims are readily apparent to those skilled in the art. For example, the blood collection tubes may be standard EDTA tubes or heparin tubes. Those skilled in the art can modify the composition of the wash buffer to achieve essentially the same results. For example, the wash buffer may simply be a 70-80% aqueous ethanol solution. Similarly, the elution buffer may be water or any standard dilute Tris-HCl or Tris-EDTA buffer. Those skilled in the art should also understand that any suitable solid phase other than silica-coated microbeads, such as glass microbeads and glass fiber membranes, which are also DNA-binding, can be used. Several alternatives for detergents and chaotropic agents are known in the art, and those skilled in the art can use them without departing from the scope of the claims.

Claims

1. 1. A method for isolating cell-free DNA from a liquid biological sample, comprising: The following process: a) providing a liquid biological sample; b) adding to said sample a solid phase capable of binding DNA, a binding buffer containing a detergent and a chaotropic agent; and adding 2-propanol to form a binding mixture thereof; c) washing the solid phase to remove unbound material; and d) eluting the bound cell-free DNA, wherein the majority of the eluted DNA is less than 400 bp. A method comprising:

2. The method of claim 1, wherein the sample is plasma, serum, or urine.

3. 3. The method of claim 2, wherein the plasma is obtained from whole blood collected in a cell-free DNA stabilizing tube.

4. The detergent is a non-ionic surfactant, e.g., Triton X-100, which has a hydrophilic polyethylene oxide chain and an aromatic hydrocarbon lipophilic or hydrophobic group (C 14 H 22 O(C 2 H 4 4. The method of claim 1, 2 or 3, wherein O) n is a surfactant having the formula n, where n=9-10), or a non-ionic surfactant such as Triton X-114, Nonidet P-40 or Igepal CA-630.

5. 5. The method according to claim 1, wherein the chaotropic agent is guanidine thiocyanate or sodium perchlorate.

6. 6. The method according to one or more of claims 1 to 5, wherein the sample is plasma, the detergent is a non-ionic surfactant such as Triton X-100, and the chaotropic agent is guanidine thiocyanate.

7. 7. The method of claim 2, wherein the plasma is about 25-40% v / v in the binding mixture.

8. 8. The method according to claim 4, wherein the non-ionic detergent or Triton X-100 is about 8.8-11.1% in the binding mixture.

9. 9. The method of claim 5, wherein guanidine thiocyanate is about 1.5 to 2.5 M in the binding mixture.

10. 10. The method according to claim 1, wherein 2-propanol is about 15-25% v / v in the binding mixture.

11. The method of claim 1, wherein the solid phase comprises magnetic microbeads, preferably silica-coated magnetic beads.

12. The method of claim 11, wherein the magnetic microbeads are in an aqueous suspension of 20 to 200 mg / ml.

13. 1. A method for size-selective isolation of cell-free DNA from a liquid biological sample, comprising: The following process: a) providing a liquid biological sample; b) adding to said sample - an aqueous suspension of silica-coated magnetic microbeads capable of binding DNA; - a binding buffer containing guanidine thiocyanate and a non-ionic detergent such as Triton X-100, and - adding 2-propanol to form a conjugation mixture thereof, said conjugation mixture comprising about 20-30% w / v of a non-ionic detergent such as Triton X-100, about 1.5-2.5 M guanidine thiocyanate, and about 15-25% v / v of 2-propanol; c) incubating the binding mixture at room temperature for about 10-30 minutes to promote binding of cell-free DNA to the magnetic microbeads; d) washing the magnetic microbeads with one or more washing buffers comprising ethanol; e) adding an elution buffer to the washed magnetic beads of step d) to release the cell-free DNA bound to the magnetic microbeads in solution; and f) optionally analyzing or quantifying the cell-free DNA obtained in step e). A method comprising:

14. 14. The method of claim 13, wherein the sample is plasma obtained from whole blood collected in a cell-free DNA stabilizing tube.

15. 15. The method of claim 14, wherein the plasma is optionally treated with proteinase K and sodium dodecyl sulfate (SDS) to form a mixture thereof, and the mixture is incubated at about 55-65°C for about 20-30 minutes.

16. 16. The method of claim 14 or 15, wherein the binding mixture is about 25-40% v / v plasma.

17. 17. The method of one or more of claims 13 to 16, wherein the wash buffer is composed of 50% ethanol and 50% of a solution containing about 2.0 M guanidine thiocyanate and about 22% w / v of Triton X-100.

18. 17. The method of one or more of claims 13 to 16, wherein the wash buffer is composed of 80% ethanol and 20% of a solution containing about 10 mM Tris-HCl, about 1.0 mM ethylenediaminetetraacetic acid (EDTA), and about 0.5% w / v of a polysorbate-type non-ionic surfactant such as TWEEN®-20.

19. 19. The method of one or more of claims 13 to 18, wherein the elution buffer contains about 10 mM Tris-HCl and about 0.5 mM EDTA, the buffer being adjusted to pH 8.

0.

20. 20. The method of any one of claims 1 to 19, wherein the binding buffer, the microbeads and the 2-propanol are pre-mixed into a single combined reagent before adding to the sample.

21. 20. The method of any one of claims 1 to 19, wherein the microbeads and the binding buffer are added to the sample before adding the 2-propanol.

22. the binding mixture comprising: a) guanidine thiocyanate, preferably in the range of 1.75 to 2.25 M, more preferably in the range of 1.9 to 2.1 M, for example about 2.0 M; b) Triton X-100, preferably in the range of 23-25% w / v, more preferably about 24% w / v, e.g., about 24.1% w / v; c) A method according to any one of claims 1 to 21, comprising 2-propanol, preferably in the range of 17-25% w / v, more preferably about 22% w / v, for example about 22.2% w / v.

23. 23. The method according to one or more of claims 1 to 22, wherein the amount of plasma used is between 0.5 ml and 4 ml.

24. 24. The method of any one of claims 1 to 23, wherein the fragment distribution of the isolated cell-free DNA ranges from about 50 to 400 bp.

25. 1. Use of guanidine thiocyanate and Triton X-100 to form a binding buffer composition for size-selective binding of cell-free DNA present in plasma to silica-coated magnetic microbeads in an aqueous suspension of 20-200 mg / ml, wherein the binding buffer is intended to be contacted with 2-propanol, plasma, and magnetic microbeads to form a binding mixture comprising about 1.5-2.5 M guanidine thiocyanate, about 20-30% w / v Triton X-100, about 15-25% v / v 2-propanol, and about 25-40% v / v plasma.

26. A kit containing silica-coated microbeads capable of binding 50-400 bp DNA from liquid biological samples in the presence of guanidine thiocyanate, Triton X-100, and 2-propanol.

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