Novel nucleic acid purification chemicals

Abstract

The present invention relates generally to the field of nucleic acid isolation on silica solid supports. In particular, disclosed herein is a novel silica-solid support binding nucleic acid buffer chemistry based on the use of small tetrameric organic compounds, such as tetramethylammonium chloride (TMAC), under acidic conditions. This novel nucleic acid purification chemistry not only purifies RNA, but also DNA, and has the potential to be implemented in a wide variety of commercial kits, from spin columns to Lab-On-A-Chip (LOC) devices, such as disposable cartridges utilizing solid phase extraction technology. Furthermore, the present method can use relatively small volumes of binding buffer and thus can be performed in such integrated or closed molecular diagnostic devices, which have the potential to allow for increased sample input volumes, which for liquid biopsy samples such as plasma or urine, can increase the chances of detecting rare nucleic acid targets.

Description

Technical Field

[0001] This invention generally relates to the field of nucleic acid isolation on silica solid supports. In particular, this document discloses novel silica-solid support-based buffer chemicals for binding nucleic acids, based on the use of small quaternary organic compounds, such as tetramethylammonium chloride (TMAC), under acidic conditions. These novel nucleic acid purification chemicals can purify not only RNA but also DNA and have the potential to be implemented in a wide variety of commercial kits, ranging from centrifuge columns to lab-on-a-chip (LOC) devices, such as disposable kits utilizing solid-phase extraction techniques. Furthermore, the methods of this invention can use relatively small volumes of binding buffers and therefore can be performed in such integrated or closed molecular diagnostic devices, which have the potential to allow for increased sample input volumes, potentially increasing the chance of detecting rare nucleic acid targets for liquid biopsy samples such as plasma or urine. Technical Background

[0002] In patent EP389063, filed in 1990, Boom et al. described a general nucleic acid purification technique based on adsorption on a solid support. Boom extraction uses a large amount of chaotropic salt to mediate the binding of nucleic acids to silica, with or without alcohol. Due to its high performance, allowing for the extraction of >50% nucleic acids from biological samples, it has rapidly become the gold standard for nucleic acid isolation and remains widely used in many commercially available extraction kits and integrated molecular diagnostic devices. For example, the Boom protocol, or slight variations thereof, forms the basis of the DNA extraction principle used in QIAGEN's QIAamp Circulating Nucleic Acid Kit or in Biocartis NV's integrated kits such as Idylla ctRAS.

[0003] The Boom protocol requires a large amount of binding buffer relative to the volume of biological sample due to the need for large amounts of cholesteric salt and subsequent alcohol. For this reason, and given the increasing prevalence of increasingly miniaturized, handheld, fully integrated lab-on-a-chip (LOC) molecular assay devices (which are typically designed to maximize sample volume and therefore lack sufficient buffer storage), there is a need to find efficient alternatives to the Boom protocol. Another reason is the high cost of cholesteric salts, their strong PCR-inhibiting properties, and the multiple challenges they pose to the final product delivery line. For these reasons, there is currently a need for efficient, cholesteric-free nucleic acid purification chemicals that enable increased sample input in integrated systems, particularly for applications in liquid biopsy, where sample size is becoming increasingly important for detecting, for example, very scarce cell-free (cf) DNA targets per milliliter of plasma.

[0004] Several attempts have been made to date to develop novel silica-based nucleic acid purification chemicals that do not require ionizing agents. Notable examples include:

[0005] The method published by Hourfar et al. in 2005 describes viral RNA purification based on acidic conditions and the use of kosmotropic salts. The publication is titled "High-Throughput Purification of Viral RNA Based on Novel Aqueous Chemistry for Nucleic Acid Isolation." This method is RNA-specific and not suitable for purifying DNA from plasma.

[0006] Lee et al. published a similar method in 2008, which involves isolating total RNA from *E. coli* using a kosmotropic salt. Additionally, Lee et al. hold patent US 7,923,551 entitled "Method of purifying RNA using kosmotropic salt." Both the publication and the patent describe and focus on RNA-selective purification chemicals based on acidic conditions and the use of kosmotropic salts, but do not provide any instruction on how to apply these chemicals to DNA.

[0007] Johns Hopkins University holds a patent application, WO2016 / 073824, entitled "Chaotrope-and volatile-free method for purifying nucleic acids from plasma." The method described therein is very similar to that of Lee et al., and includes the use of acidic conditions and hydrophilic salts to mediate the binding of RNA to silica.

[0008] MiDiagnostics holds a very similar patent application, WO 2018 / 156906 A1, entitled "System and method for purifying and amplifying nucleic acids." This patent application describes nucleic acid purification chemicals that use acidic conditions and lyophilic salts to mediate the binding of viral nucleic acids to silica. However, this method provides no evidence that it is at least as effective as the Boom protocol, nor does it demonstrate its applicability to the purification of cfDNA present in plasma.

[0009] Despite its widespread use in molecular diagnostics, little is known about the interaction between nucleic acids and silica. In fact, little has changed since Boom et al. published the first silica-based nucleic acid purification technique in 1990. To date, research attempting to elucidate the fundamental mechanisms by which DNA / RNA adsorbs onto silica is extremely limited.

[0010] Melzak et al. (1996) were among the few who attempted to elucidate the fundamental mechanisms of Boom extraction techniques. They described three effects considered to be major contributors to nucleic acid adsorption onto silica, including:

[0011] • Shielding intermolecular electrostatic forces

[0012] Dehydration of DNA and silica surfaces

[0013] • Intermolecular hydrogen bonding formation in the nucleic acid-silica contact layer (described as the least significant contributor).

[0014] In the presence of a silica solid support, these three factors can be modulated by adding different salts to the nucleic acid solution. Hofmeister categorized salts based on their ability to influence the structure of macromolecules (primarily proteins) in aqueous solution. According to this classification, ionizing salts were initially described as structure-destroying agents because they increase protein solubility (the so-called "salting-in"). Conversely, lyophilic salts were described as structure-building agents because they decrease protein solubility (the so-called "salting-out"). In the case of silica-based nucleic acid separation, ionizing salts are a natural choice because they can influence the structure of water and induce dehydration. From this perspective, ionizing ions are described by Hofmeister as large, single-charged ions with low charge density whose interaction with water is weaker than water's interaction with itself. They are believed to rarely interfere with the hydrogen bonds of surrounding water. For example, high concentrations of guanidine thiocyanate were used in the initial nucleic acid purification chemistry described by Boom et al. in 1990 due to its strong ionizing properties, its cytolytic properties, and its potential to inactivate ribonucleases. Conversely, lyophilic ions are described as small or multiply charged ions with high charge density, thus capable of breaking hydrogen bonds between water molecules.

[0015] Although guanidinium cations and thiocyanate anions are not expected to have large hydration shells, this is believed to be compensated for by the excessively high concentrations (3M-5M) used in the Boom protocol. It is speculated that this high salt concentration sufficiently reduces the free water concentration, leading to dehydration of the nucleic acid and silica membrane. Furthermore, the abundant guanidinium cations are believed to shield the electrostatic forces between the negatively charged phosphate backbone of the nucleic acid and the negatively charged silanol groups on the silica surface. It can be hypothesized that these two effects promote hydrophobic interactions between the bases and the siloxane bridges, thereby enabling the nucleic acid to be adsorbed onto the silica membrane. Subsequent modifications to the original Boom protocol included the addition of alcohol to the binding buffer to further reduce the free water concentration and enhance this dehydration effect.

[0016] Based on this change, the silica-bound nucleic acids are then washed with concentrated alcohol (typically 70%–90% ethanol). This washing procedure ensures the removal of residual non-nucleic acid compounds derived from biological samples or binding buffers. Finally, the nucleic acids are eluted with a low-ionic-strength solution at a neutral or weakly alkaline pH. This elution mechanism allows for direct compatibility with downstream applications such as PCR and NGS.

[0017] As explained above, Hourfar et al. first published a silica-based alternative method for purifying RNA from biological samples. Subsequent publications and / or patents by Samsung electronics, Johns Hopkins University, and MiDiagnostics are based on the same chemicals, using acidic conditions and hydrophilic salts to mediate the binding of RNA (and, to a much lesser extent, DNA) to silica. A possible explanation for the action of this chemical on RNA is based on the following: The pKa values ​​of the silanol groups on the silica surface are 4–8. Lowering the pH of the binding buffer-sample mixture below these values ​​promotes the protonation of the weakly acidic silanol groups, thereby eliminating the strong negative charge of the weakly acidic silanol groups. Therefore, the electrostatic repulsion with the negatively charged phosphate backbone of nucleic acids is significantly reduced or even completely eliminated.

[0018] Furthermore, a minimal amount of lyophilic salt (i.e., (NH4)2SO4) can be used to significantly reduce the amount of free water, thereby dehydrating nucleic acids and silica membranes. As explained earlier, strongly lyophilic ions can have large hydration shells, trapping large amounts of free water. In this regard, it can be assumed that only a limited amount of lyophilic salt (400 mM–1000 mM, depending on the specific salt) is needed to provide an effect similar to, for example, 5 M guanidine thiocyanate. These effects can be used to explain the binding of flexible RNA to silica, and to a much lower degree the binding of double-stranded and therefore stiffer DNA to silica.

[0019] The bound nucleic acids are then washed with a high percentage of alcohol, as in the Boom protocol, although a variant protocol in which the washing is completely alcohol-free is described. In this variant protocol, washing is performed with a buffer similar to the binding buffer or a simplified form thereof (i.e., an acidic solution (pH 4–7) containing no or a limited amount of lyophilic salts). It can be assumed that these washing procedures are primarily based on an attempt to eliminate electrostatic repulsion caused by the protonation of silanol groups, which prevents the elution of nucleic acids. The elution mechanism is then similar to that of the Boom protocol.

[0020] It is important to note that while this method has proven highly successful in RNA purification (often even described as RNA-selective), the purification of double-stranded DNA (dsDNA) remains far more challenging. dsDNA extraction yields based on these chemical methods have been shown to be as low as 1 / 100 to 1 / 10, which is clearly insufficient for DNA extraction from plasma.

[0021] In this paper, we overcome the shortcomings of nucleic acid extraction methods without strong ionizing agents by successfully using salts composed of small quaternary organic cations and very weakly ionized and highly soluble anions. Quaternary compounds are cations consisting of a central positively charged atom and four uncharged substituents (primarily alkyl and aryl). These cations are permanently charged, independent of the pH of their solution. They are generally described as inert cations. In particular, we have observed and demonstrated the effectiveness of salts composed of, for example, tetramethylammonium (TMA) + ) Cations and the chlorine (Cl) of weakly ionized liquids - ) or bromine (Br - The combination of anions forms salts that create unique conditions at acidic pH for the isolation of dsDNA on a silica solid support, with efficiencies comparable to those of ionizing agent-based schemes such as the Boom scheme.

[0022] To our knowledge, salts consisting of small quaternary organic cations and weakly ionizing anions have not been used for solid-support-based nucleic acid extraction from biological samples. Although J.M. Rosz and J.W. G. Thurmur used similar salts in 1977 to study the unwinding and renaturation properties of DNA and dsRNA (Biopolymers, Vol. 16, 1183–1977), their study did not consider the option of using these salts in solid-phase nucleic acid extraction. Furthermore, WO2015165859 describes the use of sodium salts in combination with quaternary ammonium salts in methods for enriching nucleic acids containing single-stranded poly(A) segments (i.e., primarily messenger RNA) while simultaneously removing unwanted nucleic acids (such as, for example, rRNA) from solid supports coated with immobilized oligo-dT capture probes. The teachings of WO2015165859 imply a strict and selective specificity for poly(A) nucleic acids and do not appear applicable to the isolation of any other types of nucleic acids from samples such as liquid biopsies. Then, WO1995015970 discloses a hybridization solution comprising tetramethylammonium chloride ((CH3)4NCl) and a cationic detergent for immobilizing synthetic oligonucleotides onto solid surfaces such as polystyrene. However, importantly, WO1995015970 does not teach the purification of native nucleic acids from complex biological sample backgrounds on solid supports, particularly native nucleic acids from liquid biopsy samples such as plasma. In summary, none of the above disclosures teach or imply the use of small quaternary organic compound salts as a general alternative to the Boom scheme for nucleic acid isolation.

[0023] The method presented here offers several advantages over the Boom protocol. First, the Boom protocol was originally designed for isolating long genomic and plasmid DNA / RNA. Its application in molecular diagnostics has been challenged by the highly fragmented nature of genomic material commonly found in plasma and FFPE samples. Short nucleic acid fragments naturally possess far fewer hydrophobic binding sites, thus limiting their binding efficiency to silica at the high concentrations of dissociating agents used in the Boom protocol. In contrast, using the method presented herein, based on a salt composed of quaternary cations and a mild dissociating agent, we are able to isolate both shorter and longer dsDNA fragments. Furthermore, we have observed that the extraction efficiency of short DNA (i.e., in the 10–300 bp range) decreases with increasing pH, while the extraction efficiency of high molecular weight DNA increases with increasing pH, thus indicating that the method disclosed herein opens up additional avenues for fine-tuning extraction efficiency for desired DNA target lengths by varying the pH value.

[0024] A further advantage of the method disclosed herein is that the chemical composition of the binding buffer does not induce protein aggregation, which in principle allows for the processing of plasma samples without a protein digestion step. The plasma binding buffer mixture enables a smooth flow rate, which is beneficial for its use in microfluidic devices. Therefore, for low-volume or diluted samples, the protein digestion step can be skipped when using the method disclosed herein, and even for some older plasma samples with volumes >400 μL, incorporating the protein digestion step as an optional step to increase the final extraction yield remains advantageous.

[0025] Then, as mentioned above, LOC devices or disposable kits typically lack sufficient storage space to accommodate the relatively large amounts of binding buffer used in Boom protocols. Furthermore, clonating salts are expensive, have strong PCR-inhibiting properties, and introduce numerous problems into the production line, such as issues caused by crystallization. In contrast, the methods and binding buffers presented here are free of strong clonating agents, inexpensive, and significantly reduce the volume of binding buffer required per sample, thereby enabling increased sample input in fully integrated molecular diagnostic devices. The latter offers significant advantages for processing, for example, liquid biopsies from cancer patients, where the amount of tumor-derived mutant DNA copies per milliliter of plasma is extremely sparse and difficult to detect. Last but not least, the methods presented here are versatile, meaning they enable the efficient purification of short and long dsDNA, as well as ssDNA and potential RNA, from diverse biological samples. Invention Overview

[0027] Since Boom et al. published their initial method in 1990, the chemistry for silica-based nucleic acid purification has remained largely unchanged. This is primarily due to the robust performance of the Boom extraction technique, which yields >50% of nucleic acids from biological samples. The Boom protocol mediates the binding of nucleic acids to silica using a large amount of dissociative salt and alcohol. However, given the recent advent of miniaturized laboratory-on-a-chip (LOC) devices, there is a need to minimize the buffer volume they contain in order to maximize the sample volume input they can accept. Therefore, there is a clear need for novel nucleic acid purification chemistry that is free of strong dissociative agents, economical, and enables increased sample input in integrated systems.

[0028] In this paper, we present novel purification chemicals that mediate the binding of nucleic acids to silica using only acidic conditions and a relatively small amount of salt composed of quaternary ammonium compounds. The disclosed method significantly reduces the binding buffer volume relative to the sample volume, thus enabling increased sample input, which is highly beneficial in fully integrated molecular diagnostic devices. Furthermore, the disclosed separation method provides sufficient nucleic acid yields comparable to those of strong ionizing agents such as the Boom protocol. Attached Figure Description

[0029] To gain a more comprehensive understanding of the concepts presented in this paper, please refer to the following detailed explanation in conjunction with the accompanying drawings, in which:

[0030] Figure 1 This shows the extraction efficiency of dsDNA in different ionizing and lyophilizing salt binding buffers at neutral pH.

[0031] Figure 2 This shows the extraction efficiency of dsDNA in different ionizing and lyophilizing salt binding buffers at acidic pH.

[0032] Figure 3 This shows a comparison between Boom extraction binding buffer and chloride-based buffers with or without quaternary ammonium compounds;

[0033] Figure 4 This shows a comparison of different DNA extraction chemicals at different pH ranges;

[0034] Figure 5 This shows a comparison between TMAS and TMAC;

[0035] Figure 6 This demonstrates the properties of different TMA-containing salts;

[0036] Figure 7 This demonstrates the performance of TMAC at different binding concentrations;

[0037] Figure 8 This shows the performance of TMAC at different pH values;

[0038] Figure 9 This demonstrates the performance of TMAC on different plasma batches with and without varying concentrations of CTAB.

[0039] Figure 10 This demonstrates the performance of different TMAC buffers;

[0040] Figure 11 This study demonstrates research under different elution conditions.

[0041] Figure 12 This demonstrates the potential benefits of pre-digestion of proteases;

[0042] Figure 13 The results demonstrate the performance of the method in a closed, integrated medicine box.

[0043] Figure 14 and Figure 15 This study shows a comparison between Boom extraction chemicals and TMAC+CTAB extraction chemicals for different batches of plasma in a closed integrated kit. Invention Details

[0045] This invention generally relates to a nucleic acid extraction method, which involves contacting a biological sample (possibly a liquid biopsy sample) with a silica solid support in the presence of a salt consisting of the following substances at a pH of 3 to 6:

[0046] - Small quaternary organic compounds are defined as quaternary compounds consisting of a central positively charged atom and four organic substituents R1-R4, wherein each organic substituent R1-R4 contains no more than 2 carbon atoms; and

[0047] - Bromoanion or Chloride anion.

[0048] In other words, novel binding buffer chemicals are disclosed herein, based on the use of acidic conditions and a minimal amount of salt containing small quaternary organic compounds, providing a universal, liquid-free nucleic acid purification protocol that enables efficient separation of not only RNA but also DNA. As used herein, the term "quaternary compound" is used interchangeably with the term "quaternary organic compound," which should be understood as a compound that is or has an ion, the ion being a cation consisting of a central positively charged atom and four organic substituents (i.e., alkyl and / or aryl, without regard to hydrogen atoms), the four organic substituents being further designated as organic substituents R1-R4. As used herein, the term "small quaternary organic compound" should be understood as a quaternary organic compound in which each of the four organic substituents R1-R4 contains no more than two carbon atoms. For solubility considerations, it is preferred that the organic substituent is a single carbon group, i.e., a methyl group. We believe that the more methyl groups are present in the four organic substituents R1-R4, the better the solubility, and therefore the easier and more preferred to act with small quaternary ammonium organic compounds. Nevertheless, as stated above, we also believe that organic substituents containing two carbon atoms on one or more of the four organic substituents R1-R4 are still sufficiently soluble and suitably suited for implementing the methods disclosed herein.

[0049] The most well-known quaternary compounds are quaternary ammonium salts, which are quaternary ammonium cations (R4N) containing a nitrogen atom at the center. + Therefore, in embodiments, a method is provided in which the positively charged atom of the small quaternary organic compound is nitrogen. Other possible examples and seemingly feasible embodiments may include quaternary phosphonium salts (R4P). + Arsenic salts (R4As+) such as arsenobetaine, and some arsenic-containing superconductors. Substituted antimony salts (R4Sb+) and bismuth salts (R4Bi+) have also been described as present and may function in some embodiments of the methods proposed herein.

[0050] As demonstrated in the following embodiments, the anion of the salt used in the method of the present invention also affects the final nucleic acid extraction yield. In contrast to the known RNA-specific methods described above, strongly hydrophilic anions appear unsuitable for DNA extraction. Instead, we have recognized that very weak ionizing ions such as bromide ions or even weaker ionizing agents / critical hydrophiles (kosmotrope) chloride ions generally provide the best results, with a slight bias towards the latter in most experimental settings. Therefore, in the next embodiment, a method in which the anion is a chloride ion is provided.

[0051] We hypothesize that the difference in binding of RNA and DNA to silica highlighted above may stem from the at least partially single-stranded nature of RNA. That is, we believe that RNA binds to silica more readily than dsDNA, likely due to the increased rotational mobility of bases in single-stranded nucleic acids, thereby increasing the number of available hydrophobic binding sites. Conversely, double-stranded DNA may require substantial alterations to its helical structure to facilitate hydrophobic interactions between bases and siloxane (Si-O-Si) bridges in the silica membrane.

[0052] It is believed that the DNA double helix is ​​stabilized primarily by the following factors:

[0053] • Hydrogen bonds between alkali and the aqueous environment;

[0054] • Electrostatic shielding of the negatively charged phosphate backbone;

[0055] • Base stacking interactions between adjacent bases.

[0056] The latter is described as the primary contributor to double helix stability. We have assumed that destabilization of the double helix is ​​necessary to allow efficient binding with silica membranes, and that the type and amount of ions present play a major role in defining the helical conformation of double-stranded DNA.

[0057] Based on our experience, acidic conditions and hydrophilic salts do not appear to promote the binding of dsDNA to silica. It is possible that using a limited amount of hydrophilic salt could alter the double helix conformation, potentially further reducing the affinity of dsDNA for the silica membrane. Therefore, we hypothesize that prior to this approach, hydrophilic-based methods are RNA-selective. Small cations with high charge density are theoretically capable of coordinating between the minor and major grooves of the helical structure, while strong hydrophilic anions can strongly dehydrate the double helix, potentially leading to a conformational change to rigid A-DNA, thus hypothesizing a reduction in the availability of bases to bind to the silica solid support. Following this reasoning, we hypothesize that to counteract this affinity-reducing effect and promote dsDNA binding to the silica membrane, the stabilizing effect of cations should be eliminated and the dehydrating effect of hydrophilic anions reduced. We find that this effect can be achieved by using a salt composed of a quaternary ammonium compound and a weak ionotropic / weak hydrophilic anion such as chloride, described as being on the boundary between ionotropic and hydrophilic behavior. Quaternary ammonium compounds are cations consisting of a central positively charged nitrogen atom and four uncharged substituents (primarily alkyl and aryl). These cations are permanently charged, regardless of the pH of their solution. They are typically described as inert cations.

[0058] Based on our theoretical model, we hypothesize that the success of our inventive method using small quaternary organic compounds such as TMAC is at least in part due to the inertness and sheer size of the quaternary ammonium cation preventing electrostatic shielding of the negatively charged dsDNA phosphate backbone. The methyl group of TMA+ may also cause steric hindrance, thus preventing its binding to the minor or major groove of the helical structure. By using a salt composed of such an inert cation, we believe that a conformational change that negatively affects the affinity of dsDNA for silica is prevented, while the weakly ionizing anion still provides sufficient dehydration of the silica support for efficient binding of the dsDNA double helix, as we observed.

[0059] Based on the foregoing, in a next embodiment, a method is provided in which the small quaternary organic compound is tetramethylammonium chloride (also known as TMAC). In another embodiment, as supported by the examples below, the concentration of the small quaternary organic compound, such as the TMAC concentration under silica-binding conditions, is between 0.1M and 2M, possibly between 0.5M and 1.8M, or possibly between 0.8M and 1.6M, or possibly between 1M and 1.4M, or may be about 1.2M.

[0060] One advantage of the disclosed method is that the required binding buffer volume is compared to the potential maximum sample input volume, depending on the desired application. This feature is particularly advantageous for integrated devices with defined and limited internal volumes, such as closed fluid cartridges. This feature is directly dependent on the solubility of the binding buffer components. The preferred small quaternary organic compound TMAC has excellent solubility >1000 g / L, corresponding to a 9 M TMAC stable solution at room temperature. Sodium acetate is an exemplary buffer compound used to ensure acidic pH conditions and also has a high solubility of 5.6 M in water. Therefore, for example, if 1.2 M TMAC and 0.2 M ethyl acetate are used under conditions of nucleic acid binding to silica (i.e., conditions where the sample and binding buffer are in contact with a silica solid support) in an embodiment of the disclosed method, a 6 / 1 sample to buffer ratio can be achieved under binding conditions. Such an exemplary binding buffer would contain 8.4 M TMAC and 1.4 M acetate, both concentrations of which are soluble at room temperature. Therefore, in possible embodiments, the sample / buffer ratio can range from 6 / 1 to 1 / 6, depending on what is suitable for the application.

[0061] The uniqueness of the disclosed method lies in the use of a salt composed of a small quaternary organic compound and a weak ionizing anion (i.e., chloride ions, or to some extent bromide ions) to mediate the binding of nucleic acids (not only RNA, but especially DNA, particularly dsDNA) to a silica membrane under acidic conditions. As used herein, the term "acidic conditions" should be understood to mean conditions in which the pH of the aqueous solution is at least below 7, a pH widely accepted in the art and estimated based on a standard pH value on a base-10 logarithmic scale of the molar concentration of hydrogen ions (measured in moles per liter). Therefore, in another embodiment, a method is provided in which the pH is between 4 and 5.8; between 4.2 and 5.6; between 4.4 and 5.4; between 4.6 and 5.2; and possibly approximately 5. We believe that, within these pH ranges and in the presence of a silica solid binding support, the provision of the aforementioned specific salt composition provides a universal nucleic acid purification technique that is compatible not only with RNA but, more importantly, with DNA.

[0062] After binding with silica, the nucleic acids can then be washed and eluted using standard silica washing and elution methods known in the art.

[0063] For example, washing of bound nucleic acids can be performed in a manner similar to that used in the original Boom protocol. This involves using a high concentration of alcohol, typically 90% ethanol. As previously mentioned, some known methods describe washing procedures based on acidic solutions containing little or no lyophilic salts. Based on our observations, we believe this approach is only suitable for RNA applications. We believe that rehydration of dsDNA during washing leads to double helix stabilization due to the formation of hydrogen bonds between bases, causing premature release from the silica solid support.

[0064] The elution mechanism will then be largely similar to that known from previously known methods. For example, a low ionic strength solution with a neutral or weakly alkaline pH is used. This could be water or a standard PCR buffer. We also observed that the pH and the amount of divalent cations in the elution buffer can significantly affect elution efficiency. This is likely because the charge repulsion between the negatively charged silanol groups and the negatively charged phosphate backbone may play a significant role in the elution process. Therefore, deprotonating the silanol groups (enhancing the negative charge) by increasing the pH of the elution buffer will result in increased elution efficiency. The complete absence of small and / or divalent cations also increases elution efficiency, likely due to the lack of electrostatic shielding. These mechanisms are generally known in the art, and therefore selecting a suitable washing and elution strategy will not pose a major problem for those skilled in the art, and will not be discussed further here.

[0065] In alternative implementations, abandoning the strong liquid-electrochemicals of the Boom scheme for at least some types of biological samples may introduce several challenges, which could lead to several additions to the currently disclosed methods.

[0066] In particular, when focusing on Boom-based extraction schemes, liquid salts allow:

[0067] (i) To prevent other biomolecules (e.g., proteins, lipoproteins) from precipitating on the silica solid support;

[0068] (ii) Inhibit the activity of nucleases;

[0069] (iii) Release DNA from histones to enhance interaction with silica.

[0070] As those skilled in the art will know, the same effect can be achieved by introducing a protease for performing the protein digestion step, which can be advantageous in particularly difficult (e.g., old) samples. Therefore, in another embodiment, the method is preceded by protease treatment, such as treatment with proteinase K.

[0071] In another embodiment, a method is provided in which the biological sample is a liquid biopsy sample. As used herein, the terms "liquid biopsy material" or "liquid biopsy sample" should be understood to mean any non-tissue sample obtained from a subject, especially a body fluid sample. Sources of liquid biopsies include, but are not limited to, blood, plasma, serum, urine, cerebrospinal fluid (CSF), amniotic fluid, and other body fluids such as saliva, sweat, tears, breast milk, semen, feces, pleural fluid, peritoneal fluid, or lavage fluid. Analyzing nucleic acids in liquid biopsy samples can minimize the need for expensive, invasive, and often painful tissue and / or tumor biopsies to enable dynamic monitoring of disease or other physiological states. For example, in cancer patients, cell-free tumor DNA or RNA extracted from liquid biopsies may be used to detect mutations, translocations, or copy number alterations, as well as the expression of specific cancer markers.

[0072] Blood (plasma, serum, or whole blood, etc.) is the most commonly used fluid in the analysis of human liquid biopsy samples. In cancer patients, blood is a source of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) and cell-free RNA (cfRNA) released from tumor tissue (which includes circulating tumor DNA (ctDNA) and circulating tumor RNA (ctRNA), respectively), which can be used to detect mutations present in the patient's tumor. It is noteworthy that ctDNA, however, comprises only a very small fraction of the cfDNA present in blood, highlighting the importance of maximizing the sample volume used for nucleic acid analysis to detect rare mutations. Furthermore, cfDNA is always of low quality and fragmented to approximately the size of a nucleosome (140 bp). Therefore, for certain cancer types, including kidney cancer, prostate cancer, and upper and lower urinary tract thethelial carcinomas, alternative liquid biopsy methods using urine can be a richer source of tumor-derived material. Urine also offers other unique benefits such as ease of access (no need for trained medical personnel), no patient discomfort (enhanced patient compliance), and the presence of less contaminated protein compared to blood. However, urine is still a very diluted substance, and its application in diagnostic methods, especially in PoC devices, will benefit from maximizing the sample input volume. Given the current need for nucleic acid extraction chemicals that allow for maximizing the input volume of blood or urine samples, particularly within integrated PoC devices such as fluid cartridges, and because the method of the present invention is well-suited for this purpose, another exemplary embodiment provides a method in which the liquid biopsy sample is selected from plasma, serum, whole blood, or urine.

[0073] In the relevant implementation, a method is provided in which the nucleic acid is DNA, which, although relatively diluted in liquid biopsy samples, is more stable than RNA and can be separated with efficiency similar to that of a Boom-based extraction scheme using the methods disclosed herein.

[0074] In another implementation, the DNA may be cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA), which is typically a fragmented and / or double-stranded DNA type.

[0075] It is noteworthy that, for certain whole blood, old plasma, or serum samples, we have observed that the novel binding chemicals proposed herein (e.g., involving 1 M TMAC + 0.2 M acetate, pH 5) can sometimes be challenged by excessive protein precipitation and / or blockage by silica solid support, which can lead to reduced extraction yields. Depending on the sample type, this problem can be addressed by adding an appropriate detergent. Therefore, in another alternative embodiment, a method in which contact occurs in the presence of a detergent is provided. As used herein, the term "detergent" is broadly interpreted to refer to compounds or mixtures having surfactant properties. As used herein, the term "detergent" should be understood to be synonymous with the term "surfactant," which refers to any compound or mixture of compounds having amphiphilic properties and reducing the surface tension of the liquids containing them. We also believe that detergents can further enhance the efficiency of the process by additionally enhancing the removal of DNA from histones and inhibiting nuclease activity.

[0076] In one particular embodiment, for example, in cases where the sample is a plasma sample, potentially revealing a problem stemming from the high presence of albumin, a method is provided in which the detergent is a quaternary ammonium compound detergent. We have observed that quaternary ammonium compound detergents, such as cetanetrimethylammonium bromide (CTAB), strongly promote the solubility of albumin, preventing it from penetrating the silica membrane. The quaternary nature of such detergents may also prevent them from altering the helical conformation of DNA, thus they may be advantageous since it is assumed they do not affect the binding efficiency of dsDNA to silica. In experiments using difficult plasma samples, we have recognized the effectiveness obtained with CTAB, particularly due to its efficacy observed in even very low silica binding concentrations between 0.25% and 1%. Such low concentrations are meaningful for closed integrated device applications, where maximizing the sample input volume comes at the expense of minimizing the buffer volume. Therefore, in another embodiment, a method is provided in which the quaternary ammonium compound detergent is cetanetrimethylammonium bromide (CTAB).

[0077] In another embodiment, the method is performed within a fluid cartridge, which may be a closed fluid cartridge and may be part of an automated system. In one embodiment of the above embodiments, the cartridge may be of the type that directly receives biological samples, obtains PCR-grade nucleic acids from the biological samples using the novel nucleic acid extraction chemicals presented herein, and is suitable for accommodating at least one PCR reaction.

[0078] As used herein, the term "cartridge" should be understood as a separate component of a chamber and / or channel formed as a single object that can be transferred or moved as an accessory within or outside a larger instrument suitable for receiving or connecting to such a cartridge. Cartridges and their instruments can be considered as forming automated systems or automated platforms. Some components contained within a cartridge may be rigidly connected, while others may be flexibly connected and movable relative to other components of the cartridge. Similarly, as used herein, the term "jet cartridge" should be understood as a cartridge comprising at least one chamber or channel suitable for handling, processing, discharging, or analyzing a fluid (potentially a liquid). Examples of such cartridges are given in WO2007004103. Advantageously, a jet cartridge may be a microfluidic cartridge. In the context of a jet cartridge, the terms "downstream" and "upstream" can be defined in relation to the direction of fluid flow within such a cartridge. That is, a portion of the fluid path from the cartridge to a second part within the same cartridge is interpreted as being upstream of the second part. Similarly, the portion of fluid arriving later is located downstream relative to the portion of fluid passing earlier. Generally, as used herein, the term "fluidic" or sometimes "microfluidic" refers to systems and arrangements that handle the behavior, control, and manipulation of fluids, which are geometrically confined to a small, typically sub-millimeter scale in at least one or two dimensions (e.g., width and height or channel). Such small volumes of fluid are moved, mixed, separated, or otherwise handled at microscales requiring small size and low energy consumption. Microfluidic systems include structures such as micropneumatic systems (pressure sources, liquid pumps, microvalves, etc.) and microfluidic structures (microfluidic channels, etc.) for handling microliter, nanoliter, and picoliter volumes. Exemplary fluid systems are described in EP1896180, EP1904234, and EP2419705, and are therefore applicable to certain embodiments disclosed herein. Consistent with the foregoing, the term "chamber" should be understood as any functionally defined compartment of any geometry within a fluid or microfluidic assembly, defined by at least one wall and including the means necessary to perform the functions attributed to that compartment. Following these lines of thought, an "amplification chamber" should be understood as a compartment within a (micro)fluidic assembly, adapted to perform and purposefully provided within said assembly for the amplification of nucleic acids. Examples of amplification chambers include PCR chambers and qPCR chambers.

[0079] As used herein, the term "automated system" refers to an integrated platform comprising instruments and disposable materials (such as plastics and solutions) used in an automated manner to complete a specific process. This process can be initiated by a user, but user intervention is unnecessary throughout the automated processing within the system until the process is complete. As used herein, the term "instrument" should be understood to include an onboard computer equipped with at least one user interface (e.g., including at least one start button or power plug), software, and a machine programmed to perform certain functions such as running measurements, which may include, for example, mixing, ultrasonic treatment, heating, data detection and collection, and possibly analysis. In a possible embodiment, the interface may take the form of a console comprising a computer system running user interface software capable of initiating tests, displaying test results, and communicating with external information systems. An excellent automated system readily adaptable to the methods of the present invention is the Idylla diagnostic platform manufactured by Biocartis NV. TM It uses a disposable reagent carrier cartridge that can be coupled with a cartridge handling instrument and provides sample-to-result analytical performance.

[0080] In alternative embodiments, products directly related to and / or enabling the performance of the methods described above are further provided. In the simplest embodiment of such a product, a binding buffer is provided comprising a buffer (e.g., acetate) suitable for maintaining a pH value between 3 and 6, and also comprising TMAC, both concentrations directly suitable for obtaining the desired concentration under silica solid support binding conditions after mixing with a selected sample. Advantageous examples of such binding buffers include, for example, 2.33M TMAC and 0.47M acetate, 3.6M TMAC and 0.6M acetate, 4.8M TMAC and 0.8M acetate, 6M TMAC and 1M acetate, 7.2M TMAC and 1.2M acetate, and 8.4M TMAC and 1.4M acetate.

[0081] In an alternative embodiment of the above embodiments, a binding buffer solution comprising a quaternary ammonium compound detergent and / or proteinase K at a suitable concentration as described above is also provided. Examples of solutions listed above include CTAB concentrations of 2.33 M TMAC and 0.47 M acetate and 1.17% (w / v) CTAB, and 3.6 M TMAC and 0.6 M acetate and 1.5% (w / v) CTAB.

[0082] In alternative embodiments of the products disclosed herein, kits and / or jetting cartridges comprising any of the binding buffer solutions described above may be provided. As used herein, the term "kit" should be interpreted as a group of objects including at least one article or component or an article or device required for a specific purpose such as performing a molecular biology method or assay. Kits may be provided in the form of a standard benchtop nucleic acid purification kit comprising a container having reagents such as binding buffers, washing buffers, etc., and, for example, one or more silica solid supports, such as centrifuge columns, membranes, beads, etc. Alternatively, the kit may comprise a cartridge or be simply provided in the form of a cartridge. Following these ideas, in another embodiment, a cartridge is provided containing a binding buffer solution, a buffer adapted to maintain a pH between 3 and 6, and also containing TMAC. In another embodiment, the binding buffer solution in such a cartridge may also contain CTAB. In another embodiment, such a cartridge may advantageously further contain or include silica solid supports for nucleic acid purification. In another possible embodiment, such a cartridge may be a jetting cartridge and / or may be adapted to process liquid biopsy samples, such as plasma or urine.

[0083] Finally, this document also provides the use of the methods and products described herein (such as kits, cassettes, automated systems, etc.) for extracting nucleic acids from liquid biopsy samples. In another embodiment, the use of the disclosed methods and products for extracting DNA, which may be double-stranded DNA (dsDNA), cell-free DNA (cfDNA), or even circulating tumor DNA (ctDNA), is provided.

[0084] The novel nucleic acid purification chemicals and related products described herein have the potential for application in a variety of commercial reagent kits, lab-on-a-chip (LOC) devices, or disposable kits that utilize solid-phase extraction techniques to isolate nucleic acids from biological samples. More specifically, their application in fully integrated molecular diagnostic devices could be highly valuable due to the relatively small volume of binding buffer required, enabling increased sample input relative to the necessary buffer volume. Working examples of the concepts presented herein are given below. Example

[0085] General experimental setupA silica centrifuge column (Machery-Nagel, blood column nucleospin) was mounted on a QIAvac 24plus system, which was connected to the vacuum manifold of the vacuum pump via the QIAvac connection system. The entire setup could be used as a flow-through system. Plasma samples were mixed with binding buffer at a 4:3 ratio (e.g., 1 mL plasma to 0.75 mL binding buffer) and flow-through the silica centrifuge column. Therefore, the binding buffer was typically diluted 2.33 times when mixed with plasma. The 4:3 ratio is not a requirement but merely an arbitrary choice, partly related to the design of the Idylla cartridge (the lysis chamber allows a maximum input of 7 mL), even for silica centrifuge column experiments. It is entirely possible to further increase the concentration of the binding buffer and thus reduce the required buffer volume relative to the sample volume. However, for this particular centrifuge column setup, a 1.75-fold dilution of the plasma sample seems satisfactory in terms of clogging and flow rate. The silica membrane was then washed with washing buffer before the centrifuge column was removed from the vacuum manifold. The column was then placed in a 1 mL Lo-Bind Eppendorf tube and centrifuged at 10,000 rpm for one minute. The column was then transferred to a new 1 mL Lo-Bind Eppendorf tube, and elution buffer was added. After incubating at room temperature for two minutes, the column was centrifuged again at 10,000 rpm for one minute. The elution products were then analyzed by qPCR to provide a relative quantification of the purified DNA.

[0086] Sample type and binding buffer chemicals. Plasma (Innovative Research) was incorporated with nucleosome DNA (nDNA) isolated from whole blood. Spiking was used to provide robust downstream qPCR-based target detection when processing smaller plasma volumes. Additionally, nucleosome DNA is characterized by a fragmentation pattern very similar to that of cell-free DNA (cfDNA). The presence of short fragments allowed us to assess their extraction efficiency. 20,000 copies of nDNA were incorporated into 100 μL of plasma. The spiked plasma was then mixed with 500 μL of binding buffer. This binding buffer consisted of 1.2 M tetramethylammonium chloride (TMAC) dissolved in 0.24 M sodium acetate pH 5 buffer. This resulted in a final concentration of 1 M TMAC and 0.2 M sodium acetate when mixed with the plasma sample. The total volume of this acidic mixture (600 μL) was then centrifuged on a silica column as described above.

[0087] Washing buffer chemicals.The silica membrane was washed by running 1000 μL of 90% ethanol through a centrifuge column. Any remaining ethanol traces were then removed by centrifuging the column for one minute at 10,000 rpm.

[0088] Elution buffer chemicals. DNA was eluted by rehydrating the silica membrane with water or Tris-HCl pH 8.6 buffer. Importantly, the elution buffer was at room temperature and in contact with the silica membrane for at least two minutes. The column was then subjected to a final centrifugation step (one minute, 10,000 rpm). The eluted product was then recovered in a 1 mL Lo-Bind Eppendorf tube.

[0089] qPCR design and conditions. To evaluate the extraction efficiency of short and long DNA fragments, a triplet design consisting of three amplicons of varying sizes was used. The target amplicons were 62 bp, 98 bp, and 136 bp in length. The difference in Ct values ​​between the shortest and longest amplicons indicated the presence of a short target fragment. Primer and probe sequences are available upon request. 20 μL of elution product was mixed with 5 μL of PCR buffer. The final PCR reaction consisted of: 50 mM KCl, 20 mM Tris-HCl pH 8.6, 2 mM MgCl2, 0.2 mM dNTP mixture, 300 nM of each primer and probe, and 5 units of Gotaq DNA polymerase. The qPCR reaction was performed in a Biorad CFX96 Touch PCR machine. TM The assay was performed on a real-time PCR system. The total reaction volume was 25 μL. The cycling protocol consisted of a hot start (5" 95°C), followed by 50 cycles of denaturation (3" 95°C) and annealing (30" 64°C). Fluorescence was measured after each cycle.

[0090] result. We first investigated the extraction efficiency of dsDNA in binding buffers with different ionizing salts and hydrophilic or mild ionizing salts at neutral pH. The Ct values ​​for PCR of 62bp and 136bp amplicons extracted in different binding buffers were shown... Figure 1The Y-axis shows the composition of the binding buffer at neutral pH. The X-axis shows the composition of the binding buffer at different pH values. "Input" is the reference point, reflecting the Ct value obtained when the total amount of spiked nDNA is targeted. Therefore, ΔCt for the reference point represents the extraction efficiency (i.e., Δ = 50% extraction efficiency for 1 Ct). If the ΔCt between small and large amplicon remains the same as the reference point, this indicates that small fragments (62bp-136bp) are not lost. The results show that at neutral pH, the binding efficiency of dsDNA to silica decreases as the amount of lyophilic salt in the binding buffer (NaCl or (NH4)2SO4) increases. As mentioned earlier, we hypothesize that this may be due to the stabilizing effect of small lyophilic cations (Na+ and NH4+) on the DNA double helix. Clearly, under neutral conditions, a high concentration of lyophilic salt is preferred to mediate the binding of DNA to the silica membrane.

[0091] We then repeated the experiment under acidic conditions (pH 5). The results are as follows. Figure 2 As shown in the diagram. As previously described, the Y-axis displays the Ct values ​​for all three different amplicon sizes, while the X-axis displays the different binding buffer compositions. The data show that, as described in the prior art, the use of acidic conditions (pH 5) and hydrophilic salts cannot effectively mediate the binding of nDNA to the silica membrane. 0.1M NaCl at pH 5 performed best, with an extraction efficiency of approximately 6.25% (ΔCt = 4).

[0092] We then compared the performance of the ionized Boom-binding buffer (3.68 M GuSCN and butanol) with buffers containing chloride-based salts (with or without quaternary ammonium compounds). The results are as follows: Figure 3 As shown in the figure. The Y-axis displays the Ct values ​​for all three different amplicon sizes. The X-axis displays the different binding buffer compositions. The data show that using a salt composed of a quaternary ammonium cation (TMA+) and a hydrophilic anion (Cl-) effectively mediates the binding of DNA to the silica membrane. The results support the hypothesis that the inertness of the quaternary cation destabilizes the helical structure of DNA, thereby increasing the number of available silica binding sites.

[0093] We then investigated the optimal pH range for plasma samples. The results are as follows: Figure 4As shown. The Y-axis displays the Ct values ​​for all three different amplicon sizes. The X-axis displays the different binding buffer compositions. This experiment shows that lowering the pH of the binding buffer to 4 is incompatible with native plasma samples. Protein aggregation becomes so severe with the addition of just 0.3 M TMAC that it becomes almost impossible to process the sample successfully. This is likely related to the isoelectric point (pI) (4.7) of albumin, which is abundant in plasma. Once the pH of the solution approaches the pI of the protein, the charge repulsion between individual protein molecules decreases, potentially leading to precipitation. At this point, it appears that only slight dehydration by anions is sufficient to promote protein aggregation.

[0094] As a next step, we compared the performance of TMA sulfate (TMAS) with that of TMAC, and the results are as follows: Figure 5 As shown. The Y-axis displays the Ct values ​​for all three different amplicon sizes. The X-axis displays the different binding buffer compositions. The experiments demonstrate that TMAS cannot effectively mediate the binding of DNA to silica. These results were initially surprising based on our initial hypothesis that the key binding mechanism was:

[0095] (i) Dehydration of silica membranes and nucleic acids. This is achieved by providing sufficient hydrophilic anions and thus reducing the amount of free water.

[0096] (ii) Shielding intermolecular electrostatic forces. This is achieved by using acidic conditions and protonating negatively charged silanol groups.

[0097] Based on the above, we anticipate that sulfate, being a stronger lyophile than chloride ions due to its double charge, will provide a stronger dehydrating effect, thus enabling a more efficient binding of DNA to silica. As confirmed in this experiment and many other experiments, we conclude that other mechanisms must exist besides those described above. Melzak et al. (1996) described a third effect that may influence DNA-silica interactions: (iii) the formation of intermolecular hydrogen bonds in the nucleic acid-silica contact layer. The data appear to suggest that these hydrogen bonds may be more important than initially thought, and that their formation is strongly disrupted or even prevented by the use of the strongly lyophilic sulfate anion. Therefore, the properties of chloride anions (which are specifically described as weak lyophiles or critical lyophiles / liquidophiles) may be uniquely characterized by their interaction with water being no stronger than the interaction of water with itself.

[0098] To further investigate this hypothesis, we compared the performance of different TMA-containing salts in mediating the binding of spiked nDNA to silica in PBS and plasma samples. The results are as follows: Figure 6As shown. The Y-axis displays the Ct value for a 62bp amplicon. The X-axis displays different binding buffer compositions. "Input" shows the reference point and reflects the Ct value obtained when targeting the total amount of spiked nDNA. Therefore, ΔCt for the reference point represents the extraction efficiency (i.e., Δ = 50% extraction efficiency for 1 Ct). The data illustrate the importance of the selected anion. The charge density of chloride allows for the highest extraction efficiency at the lowest concentration. More hydrophilic anions with higher charge densities, such as sulfate, may rapidly reduce extraction efficiency by mediating the conformational transition from B-DNA to A-DNA or any other unknown mechanism. On the other hand, more hydrophilic anions with lower charge densities, such as bromide, may prevent this conformational transition but are significantly less efficient at dehydrating silica membranes, thus requiring higher molar concentrations to achieve performance equivalent to that conferred by the selection of chloride. It should also be noted that the reduced charge density of more hydrophilic anions also negatively impacts the solubility of quaternary ammonium salts. From this perspective, TMAC is superior in both performance and solubility.

[0099] TMAC has been identified as the most promising quaternary ammonium salt, and we then investigated its optimal concentration in our specific experimental setup. The results are as follows: Figure 7 As shown in the figure. The Y-axis displays the Ct values ​​for all three different amplicon sizes. The X-axis displays the different binding buffer compositions. The results indicate that increasing the concentration of TMAC in the binding buffer above 1 M has no beneficial effect on the binding efficiency of DNA to silica. In fact, the binding efficiency even slightly decreases when the concentration of TMAC increases.

[0100] Then, we investigated the dsDNA extraction performance using TMAC at different pH values. The results are as follows: Figure 8 As shown. The Y-axis displays the Ct values ​​for all three different amplicon sizes. The X-axis displays the different binding buffer compositions. The results demonstrate the importance of the binding buffer pH. As previously mentioned, lowering the binding buffer pH to near the isoelectric point of albumin (4.7) when processing undigested plasma leads to severe protein aggregation, making sample processing impossible in centrifuge columns or microfluidic channels. Additionally, raising the binding buffer pH above 5 slightly increases the negative charge repulsion between the silanol groups of DNA and the phosphate backbone, resulting in reduced DNA binding efficiency to silica. Surface silanol groups on silica membranes have been described to have pKa values ​​of 4–8. Increasing the pH may cause deprotonation of silanol groups with the lowest pKa values, thus making them negatively charged.

[0101] We then investigated the molar concentration of TMAC and the pH of the acetate buffer in a more extensive manner across multiple and different batches of plasma. Additionally, we added the quaternary ammonium detergent cetanetrimethylammonium bromide (CTAB) to the binding buffer. The results are as follows: Figure 9 As shown. The Y-axis displays the Cq value of the 62bp amplicon. The X-axis displays the binding conditions, including different amounts of added CTAB. Unlike previous experiments where a limited amount of plasma was treated after incorporation with nDNA, this experiment focused on extracting cfDNA from 1 mL of unspecified plasma. To obtain additional improvements, a protein digestion step was performed on the plasma sample at 37°C for 10 minutes using 1 mg / mL proteinase K. 1 mL of plasma sample was treated with 0.75 mL of binding buffer (2.33 M TMAC, 0.47 M acetate, 1.17% CTAB, pH 5). Subsequently, the membrane was washed with 1 mL of first wash buffer (1 M TMAC, 0.2 M acetate, pH 5), and finally with another 1 mL of 90% EtOH. Ten different samples were treated for each binding condition. Figure 9 The box plots show the mean and median Cq values ​​and their variations. Clearly, the addition of CTAB has a beneficial effect, as it reduces inter-sample variability and increases DNA yield. We believe this effect is due to CTAB promoting the solubility of albumin, thus preventing its precipitation on silica membranes. It may also inhibit nuclease activity and may facilitate the removal of cfDNA from histones.

[0102] Then, we observed different TMAC molar concentrations at different pH values. By evaluating multiple batches of plasma under different binding conditions, it became clear that different samples responded very differently to different conditions, such as... Figure 10 As shown in the figure. The Y-axis displays the Ct value of the 62bp amplicon. The X-axis displays different pH values ​​for binding conditions. The shape of the graph represents different TMAC molar concentrations (as explained in the example).

[0103] We then investigated the efficiency of the elution conditions. The results are as follows: Figure 11 As shown. The Y-axis displays the Ct values ​​for all three different amplicon sizes. The X-axis displays the different elution buffer compositions and incubation times at room temperature. The data show that the elution step is most efficient when using a weakly alkaline buffer. Similarly, this may be related to the pKa value of the surface silanol groups. We believe that the negative charge repulsion between the silanol groups and the phosphate backbone is the main driving force in the elution process, accompanied by rehydration. Therefore, providing an elution buffer with pH > the highest silanol pKa value (8) will ensure that all silanol groups are negatively charged. Figure 11It can be seen that the presence of 50 mM K+ and 2 mM Mg++ negatively impacts elution efficiency. We hypothesize that this effect may be caused by these strongly hydrophilic cations shielding the negative charge repulsion between the silanol groups and the phosphate backbone. This observation should be kept in mind when applying this approach to fully integrated molecular diagnostic devices, where silica-bound nucleic acids are eluted directly with amplification buffer.

[0104] Next, we investigated the potential beneficial effects of incorporating proteinase K predigestion in relation to the volume of the starting material, particularly for recalcitrant plasma samples. The results are as follows: Figure 12 As shown. The Y-axis displays the Ct value of the 62bp amplicon. The X-axis shows the different plasma volumes processed. The results indicate that incorporating a proteinase K digestion step improved DNA yield when processing plasma samples >400 μL. The plasma was digested at 56 °C for 10 min. We suspect that protein binding to the silica membrane also occurs to some extent under the binding buffer chemistry conditions studied in this paper. Acidic conditions reduced the negative charge of albumin with pI = 4.7, which significantly reduced the charge repulsion between this abundant plasma protein and the silica membrane. Therefore, it is expected that proteins and nucleic acids will compete for binding sites, while the surface area of ​​the silica membrane remains limited. In addition, the reduced charge repulsion between individual protein molecules allows them to pack more tightly together when binding to the membrane. Therefore, digestion or removal of albumin from plasma may be beneficial to improve nucleic acid binding to the silica membrane when processing larger samples.

[0105] Then, we belong to Idylla TM Plasma sample volume scaling was evaluated in the single-use, patented product of the Biocartis NV kit integrated system. Results are as follows: Figure 13 As shown. The Y-axis displays the Ct value of the 62bp amplicon. The X-axis displays different plasma volumes treated with different binding buffer chemicals. The samples used were unspiked plasma samples. The results encouragely demonstrate that the novel binding buffer chemicals presented in this paper allow for substantial sample scale-up in the Idylla kit, resulting in increased linear yields. The binding conditions applied were as follows: 1.2M TMAC, 0.2M acetate, 0.5% CTAB, pH 5. The final buffer layout used in the kit-based system is as follows:

[0106] • Binding buffer (3 mL): 2.8 M TMAC, 0.47 M acetate, 1.17% CTAB, pH 5 (diluted 2.33 times with sample)

[0107] • Plasma sample (4 mL) (treated with 1 mg / mL proteinase K for 10 minutes at room temperature)

[0108] • First wash buffer (1.25 mL): 1.2 M TMAC, 0.2 M acetate, pH 5

[0109] • Second wash buffer (2.4 mL): 90% ethanol

[0110] • Elution buffer: H2O (volume can be scaled up as needed; for Idylla, the minimum elution volume is 160 μL and the maximum elution volume is 250 μL)

[0111] Finally, we decided to compare the performance of a kit using the disclosed novel extraction chemical with that using a reference chemical that was separated from the plasma. For this purpose, we used seven different batches of plasma, and performed five kit replicates per batch for each extraction chemical. All runs of 70 kits were completed successfully without any clogging errors. The results are as follows: Figure 14 and Figure 15 As shown. In Figure 14 In the diagram, the Y-axis displays the Ct value of the 62bp target amplicon. The X-axis specifies the extraction chemical (left-hand side for each group is the liquid-free reference, right-hand side is the novel TMAC+CTAB chemical) and sample volume (1 ml and 4 ml, respectively). Each group represents a different batch of plasma. Figure 15 In the diagram, the Y-axis displays the Ct values ​​for the same amplicon, while the X-axis lists different batches of plasma. Each group represents a different extraction chemical (left-hand side is the liquid reference, right-hand side is the novel TMAC+CTAB chemical). Hollow dots are 10-fold dilutions of solid dots, thus indicating PCR inhibition. Figure 14 and Figure 15 The data showed that increased sample input (4 ml) through the use of the new extraction chemical resulted in a significant yield increase. The actual measured yield increases are listed in Table 1 below. Figure 15 The results also showed that none of the sample extracts contained any components that inhibited PCR. The results indicate that the novel binding chemical is robust, with a standard deviation comparable to that of the reference chemical used in the ionization process. Furthermore, for all plasma batches studied, at least a 2-fold yield gain was achieved due to the ability to increase the sample input volume per kit, as shown in Table 1. In addition to this increased detection sensitivity, it should be noted that the novel buffer chemical is completely free of ionization agents and is cost-effective.

[0112] Table 1: Figure 14 The average Ct value obtained for each batch of plasma. The ΔCt between the two extraction chemicals reflects the increase in cfDNA yield.

[0113]

Claims

1. A method for extracting double-stranded DNA, comprising treating a liquid biopsy sample with a protease and contacting the liquid biopsy sample with a silica solid support at a pH between 3 and 6 and in the presence of a salt consisting of: - Small quaternary organic compounds are defined as quaternary compounds consisting of a central positively charged atom and four organic substituents R1-R4, wherein each organic substituent R1-R4 contains no more than 2 carbon atoms; and - Bromine anion or chloride anion; The positively charged atom in the small quaternary organic compound is nitrogen; and The concentration of the small quaternary organic compounds contained therein is between 0.1 M and 2 M.

2. The method of claim 1, wherein the pH value is between 4 and 5.

8.

3. The method of claim 1, wherein the pH value is between 4.2 and 5.

6.

4. The method of claim 1, wherein the pH value is between 4.4 and 5.

4.

5. The method of claim 1, wherein the pH value is between 4.6 and 5.

2.

6. The method according to claim 1, wherein the pH value is 5.

7. A method for extracting double-stranded DNA, comprising treating a liquid biopsy sample with a protease and contacting the liquid biopsy sample with a silica solid support at a pH between 5 and 6 and in the presence of a salt consisting of: - Small quaternary organic compounds are defined as quaternary compounds consisting of a central positively charged atom and four organic substituents R1-R4, wherein each organic substituent R1-R4 contains no more than 2 carbon atoms; and - Bromine anion or chloride anion; The positively charged atom in the small quaternary organic compound is nitrogen; and The concentration of the small quaternary organic compounds contained therein is between 0.1 M and 2 M.

8. The method according to claim 1 or 7, wherein the anion is a chloride ion.

9. The method according to claim 1 or 7, wherein the small quaternary organic compound is tetramethylammonium chloride (TMAC).

10. The method according to claim 1 or 7, wherein the concentration of the small quaternary organic compound contained therein is between 0.5 M and 1.8 M.

11. The method of claim 10, wherein the concentration of the small quaternary organic compound contained therein is between 0.8 M and 1.6 M.

12. The method of claim 10, wherein the concentration of the small quaternary organic compound contained therein is between 1 M and 1.4 M.

13. The method of claim 10, wherein the concentration of the small quaternary organic compound contained therein is 1.2 M.

14. The method according to claim 1 or 7, wherein the liquid biopsy sample is selected from plasma, serum, whole blood or urine.

15. The method according to claim 1 or 7, wherein the double-stranded DNA is cell-free double-stranded DNA.

16. The method according to claim 1 or 7, wherein the double-stranded DNA is circulating tumor double-stranded DNA.

17. The method of claim 1 or 7, wherein the contact occurs in the presence of a detergent.

18. The method of claim 17, wherein the detergent is a quaternary ammonium compound detergent.

19. The method of claim 18, wherein the quaternary ammonium compound detergent is cetanetrimethylammonium bromide (CTAB).

20. The method according to claim 1 or 7, wherein the method is performed inside the medicine box.

21. The method according to claim 20, wherein the medicine box is a jet box.

22. A kit comprising a silica solid support, a binding buffer solution, said binding buffer solution comprising a buffer suitable for maintaining a pH between 4.8 and 5.6, and further comprising TMAC at a concentration between 0.1 M and 2 M, and The binding buffer solution also contains CTAB.

23. The medicine box according to claim 22, wherein the medicine box is a jet box.

24. Use of the method according to any one of claims 1-21 or the kit according to any one of claims 22-23 for extracting cell-free double-stranded DNA from liquid biopsy samples.

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