Nucleic acid purification from fixed biological samples

Abstract

To provide a method for lysing a fixed biological sample and / or for obtaining purified nucleic acid from a fixed biological sample.SOLUTION: Disclosed herein is a method for lysing a fixed biological sample, where the fixed biological sample comprises crosslinks between nucleic acid molecules and protein molecules due to the fixation, the method comprising: (a) lysing the fixed biological sample, where lysis involves digestion with a proteolytic enzyme; (b) heating the lysed sample to reverse crosslinks; (c) adding a proteolytic enzyme and performing a proteolytic digestion; optionally one or more additional treatment steps are performed between step (b) and step (c). The provided nucleic acids are of high yield and quality and can be purified from the lysed sample.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Field of the Disclosure The present invention relates to methods for lysing fixed biological samples and / or obtaining purified nucleic acids from fixed biological samples. [Background technology]

[0002] background Fixation of biological samples allows for long-term storage and archiving of the samples. Fixation is usually achieved with acids, alcohols, ketones, or other organic substances, such as protein-precipitating or protein-crosslinking compounds, such as glutaraldehyde, formaldehyde, and / or paraformaldehyde. Such fixatives are well known in the art. Fixation with formaldehyde (e.g., used in the form of a 35 weight percent aqueous solution called "formalin") can be followed by embedding the fixed material in paraffin (referred to as "formalin-fixed, paraffin-embedded" (FFPE) material) and is a widely used fixation technique. Formaldehyde-based fixation has the advantage that tissue structure is relatively well maintained under fixation. Fixation involving crosslinking fixatives is also used to fix and thereby preserve liquid samples. A commonly used preservative containing ethanol and formaldehyde is SurePath®.

[0003] Fixed biological samples (e.g., FFPE samples) are a valuable resource for investigating disease. However, such samples, primarily used for histopathology, are unsuitable for nucleic acid extraction and analysis due to extensive DNA damage and cross-linking resulting from exposure to and storage in formaldehyde. The most significant quality issue with DNA extracted from fixed biological samples is cross-linking of nucleic acids to proteins and between nucleic acids. This cross-linking renders the DNA inaccessible to enzymes during reactions such as PCR, severely reducing performance and potentially resulting in erroneous test results. As a result, efficient release and purification of nucleic acids (DNA or RNA) from fixed biological samples (solid or liquid) is challenging. However, nucleic acid analysis is crucial for many molecular-level investigations, especially for clinical or diagnostic applications. Methods for extracting nucleic acids, such as DNA, from fixed biological samples must reverse the crosslinks introduced by fixation while preventing any further damage to the nucleic acids. A standard method for decrosslinking DNA is to heat-treat crude lysates, for example, at 90°C for 1 hour or at 80°C for 4 hours; these decrosslinking steps are all used to eliminate crosslinks present in fixed biological samples. Numerous commercially available kits and methods for FFPE extraction are available in the art. All kits use a combination of heat, enzymes, and chemical lysis to remove tissue from paraffin, digest the tissue, and purify DNA. Additional methods for isolating / releasing nucleic acids from fixed samples are described in WO2007 / 068764, WO2014 / 072366, WO2005 / 075642, WO2001 / 46402, and US2005 / 0014203. Most available kits and methods must compromise on the total yield and / or quality of the extracted DNA (i.e., higher DNA yields are often accompanied by fragmentation, while largely intact high-molecular-weight DNA is often not fully decrosslinked and is of poorer quality in PCR applications). Furthermore, an important indicator for assessing the quality of extracted nucleic acids is their performance in PCR and / or NGS applications. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2007 / 068764 [Patent Document 2] International Publication No. 2014 / 072366 [Patent Document 3] International Publication No. 2005 / 075642 Summary of the Invention [Means for solving the problem]

[0005] An object of the present invention is to provide a method for lysing a fixed biological sample that effectively releases contained nucleic acids, such as DNA and / or RNA, particularly DNA. A further object of the present invention is to provide a method for purifying nucleic acids, such as DNA and / or RNA, from a fixed biological sample. In embodiments, the present invention aims to avoid the drawbacks of prior art methods. In embodiments, the methods of the present invention provide improvements in at least one criterion, such as yield, fragmentation, and / or performance in subsequent nucleic acid analysis methods, such as PCR and / or next-generation sequencing.

[0006] Summary of the Invention The present invention provides an improved method for lysing fixed biological samples, where the fixed biological samples contain crosslinks between nucleic acid molecules and protein molecules. These crosslinks exist due to the fixation used (e.g., formaldehyde- and / or paraformaldehyde-based fixation). It has been discovered that a stepwise, sequential lysis process can significantly improve the lysis and digestion of fixed biological samples by digesting the fixed biological sample with a proteolytic enzyme, then heating to reverse the crosslinks, and then adding a proteolytic enzyme to perform an additional proteolytic digestion. The examples show that performing a crosslink reversal step followed by a second proteolytic digestion provides significant and unexpected improvements.

[0007] According to a first aspect of the present invention there is provided a method for lysing a fixed biological sample, wherein the fixed biological sample contains crosslinks between nucleic acid molecules and protein molecules due to fixation, the method comprising: (a) lysing the fixed biological sample, wherein the lysis comprises digestion with a proteolytic enzyme; (b) heating the dissolved sample to reverse the crosslinks; (c) adding a proteolytic enzyme and performing proteolytic digestion; Including, Optionally, methods are provided in which one or more additional processing steps are performed between steps (b) and (c).

[0008] As demonstrated by the examples, the method according to the first aspect significantly improves the release of nucleic acids, such as DNA, compared to prior art methods. The fixed biological sample may be a solid biological sample (e.g., a fixed tissue sample) or a liquid biological sample (e.g., a liquid sample containing fixed cells).

[0009] According to a second aspect of the present invention, there is provided a method for obtaining purified nucleic acids from a fixed biological sample, the fixed biological sample containing crosslinks between nucleic acid molecules and protein molecules due to fixation, the method comprising the step of lysing the fixed biological sample according to steps (a) to (c) of the lysis method of the first aspect, wherein after step (c) of the method of the first aspect: (d) purifying nucleic acids from the lysed sample. A method is provided, comprising:

[0010] As disclosed herein, one or more additional processing steps (e.g., at least one further enzymatic treatment step different from the proteolytic digestion step) may optionally be performed between steps (b) and (c) of the method according to the first aspect. The stepwise lysis / digestion procedure according to the first aspect improves the release of high-quality nucleic acids (such as DNA) from the fixed biological sample, whereby the released nucleic acids (such as DNA) can then be purified with high quality and / or yield from the digested sample in step (d) of the method according to the second aspect. Thereby, an improved method for purifying nucleic acids, such as DNA, from a fixed biological sample is provided.

[0011] A third aspect of the present invention relates to the use of a proteolytic enzyme, such as proteinase K, to lyse a fixed biological sample prior to proteolytic digestion, preferably comprising proteolytic digestion of the fixed biological sample in a method according to the first or second aspect of the present invention, and heating the lysed sample to reverse crosslinks. As disclosed herein, the fixed biological sample contains crosslinks between nucleic acid molecules and protein molecules resulting from fixation. According to one embodiment, the fixation involved the use of formaldehyde or paraformaldehyde.

[0012] A fourth aspect of the present invention relates to the use of a glycosylase, such as a DNA glycosylase, preferably a uracil DNA glycosylase, to perform enzymatic treatment, wherein the enzymatic treatment is completed in 30 minutes or less, 20 minutes or less, 15 minutes or less, or 10 minutes or less. Such a use may be carried out in a method according to the first or second aspect of the present invention. In a preferred embodiment, the uracil glycosylase is uracil-N-glycosylase.

[0013] Other objects, features, advantages and aspects of the present application will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, while indicating preferred embodiments of the present application, are given by way of illustration only. [Brief explanation of the drawings]

[0014] [Figure 1-1] Figure 1 shows DNA yields after extraction from various human FFPE tissues, including prostate (Figure 1A), lung (Figure 1B), kidney (Figure 1C), spleen (Figure 1D), and breast cancer (Figure 1E). DNA yields were determined using a QIAxpert ("UV-Vis," dark-shaded columns) and Qubit instruments ("dsDNA (Qubit)," light-shaded columns). Extraction using a high-Tris lysis composition ("New GR-High Tris") was compared to a low-Tris lysis composition ("New GR-Low Tris") with ("+") or without ("-") an additional proteinase K digestion step (15 min, 65°C). [Figure 1-2] Figure 1 shows DNA yields after extraction from various human FFPE tissues, including prostate (Figure 1A), lung (Figure 1B), kidney (Figure 1C), spleen (Figure 1D), and breast cancer (Figure 1E). DNA yields were determined using a QIAxpert ("UV-Vis," dark-shaded columns) and Qubit instruments ("dsDNA (Qubit)," light-shaded columns). Extraction using a high-Tris lysis composition ("New GR-High Tris") was compared to a low-Tris lysis composition ("New GR-Low Tris") with ("+") or without ("-") an additional proteinase K digestion step (15 min, 65°C). [Figure 1-3]Figure 1 shows DNA yields after extraction from various human FFPE tissues, including prostate (Figure 1A), lung (Figure 1B), kidney (Figure 1C), spleen (Figure 1D), and breast cancer (Figure 1E). DNA yields were determined using a QIAxpert ("UV-Vis," dark-shaded columns) and Qubit instruments ("dsDNA (Qubit)," light-shaded columns). Extraction using a high-Tris lysis composition ("New GR-High Tris") was compared to a low-Tris lysis composition ("New GR-Low Tris") with ("+") or without ("-") an additional proteinase K digestion step (15 min, 65°C).

[0015] [Figure 2] Figure 2 shows an electrophoresis gel of DNA extracted from FFPE tissue samples of human kidney and human breast cancer. Extractions were performed using a high Tris lysis composition ("High Tris") or a low Tris lysis composition ("Low Tris"), with ("+") or without ("-") an additional proteinase K digestion step (15 minutes, 65°C).

[0016] [Figure 3] Cq values ​​of DNA extracted from breast cancer (top) or kidney FFPE tissue (bottom) measured by quantitative real-time PCR using a large amplicon (500 bp, right) or a short amplicon (66 bp, left). Extraction using a high Tris lysis composition ("New GR-High Tris") was compared to a low Tris lysis composition ("New GR-Low Tris"), with or without an additional proteinase K digestion step (15 min, 65°C). Results shown in dark-shaded columns correspond to the same amount of DNA per reaction mixture; light-shaded columns correspond to the same volume of diluted eluate per reaction mixture.

[0017] [Figure 4]Figure 4 shows the results of NGS of DNA extracted from various human tissues. The number of reads per UMI (Unique Molecular Identifier, also called Unique Molecular Index) of the extracted DNA is shown. A value greater than 10 indicates that the same molecule was read more than 10 times, indicating over-amplification / insufficient complexity of the starting material. Extractions were performed using a high Tris lysis composition ("New GR-High Tris") or a low Tris lysis composition ("New GR-Low Tris"), with or without an additional proteinase K digestion step ("2nd PK").

[0018] [Figure 5] Figure 5 shows DNA yields after extraction from various FFPE tissues (Figure 5A: human lung cancer tissue; 5B: human atrial tissue). DNA yields were determined using a QIAxpert ("UV-Vis," dark-shaded columns) and Qubit instruments ("dsDNA (Qubit)," light-shaded columns). Extraction using a high-Tris lysis composition ("New GR-High Tris") was compared to a low-Tris lysis composition ("New GR-Low Tris") with or without an additional proteinase K digestion step ("15 min, 65°C PK") ("std")). An additional control was performed by performing a first proteinase K step overnight at 56°C ("o / n 56°C"). As a reference, extraction was also performed using the QIAamp® FFPE DNA kit ("QA") as well as the Promega Maxwell RSC DNA FFPE kit and the Maxwell RSC FFPE Plus DNA kit, where proteinase K digestion took 1 hour at 70°C.

[0019] [Figure 6]Figure 6 shows an electrophoresis gel of DNA extracted from various FFPE tissues (Figure 6A: human lung cancer tissue; 6B: human atrial tissue). Extractions were performed using a high Tris lysis composition ("New GR-High Tris") or a low Tris lysis composition ("New GR-Low Tris") with ("+65°C") or without ("std") an additional proteinase K digestion step (15 min, 65°C). Additionally, an additional control was performed by performing a first proteinase K step overnight ("o / n") at 56°C. As a reference, extractions were also performed using the QIAamp® FFPE DNA Kit ("QA FFPE"), as well as the Promega Maxwell RSC DNA FFPE Kit and Maxwell RSC FFPE Plus DNA Kit, where proteinase K digestion took 1 hour at 70°C.

[0020] [Figure 7-1] Cq values ​​of DNA extracted from various FFPE tissues (left: human lung cancer tissue; right: human atrial tissue) measured by quantitative real-time PCR. In Figure 7A, a short amplicon (66 bp) was used, and in Figure 7B, a large amplicon (500 bp) was used. Extraction using a high Tris lysis composition ("New GR-High Tris") was compared with a low Tris lysis composition ("New GR-Low Tris"), with or without an additional proteinase K digestion step (15 min, 65°C). Furthermore, an additional control was performed by performing a first proteinase K step overnight at 56°C ("o / n 56°C"). As a reference, extractions were also performed using the QIAamp® FFPE DNA Kit ("QA") and the Promega Maxwell RSC DNA FFPE Kit and Maxwell RSC FFPE Plus DNA Kit, where proteinase K digestion was performed at 70°C for 1 hour. Results shown in darkly shaded columns correspond to the same amount of DNA per reaction mixture, and lightly shaded columns correspond to the same volume of diluted eluate per reaction mixture. [Figure 7-2]Cq values ​​of DNA extracted from various FFPE tissues (left: human lung cancer tissue; right: human atrial tissue) measured by quantitative real-time PCR. In Figure 7A, a short amplicon (66 bp) was used, and in Figure 7B, a large amplicon (500 bp) was used. Extraction using a high Tris lysis composition ("New GR-High Tris") was compared with a low Tris lysis composition ("New GR-Low Tris"), with or without an additional proteinase K digestion step (15 min, 65°C). Furthermore, an additional control was performed by performing a first proteinase K step overnight at 56°C ("o / n 56°C"). As a reference, extractions were also performed using the QIAamp® FFPE DNA Kit ("QA") and the Promega Maxwell RSC DNA FFPE Kit and Maxwell RSC FFPE Plus DNA Kit, where proteinase K digestion was performed at 70°C for 1 hour. Results shown in darkly shaded columns correspond to the same amount of DNA per reaction mixture, and lightly shaded columns correspond to the same volume of diluted eluate per reaction mixture.

[0021] [Figure 8] Figure 8 shows the results of NGS of DNA extracted from human atrial FFPE tissue. The number of reads per UMI (unique molecular identifier, also called unique molecular index) of the extracted DNA is shown. A value greater than 10 indicates that the same molecule was read more than 10 times, indicating over-amplification / insufficient complexity of the starting material. Extraction was performed using a high Tris lysis composition ("New GR-High Tris") or a low Tris lysis composition ("New GR-Low Tris") with or without an additional proteinase K digestion step ("2nd PK"). As a reference, extraction was also performed using the QIAamp® FFPE DNA Kit ("QA FFPE"), as well as the Promega Maxwell RSC DNA FFPE Kit and Maxwell RSC FFPE Plus DNA Kit, where proteinase K digestion took 1 hour.

[0022] [Figure 9] Figure 9 shows an electrophoresis gel of DNA extracted from FFPE tissue samples. The left photo shows DNA extracted from FFPE rat heart, while the right photo shows DNA extracted from FFPE rat lung. The average size of DNA extracted by the extraction method using diluted lysis composition is significantly smaller than that of the QIAamp® DNA FFPE Tissue reference protocol. ("GR std" = diluted lysis composition; "QA std" = reference lysis composition; L1-L3 = DNA ladder shown below).

[0023] [Figure 10] Cq values ​​of extracted DNA measured by quantitative real-time PCR using a large amplicon (727 bp). DNA was extracted using a diluted lysis composition ("Fragmented") and a reference protocol ("Standard") from FFPE rat heart tissue.

[0024] [Figure 11] Cq values ​​of extracted DNA measured by quantitative real-time PCR using a short amplicon (78 bp). DNA was extracted using a diluted lysis composition ("Fragmented") and a reference protocol ("Standard") from FFPE rat heart tissue.

[0025] [Figure 12] Figure 12 shows an electrophoresis gel of DNA extracted from FFPE tissue samples using a high Tris lysis composition ("GR-High Tris," "w / o") or a low Tris lysis composition ("GR-Low Tris," "GR std"), optionally containing additives such as spermidine, spermine, DTT, or glycine (L1-L3 = DNA ladder).

[0026] [Figure 13]Ct values ​​of bisulfite DNA measured by quantitative real-time PCR using a short amplicon (110 bp). Various amounts of DNA were applied: 5 ng (dark-shaded column) or 10 ng (light-shaded column). The uracil nucleobases of bisulfite DNA were removed in the enzymatic treatment by adding uracil-N-glycosylase (UNG). Thus, a higher Ct value indicates a higher UNG activity. 5 or 10 ng of DNA was measured for the lysis composition of Example 4 in a 5-minute UNG digestion step compared to a 60-minute UNG digestion step ("GR FFPE std"). An additional control was performed without UNG ("w / o UNG").

[0027] [Figure 14] Yields were determined by UV VIS and Qubit dsDNA BR measurements for human kidney and human breast samples (see Example 5). Means and standard deviations from two samples per condition are shown.

[0028] [Figure 15] qPCR performance was determined by adding the same volume, adjusted for the actual elution volume, to each reaction. The 66-bp and 500-bp human 18S rRNA genes were amplified from the eluate after extraction using either protocol option. Means and standard deviations from duplicate samples per condition are shown.

[0029] [Figure 16] Yields were determined for human heart samples by UV VIS and Qubit dsDNA BR measurements (see Example 5). Means and standard deviations from two samples per condition are shown.

[0030] [Figure 17]qPCR performance was determined by adding the same volume, adjusted for the actual elution volume, to each reaction. The 66-bp and 500-bp human 18S rRNA genes were amplified from the eluate after extraction using either protocol option. Means and standard deviations from duplicate samples per condition are shown. DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed Description The present invention provides improved methods for lysing fixed biological samples, where the fixed biological samples contain crosslinks between nucleic acid molecules and protein molecules resulting from fixation. Additionally, improved methods are described for obtaining purified nucleic acids, such as DNA and / or RNA, from the fixed biological samples.

[0032] The present invention provides improvements in the yield and / or quality of nucleic acids extracted from fixed biological samples, such as FFPE tissue, allowing for better analysis of the material using, for example, PCR and NGS sequencing methods. It has been particularly found that an additional round of proteolytic digestion after the decrosslinking step (performed after the first proteolytic digestion step) results in significant improvements.

[0033] Additionally, the present disclosure provides a method that allows for control of core parameters related to the analysis of fixed biological samples. In particular, it has been found that by adjusting the lysis conditions in the first step, it is possible to control the released nucleic acid and the size of the resulting nucleic acid. This important discovery makes it possible to optimize the method for either short-amplicon or long-amplicon PCR systems. Furthermore, adjusting the lysis conditions disclosed herein also improves the performance of enzymatic steps during extraction, thereby enabling the in-process use of uracil-n-glycosylase to remove artifacts caused by formalin cross-linking, for example.

[0034] As such, the various aspects and embodiments of the invention disclosed herein represent important contributions to the art.

[0035] Method according to the first aspect According to a first aspect of the present invention there is provided a method for lysing a fixed biological sample, wherein the fixed biological sample contains crosslinks between nucleic acid molecules and protein molecules due to fixation, the method comprising: (a) lysing the fixed biological sample, wherein the lysis comprises digestion with a proteolytic enzyme; (b) heating the dissolved sample to reverse the crosslinks; (c) adding a proteolytic enzyme and performing proteolytic digestion; Including, Optionally, methods are provided in which one or more additional processing steps are performed between steps (b) and (c).

[0036] The individual steps and preferred embodiments are now described in detail.

[0037] Step (a) The method according to the first aspect comprises, in step (a), lysing the fixed biological sample, wherein the lysis comprises digestion with a proteolytic enzyme. During the lysis step (a), the fixed biological sample is degraded so as to release nucleic acids.

[0038] Step (a) can be carried out using dissolution conditions and proteolytic digestion conditions known in the art. Supporting the dissolution of fixed biological samples by digestion with proteolytic enzymes is highly advantageous because it allows proteins and peptides cross-linked to nucleic acids to be degraded. As disclosed herein and known in the art, for example, when a cross-linking fixative is used, such cross-links in fixed biological samples are induced due to the fixation used. For example, the use of a cross-linking fixative such as an aldehyde-containing fixative, such as formaldehyde, results in cross-links between proteins and nucleic acids and between nucleic acids. By carrying out dissolution step (a), proteins cross-linked to nucleic acids can be degraded and cross-links between nucleic acids can be partially degraded.

[0039] According to one embodiment, the lysis step (a) comprises preparing a lysis mixture comprising (i) a fixed biological sample and (ii) a lysis composition comprising a protease. Any order of contacting the fixed biological sample with the components of the lysis composition is encompassed within this embodiment for preparing the lysis mixture in step (a). For example, the lysis composition may be prepared separately, and then the prepared lysis composition may be contacted with the fixed biological sample, or vice versa. Thus, the lysis composition may be first adjusted without affecting the fixed biological sample. Furthermore, it is within the scope of this embodiment to prepare a lysis mixture by contacting the fixed biological sample with a protease and / or additional components of the lysis composition in any order to prepare the lysis mixture.

[0040] Proteolytic enzymes aid in the digestion of fixed biological samples, improving the release of contained nucleic acids, such as DNA and / or RNA. In embodiments, the proteolytic enzyme is a protease, such as proteinase K. Proteases can be either endopeptidases, which cleave peptide bonds within proteins, and / or exopeptidases, which cleave amino acids from the ends of protein chains. Typically, proteases are further classified by mechanism, such as serine proteases (e.g., chymotrypsin, trypsin, elastase, subtilisin, and proteinase K); cysteine ​​(thiol) proteases (e.g., bromelain, papain, cathepsin, parasitic proteases, and bacterial virulence factors); aspartic proteases (e.g., pepsin, cathepsin, rerun, fungal and viral proteases); and metalloproteases (e.g., thermolysin). Such proteases can be used in the context of the present invention. In a central embodiment, the protease used in step (a) is a serine protease. According to one embodiment, the protease used in step (a) is a protease of the subtilisin family. A particularly suitable and preferred serine protease used in step (a) is proteinase K. Proteinase K is commonly used in the art for digestion of fixed biological samples containing cross-links due to the applied fixation. Proteinase K advantageously remains active even at higher temperatures and in the presence of denaturants such as detergents, urea, and chaotropic agents and salts. Proteinase K was also used in this example. All disclosures herein regarding protease or proteases generally apply specifically to and refer specifically to the preferred embodiment of proteinase K. As is clear from the listed examples, the protease may be a thermostable protease. This allows for lysis and digestion in step (a) to be assisted by heating the lysis mixture, as also described below.Examples of suitable thermostable proteases are proteinase K, trypsin, chymotrypsin, papain, pepsin, pronase, and endoproteinase Lys-C. Thermostable proteases can be inactivated at or above their inactivation temperature, which can range from 85° C. to 120° C. Suitable inactivation temperatures for various proteases are described in the art for various proteases.

[0041] According to one embodiment, step (a) comprises heating to support protease digestion. Thus, the prepared lysis mixture may be heated to a suitable elevated temperature to support lysis / digestion of the fixed biological sample. The lysis / digestion temperature is selected so that the proteases are active. In an embodiment, heating in step (a) is carried out at a temperature ranging from 35 to 75°C, e.g., 40 to 70°C or 45 to 65°C. As is known in the art, heating the lysis enhances protease activity, thereby enabling rapid and efficient lysis and digestion of the sample in step (a). According to one embodiment, protease digestion in step (a) comprises heating to a temperature of at least 30°C, particularly at least 35°C, at least 40°C, at least 45°C, and preferably at least 50°C.

[0042] According to one embodiment, the lysis mixture is incubated in step (a) for at least 15 minutes, for example, at least 20 minutes, at least 25 minutes, or at least 30 minutes. In an embodiment, the lysis mixture is incubated in step (a) for at least 45 minutes or at least 50 minutes. Advantageously, step (a) can be carried out within a short time frame. According to one embodiment, step (a) is completed in 120 minutes or less. Step (a) may be completed in 100 minutes or less, 90 minutes or less, or 70 minutes or less, for example, about 60 minutes. Incubation may be carried out at an elevated temperature as described above. Furthermore, the incubation, preferably assisted by heating as disclosed herein, may be assisted by stirring. Thus, the lysis mixture may be stirred during incubation in step (a). According to one embodiment, the lysis and digestion with the proteolytic enzyme in step (a) comprises stirring and heating the lysis mixture for 15 to 120 minutes, for example, 20 to 100 minutes, 30 to 90 minutes, or 45 to 75 minutes, at a temperature in the range of 35 to 75° C., for example, 40 to 70° C. or 45 to 65° C. Stirring may be performed by any method, for example, shaking, rotating, inverting, etc.

[0043] Those skilled in the art can select suitable concentrations of the protease in the lysis composition and lysis mixture; suitable concentrations are known in the art. According to one embodiment, the protease in step (a) is present in the lysis mixture and / or lysis composition at a concentration of at least 0.5 mg / mL, e.g., at least 1 mg / mL, at least 1.5 mg / mL, or at least 2 mg / mL. Preferably, the concentration is at least 2.5 mg / mL or at least 3 mg / mL. According to one embodiment, the protease used in step (a) is a serine protease, e.g., proteinase K, present in the lysis mixture and / or lysis composition at a concentration selected from 1 to 10 mg / mL, e.g., 1.5 to 7.5 mg / mL, 2 to 7 mg / mL, 3 to 6 mg / mL, or 3.5 to 5 mg / mL. Such concentrations can be used when the fixed biological sample is a solid fixed biological sample, e.g., a fixed tissue sample. Such concentrations can also be used in the lysis mixture when processing a fixed liquid biological sample. In such cases, the lysis composition is adapted to take into account any dilution effects caused by the fixed liquid biological sample. For example, dilution of the protease by the liquid fixed biological sample may be compensated for to provide a higher concentration of the protease in the lysis composition. The suitable concentration can be easily calculated by one skilled in the art, taking into account the suitable concentration of the protease in the lysis mixture in step (a).

[0044] As disclosed above, step (a) in a central embodiment comprises preparing a lysis mixture, the lysis mixture comprising (i) a fixed biological sample and (ii) a lysis composition comprising a proteolytic enzyme.

[0045] In embodiments, the lysis mixture contains the components of the lysis composition at concentrations similar to or equal to those of the lysis composition (e.g., allowing for deviations of up to 50%, e.g., up to 40%, up to 30%, or up to 20% or up to 10%). This is particularly true when providing a fixed biological sample that is a solid sample, such as a fixed tissue sample. For example, a solid, fixed biological sample does not dilute the compounds present in the lysis composition, i.e., the concentrations of the compounds present in the lysis composition are the same or approximately the same as those in the lysis mixture (including the solid, fixed biological sample). When the sample is a liquid, fixed biological sample, the liquid sample dilutes the compounds in the lysis composition; therefore, typically, higher concentrations of the compounds are used in the lysis composition to account for this dilution effect. For example, to establish suitable conditions for the lysis mixture in step (a), a higher concentration of the compound may be provided in the lysis composition and / or a higher volume ratio of the lysis composition may be provided.

[0046] According to a preferred embodiment, the lysis composition in step (a) has a pH in the range of 6.0 to 9.5, preferably 6.5 to 9.0 or 7.0 to 9.0. This pH range has been found to be particularly suitable for the lysis step (a) according to the methods of the present disclosure, as demonstrated in the Examples. In an embodiment, the pH is in the range of 7.0 to 8.0, for example. In a further embodiment, the pH is in the range of 8.0 to 9.0, for example, 8.2 to 8.8. In particular, the application of such alkaline pH in the lysis composition, and thus in the lysis mixture, resulted in less fragmentation compared to more acidic conditions.

[0047] According to a preferred embodiment, the dissolution composition in step (a) further comprises one or more, preferably all, of the following compounds: (i) salt; (ii) surfactants; (iii) Buffering agents.

[0048] In certain embodiments, the dissolution composition comprises salt, surfactant and buffering agent.Optionally, the dissolution composition further comprises chelating agent, for example, EDTA.Dissolution composition is found to be suitable for the method of the present disclosure, as demonstrated in Examples.The individual embodiments of the compound of dissolution composition are disclosed below.

[0049] According to a preferred embodiment, the dissolution composition comprises at least one reactive compound that can act as a formaldehyde scavenger.The reactive compound is particularly capable of reacting with the fixative and / or reacting with the crosslinking induced by the fixative.Preferably, one or two reactive compounds are included in the dissolution composition in step (a).However, the reactive compound may also be added separately to the dissolution mixture, for example, in the form of a solution or solid containing the reactive compound.

[0050] According to a preferred embodiment, the reactive compound reacts with the fixative or chemical moiety released in the heating step (b), and / or with the crosslinks induced by the fixative, such as an aldehyde-containing fixative, such as formaldehyde. The biological sample can be fixed with a fixative, such as an aldehyde-containing fixative, such as formaldehyde or a formaldehyde derivative, that induces crosslinks between nucleic acid molecules and protein molecules, or between protein molecules or between nucleic acid molecules. The reactive compound of the present invention can advantageously react with a fixative, such as formaldehyde. The fixative or chemical moiety derived therefrom that reacts with the reactive compound can be released during the heating step (b). The reactive compound reacts with the released fixative, which has the advantage that the released fixative is removed from the equilibrium state, which is highly favorable for decrosslinking (see Kawashima et al., 2014, Clinical Proteomics, 2014, Vol. 11(4), "Efficient extraction of proteins from formalin-fixed paraffin-embedded tissues requires higher concentration of tris(hydroxymethyl)aminomethane"). Thus, the reactive compound functions as a scavenger, capturing the fixative or chemical moieties derived therefrom that are released in the decrosslinking step (b). Alternatively or additionally, the reactive compound may react with fixative-induced crosslinks. In particular, the reactive compound may directly degrade fixative-induced crosslinks contained in the fixed biological sample, preferably in step (a) and / or step (b). The reaction of the reactive compound with fixative-induced crosslinks preferably occurs during step (b) of the method of the present disclosure. Additionally, reactive compounds can react with fixative-induced crosslinks through reversible crosslinking reactions. For example, aldehyde-containing fixatives, such as formaldehyde, can lead to the formation of aminal groups between two biomolecules, such as proteins and DNA.This aminal group can release the protonated amine group of one of the biomolecules to reversibly form an imine group.The imine group can then react with the reactive compound according to the present disclosure, thereby reacting with the crosslink induced by the fixative.Further conversion can then release the second biomolecule, resulting in the formation of an imine base in the reactive compound.Therefore, the crosslink induced by the fixative is reversed, and the fixative is bound to the reactive compound.This has the advantage that biomolecules, especially nucleic acids, are released.

[0051] According to a preferred embodiment, the reactive compound comprises a nucleophilic group, preferably an amine group. The reactive compound may be selected from the nucleophiles described in WO 2007 / 068764 A1. It has been found that reactive compounds comprising a nucleophilic group are particularly suitable for reacting with the fixative or chemical moiety released in heating step (b) and / or crosslinks induced by the fixative, in particular crosslinks induced by an aldehyde-containing fixative, such as formaldehyde.

[0052] According to a preferred embodiment, the reactive compound contains one or more primary amine groups, optionally one primary amine group and one or more hydroxyl groups, preferably three hydroxyl groups. The reactive compound further contains two primary amine groups and optionally one secondary amine group. As demonstrated in the examples, such reactive compounds have been found to be particularly suitable for the present invention. In particular, nucleic acid crosslinks were efficiently removed by the method of the present invention using such reactive compounds, resulting in high quality, which is particularly suitable for analytical methods such as PCR or NGS. Exemplary reactive compounds that can be advantageously used are 2-amino-2-(hydroxymethyl)propane-1,3-diol or a derivative thereof, spermidine or a derivative thereof, or a combination thereof. 2-amino-2-(hydroxymethyl)propane-1,3-diol may also be referred to as tris(hydroxymethyl)aminomethane or Tris.

[0053] According to a preferred embodiment, the reactive compound contains at least two nucleophilic groups, preferably nucleophilic groups with different nucleophilic strengths. Such an embodiment has been found to be advantageous in the examples, resulting in high-quality nucleic acids that are particularly suitable for analytical methods, such as PCR or NGS. The first nucleophilic group of the reactive compound may be stronger than the second nucleophilic group. In such a reactive compound, one or more types of nucleophilic groups may be present. For example, one or two first nucleophilic groups and one, two, or three second nucleophilic groups. According to one embodiment, the reactive compound contains a first nucleophilic group that is a primary amine group and a second nucleophilic group that is a hydroxyl group or a secondary amine group. According to a specific embodiment, the reactive compound contains a primary amine group and a hydroxyl group, preferably three hydroxyl groups. According to another embodiment, the reactive compound contains a primary amine group, preferably two primary amine groups and a secondary amine group. According to an exemplary embodiment, the reactive compound is selected from 2-amino-2-(hydroxymethyl)propane-1,3-diol or a derivative thereof or spermidine or a derivative.

[0054] According to one embodiment, the reactive compound comprises a polyamine, preferably a natural polyamine, such as spermidine. As demonstrated in the examples, such reactive compounds have been found to be advantageous for modifying the fragmentation of nucleic acids, in particular by increasing the size of the fragments.

[0055] A preferred reactive compound of the present disclosure is 2-amino-2-(hydroxymethyl)propane-1,3-diol, also known as Tris. Kawashima et al., 2014, described that Tris acts as a formaldehyde scavenger by generating Schiff bases, cyclic hemiaminals, and cyclic acetal adducts. Furthermore, as a type of transamination catalyst, Tris may directly participate in breaking crosslinks. Furthermore, Tris can form cyclic compounds with aldehyde-containing fixatives, such as formaldehyde, thus offering the advantage that one molecule of Tris captures one molecule of the aldehyde-containing fixative. As demonstrated in the Examples, 2-amino-2-(hydroxymethyl)propane-1,3-diol also enables nucleic acid fragmentation and modifies suitability for subsequent nucleic acid analysis methods. For example, the use of 2-amino-2-(hydroxymethyl)propane-1,3-diol resulted in high PCR and NGS performance.

[0056] According to one embodiment, the dissolution composition comprises two or more reactive compounds.The dissolution composition may comprise two reactive compounds, and where necessary, the reactive compounds are selected from (i) a reactive compound comprising one or more primary amine groups, preferably one primary amine group, and one, two or three hydroxyl groups, preferably three hydroxyl groups, such as 2-amino-2-(hydroxymethyl)propane-1,3-diol, and (ii) a reactive compound comprising two primary amine groups and optionally one secondary amine group, such as spermidine.

[0057] According to one embodiment, the lysis mixture comprises a reactive compound at a concentration suitable to react with at least a portion of the fixative, particularly during heating step (b). According to one embodiment, the lysis mixture comprises one or more reactive compounds at a concentration suitable to react with at least a portion of the fixative, particularly during heating step (b). In an embodiment, the portion of the fixative represents at least 15% of the fixative present in the fixed biological sample, particularly at least 25%, at least 35%, at least 45%, at least 55%, at least 65%, or at least 75% of the fixative present in the fixed biological sample.

[0058] According to one embodiment, the reactive compound is present in the lysis composition and / or lysis mixture at a concentration ranging from 1 mM to 500 mM or 5 to 500 mM in the lysis composition and optionally also in the lysis mixture.

[0059] According to one embodiment, the lysis mixture and / or lysis composition contains a reactive compound, such as spermidine, containing two primary amine groups and preferably one secondary amine group. The reactive compound (e.g., spermidine) may be present at a concentration of at least 0.5 mM, e.g., at least 1 mM, at least 1.5 mM, or at least 2 mM. The concentration may be selected from 0.25-25 mM, e.g., 0.5-20 mM, 1-15 mM, 1.25-10 mM, or 1.5-7 mM, e.g., about 2.5 mM. Such concentrations have been found to be particularly advantageous for sample lysis, resulting in highly fragmented nucleic acids. Furthermore, by varying the concentration, fragment size can be flexibly controlled. The lysis composition may also contain a buffering agent or a reactive compound that is also a buffering agent.

[0060] According to one embodiment, the lysis mixture and / or lysis composition contains a reactive compound containing one or more primary amine groups, preferably one primary amine group, and one, two, or three hydroxyl groups, preferably three hydroxyl groups, such as 2-amino-2-(hydroxymethyl)propane-1,3-diol. In an embodiment, this is present in the lysis mixture and / or lysis composition at a concentration of at least 3 mM, e.g., at least 5 mM, at least 7 mM, or at least 10 mM. Suitable concentrations may be selected from 3 mM to 100 mM, particularly 5 mM to 50 mM, 7 mM to 30 mM, 9 mM to 25 mM, or preferably 10 mM to 20 mM, e.g., 10 mM to 15 mM. Such concentrations are advantageous for sample lysis, which results in small fragment sizes and high-quality nucleic acids, particularly for nucleic acid analysis methods including small fragment amplification, e.g., short-amplicon PCR, as disclosed herein. Furthermore, high-quality nucleic acids result in enhanced NGS performance. Such applications are also disclosed in conjunction with the method according to the fifth aspect.

[0061] According to one embodiment, the dissolution mixture and / or dissolution composition contains a reactive compound containing one or more primary amine groups, preferably one primary amine group, and one, two, or three hydroxyl groups, preferably three hydroxyl groups, such as 2-amino-2-(hydroxymethyl)propane-1,3-diol. In an embodiment, this is present in the dissolution mixture and / or dissolution composition at a concentration of at least 10 mM, such as at least 20 mM, at least 40 mM, at least 60 mM, at least 75 mM, or at least 100 mM. The concentration may be selected from the range of 10 mM to 750 mM, such as 20 mM to 500 mM, 30 mM to 300 mM, 50 mM to 250 mM, or preferably 75 mM to 200 mM, such as about 100 mM to 150 mM. This concentration has been found to be advantageous for sample lysis, resulting in high-quality nucleic acids, particularly for nucleic acid analysis methods involving the amplification of large and / or small fragments, in the particular large amplicon PCR disclosed herein. This refers to the method according to the sixth aspect. Furthermore, the high quality of nucleic acids leads to enhanced NGS performance.

[0062] According to one embodiment, the reactive compound contained in the lysis composition is also a buffering agent. Such a compound advantageously fulfills both functions, in particular buffering the lysis mixture and providing a reactive compound that reacts with the fixative or chemical moiety released in heating step (b) and / or fixative-induced crosslinks, particularly those induced by aldehyde-containing fixatives such as formaldehyde. Such a compound may preferably contain one or more primary amine groups, preferably one primary amine group, and one, two, or three hydroxyl groups, preferably three hydroxyl groups (e.g., 2-amino-2-(hydroxymethyl)propane-1,3-diol). According to one embodiment, the lysis mixture and / or lysis composition contains a compound that is both a reactive compound and a buffering agent at a concentration selected from the range of 3 to 500 mM. Suitable concentration ranges are described above. As disclosed herein, selecting the concentration of the reactive compound allows for control of the fragment size of nucleic acid molecules released during lysis.

[0063] Optionally, the lysis composition comprises two reactive compounds.In this embodiment, preferably, the first reactive compound comprises two primary amine groups and preferably one secondary amine group, such as spermidine, and the second reactive compound comprises one or more primary amine groups, preferably one primary amine group, and one, two or three hydroxyl groups, preferably three hydroxyl groups, such as 2-amino-2-(hydroxymethyl)propane-1,3-diol.These embodiments are particularly advantageous for dissolving samples and obtaining nucleic acids with increased size or less fragmentation.

[0064] According to one embodiment, the lysis composition contains a reactive compound at a concentration that results in a concentration in the lysis mixture suitable for reacting with at least a portion of the fixative, particularly during heating step (b). According to one embodiment, the above-disclosed concentration of the reactive compound in the lysis mixture corresponds to the concentration of the reactive compound in the lysis composition, particularly when the fixed biological sample is a solid, fixed biological sample, such as a fixed tissue sample. According to one embodiment, the lysis composition contains a reactive compound at a concentration selected from the range of 0.25 to 500 mM, where the fixed biological sample is a solid, fixed biological sample, particularly a fixed tissue sample. As demonstrated in the Examples, such concentrations of reactive compounds, such as 2-amino-2-(hydroxymethyl)propane-1,3-diol and / or spermidine, have been found to be advantageous for lysis of fixed tissue samples, particularly by modifying the size / fragmentation of nucleic acids and obtaining high-quality nucleic acids that are particularly suitable for nucleic acid analysis methods, such as PCR and NGS.

[0065] When the fixed biological sample is a liquid fixed biological sample, the concentration of the reactive compound in the lysis composition is adjusted to establish the concentration disclosed above in the lysis mixture. Alternatively, or in addition, a higher volumetric ratio of the lysis composition may be added to the liquid fixed biological sample to establish the concentration of the reactive compound in the lysis mixture disclosed above. According to one embodiment, the lysis composition contains the reactive compound at a concentration suitable for being present in the lysis mixture at a concentration selected from the range of 0.25 to 500 mM, wherein the fixed biological sample is a liquid fixed biological sample. Corresponding adjustment of the reactive compound concentration is well within the capabilities of one skilled in the art.

[0066] According to one embodiment, the lysis composition and / or lysis mixture has a pH selected from the range of 6.0 to 9.5. The lysis composition further comprises a reactive compound, particularly one that reacts with the fixative and / or crosslinks induced by the fixative, wherein the reactive compound comprises a nucleophilic group, particularly a primary amine group. Exemplary reactive compounds are 2-amino-2-(hydroxymethyl)propane-1,3-diol or a derivative thereof and spermidine or a derivative thereof. According to one embodiment, the reactive compound is present in the lysis mixture and / or lysis composition at a concentration of 5 to 50 mM, preferably 10 to 20 mM, and the lysis composition has a pH of 6.0 to 9.5, preferably 7.0 to 8.0. According to another embodiment, the reactive compound is present in the lysis mixture and / or lysis composition at a concentration of 60 to 250 mM, preferably 75 to 200 mM, and the lysis composition has a pH of 7.0 to 9.5, preferably 8.0 to 9.0. These embodiments are particularly advantageous because they allow for improved release of nucleic acids, e.g., DNA, from fixed biological samples. By providing a neutral or slightly alkaline pH and such reactive compounds, the fixative of the fixed biological sample can be advantageously removed by reaction equilibrium by reacting with the reactive compounds, releasing nucleic acids that are less fragmented than in acidic conditions.

[0067] According to a preferred embodiment, the dissolution composition includes a salt. The salt assists dissolution. The salt is preferably provided to the dissolution mixture by the dissolution composition. The salt may preferably be a monovalent or divalent salt. Preferably, the salt is a chaotropic salt or a non-chaotropic salt. According to a preferred embodiment, the salt is a non-buffering salt. A mixture of salts may be used. The specific salt may be selected from alkali metal salts, optionally alkali metal halides. According to a preferred embodiment, the salt is a chloride salt, optionally selected from sodium chloride, potassium chloride, lithium chloride, and cesium chloride, where preferably, the salt is sodium chloride. As demonstrated by the examples, such salts have been found to be suitable.

[0068] The salt concentration in the lysis composition depends on the type of fixed biological sample from which nucleic acids are to be released and can be flexibly adjusted by those skilled in the art. According to a preferred embodiment, the salt is present in the lysis composition and / or lysis mixture at a concentration of at least 15 mM, particularly at least 30 mM, at least 50 mM, and preferably at least 100 mM. A suitable concentration range for the salt in the lysis composition and / or lysis mixture may be selected from 15-500 mM, particularly 30-440 mM, 50-300 mM, or preferably 100-250 mM, for example, about 150 mM. According to one embodiment, the salt is present in the lysis composition and / or lysis mixture at a concentration of less than 500 mM, particularly less than 400 mM, less than 300 mM, or preferably less than 250 mM, for example, less than 200 mM. As demonstrated in the examples for sample lysis, such salt concentrations have been found to be advantageous. For example, by applying such a salt concentration, digestion using a DNA glycosylase, such as uracil DNA glycosylase, preferably uracil-N-glycosylase, can be advantageously completed in 30 minutes or less, 20 minutes or less, 15 minutes or less, or 10 minutes or less. As disclosed above, when a solid fixed biological sample is provided, the salt concentration in the lysis mixture corresponds to or is approximately the same as the salt concentration in the lysis composition, while a liquid fixed biological sample dilutes the salt concentration, resulting in a lower concentration in the lysis mixture than in the lysis composition. Therefore, for a liquid fixed biological sample, the salt concentration can be adjusted to establish the above-disclosed concentration in the lysis mixture, particularly by providing a higher salt concentration in the lysis composition and / or providing a higher volume ratio of the lysis composition.

[0069] According to a preferred embodiment, the lysis composition comprises a surfactant, which aids in lysis of the sample and dissolves protein aggregates, and which is preferably provided to the lysis mixture by the lysis composition.

[0070] According to a preferred embodiment, the surfactant is an ionic or nonionic surfactant. Exemplary surfactants are known to those skilled in the art. According to one embodiment, the surfactant is an ionic surfactant, preferably an anionic surfactant. This is particularly suitable when the fixed biological sample is a solid fixed biological sample, such as a fixed tissue sample. The surfactant may be, in particular, a sulfate or sulfonate salt of a fatty alcohol, such as sodium dodecyl sulfate, sodium dodecyl sulfonate, or dodecylbenzenesulfonic acid, and preferably, the surfactant is sodium dodecyl sulfate (SDS). A mixture of surfactants may also be used.

[0071] According to preferred embodiments, the surfactant is present in the lysis composition and / or lysis mixture at a concentration of at least 0.01%, at least 0.02%, and preferably at least 0.03%. Suitable surfactant concentration ranges in the lysis composition and / or lysis mixture may be selected from 0.01-3.0%, 0.02-2.75%, preferably 0.03-2.5%, or 0.04-2.0%. In embodiments, the concentration is in the range of 0.03-1%. As disclosed above, when a solid fixed biological sample is provided, the surfactant concentration in the lysis mixture corresponds to the surfactant concentration in the lysis composition, whereas a liquid fixed biological sample dilutes the surfactant concentration, resulting in a lower surfactant concentration in the lysis mixture than in the lysis composition. Therefore, for liquid fixed biological samples, the surfactant concentration can be adjusted to achieve the above-disclosed concentration in the lysis mixture, particularly by providing a higher surfactant concentration in the lysis composition and / or by providing a higher volumetric ratio of the lysis composition.

[0072] According to one embodiment, the dissolution composition comprises a buffering agent. Preferably, the buffering agent is selected from the group consisting of 2-amino-2-(hydroxymethyl)propane-1,3-diol (also known as Tris), MOPS, HEPES, phosphate, and borate, preferably Tris. The buffering agent is preferably provided to the dissolution mixture by the dissolution composition. The buffering agent advantageously facilitates the maintenance of pH. As disclosed above, providing a suitable pH is particularly advantageous for the method according to the present disclosure, and suitable pH ranges are disclosed above. A mixture of buffering agents may also be used.

[0073] According to a preferred embodiment, the reactive compound is a buffering agent or the lysis composition or lysis mixture comprises a buffering agent, optionally with a pKa value in the range of 5.0 to 10.5, optionally selected from 5.5 to 10.0, 6.0 to 10.0, 6.5 to 10.0, 7.0 to 9.8 or 7.2 to 9.8.

[0074] According to one embodiment, the buffering agent is a reactive compound capable of reacting with the fixative and / or with crosslinks induced by the fixative, and optionally includes a nucleophilic group, such as a primary amine group. For example, the buffering agent may be 2-amino-2-(hydroxymethyl)propane-1,3-diol or a derivative thereof. In such an embodiment, the concentrations disclosed above for the reactive compound correspond to the concentrations of the buffering agent.

[0075] According to one embodiment, the lysis composition includes a chelating agent. The chelating agent can advantageously prevent nucleases from degrading target nucleic acids, such as DNA. According to a preferred embodiment, the lysis composition further includes a chelating agent, optionally an aminopolycarboxylic acid, preferably ethylenedinitrilotetraacetic acid (EDTA). According to one embodiment, the chelating agent is suitable for chelating divalent cations. Suitable chelating agents include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA), ethylenedinitrilotetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), and N,N-bis(carboxymethyl)glycine (NTA). According to a preferred embodiment, EDTA is used. As used herein, the term "EDTA" refers to the EDTA moiety of an EDTA compound, such as K2EDTA, K3EDTA, or Na2EDTA, among others. The use of a chelating agent such as EDTA advantageously inhibits nucleases, such as DNase and RNase.

[0076] The chelating agent may be used in the lysis mixture and / or lysis composition at a concentration of 0.05 mM to 5 mM, e.g., 0.075 mM to 2 mM, 0.1 mM to 1.5 mM. The suitable concentration of the chelating agent in the lysis composition may depend on the type of sample applied (solid or liquid fixed biological sample) and can be readily determined by one of skill in the art in view of the suitable concentrations disclosed above for the lysis mixture.

[0077] According to one embodiment, the lysed composition is prepared by adding a reactive compound, wherein the reactive compound is: reactive compounds containing a primary amine group and at least one hydroxyl group, preferably three hydroxyl groups, in particular 2-amino-2-(hydroxymethyl)propane-1,3-diol, and / or - reactive compounds containing at least one primary amine group, preferably two primary amine groups, and a secondary amine group, in particular spermidine is selected from.

[0078] This embodiment is advantageous for flexibly modifying the type and concentration of reactive compound independently of the additional compounds of lysis composition.As demonstrated in Examples, by adding at least one of the reactive compounds, the size / fragmentation of nucleic acid can be modified, for example, to optimize nucleic acid for nucleic acid analysis method.For example, the fragment size of nucleic acid is increased, and nucleic acid is highly suitable for the large nucleic acid amplification disclosed herein, for example, large amplicon PCR.

[0079] According to one embodiment, the dissolution composition is aqueous and contains: a reactive compound containing a primary amine, preferably selected from 2-amino-2-(hydroxymethyl)propane-1,3-diol and spermidine or a combination thereof, and a proteolytic enzyme which is a protease, preferably a serine protease, more preferably proteinase K; and optionally the dissolution composition comprises: a surfactant, preferably an anionic surfactant, more preferably the surfactant is sodium dodecyl sulfate; a salt, preferably a monovalent salt, more preferably the salt is sodium chloride; optionally a chelating agent, preferably an aminopolycarboxylic acid, more preferably EDTA Further includes:

[0080] According to one embodiment, the dissolution composition is aqueous and contains: (i) a reactive compound comprising a primary amine, preferably 2-amino-2-(hydroxymethyl)propane-1,3-diol, which is comprised in the lysis mixture and / or lysis composition at a concentration of (aa) at least 3 mM, in particular at least 5 mM, at least 7 mM, at least 9 mM or preferably at least 10 mM, for example 3 to 100 mM, in particular 5 to 50 mM, 7 to 30 mM, 9 to 25 mM or preferably 10 to 20 mM; or (bb) at least 10 mM, in particular at least 20 mM, at least 40 mM, at least 60 mM or preferably at least 75 mM, for example 10 to 1000 mM, in particular 20 to 500 mM, 40 to 300 mM, 60 to 250 mM or preferably 75 to 200 mM, for example about 100 mM to 150 mM; or (ii) a reactive compound comprising two primary amine groups and preferably one secondary amine group, which is contained in the lysis mixture and / or lysis composition at a concentration of at least 0.25 mM, in particular at least 0.5 mM, at least 1 mM, at least 1.25 mM or preferably at least 1.5 mM, for example 0.25-25 mM, in particular 0.5-12.5 mM, 1-10 mM, 1.25-7.5 mM or preferably 1.5-5 mM, for example about 2.5 mM; and (iii) a protease enzyme, optionally a serine protease, preferably proteinase K; Includes Optionally, the dissolution composition further comprises: (iv) a surfactant, preferably an anionic surfactant, more preferably, the surfactant is sodium dodecyl sulfate; (v) a salt, preferably a monovalent salt, more preferably the salt is sodium chloride; and / or, preferably, and (vi) a chelating agent, preferably an aminopolycarboxylic acid, more preferably EDTA Includes:

[0081] This combination of characteristics of the lysis composition has been found to be advantageous for sample lysis, as demonstrated in the Examples and disclosed herein.

[0082] According to one embodiment, the lysis composition contains a reactive compound, 2-amino-2-(hydroxymethyl)propane-1,3-diol, at a concentration of 5 to 50 mM, preferably 10 to 20 mM, in the lysis mixture and / or lysis composition, and the lysis composition has a pH in the range of 6.0 to 9.5, preferably 7.0 to 8.0. This embodiment has been found to be advantageous, as demonstrated in the Examples. In particular, high-quality nucleic acids can be obtained using this lysis composition, which is suitable for nucleic acid analysis methods involving the amplification of small nucleic acids. Thus, in one embodiment using the lysis composition of this embodiment, nucleic acids are analyzed by amplifying nucleic acids smaller than 500 nt, sometimes referred to as short-amplicon PCR. Details are disclosed elsewhere herein, and reference is made to each disclosure.

[0083] According to one embodiment, the lysis composition comprises a reactive compound, 2-amino-2-(hydroxymethyl)propane-1,3-diol, in a concentration of 60-250 mM, preferably 75-200 mM, in the lysis mixture and / or lysis composition. Optionally, the lysis composition has a pH in the range of 7.0-9.5, e.g., 8.0-9.0. This embodiment has also been found to be highly advantageous, as demonstrated in the Examples. In particular, high-quality nucleic acids can be obtained using this lysis composition, which is suitable for nucleic acid analysis methods involving the amplification of small and / or large nucleic acid molecules. Details of short and long amplicon PCR are disclosed elsewhere.

[0084] According to one embodiment, the lysis composition comprises a reactive compound, spermidine, in a concentration of 0.5 to 12.5 mM, preferably 1.5 to 5 mM, in the lysis mixture and / or lysis composition. The lysis composition further comprises 2-amino-2-(hydroxymethyl)propane-1,3-diol in a concentration of 5 to 50 mM, preferably 10 to 20 mM, in the lysis mixture and / or lysis composition. This embodiment has been found to be highly advantageous, as demonstrated in the Examples. In particular, high-quality nucleic acids, exhibiting larger sizes and less fragmentation, were obtained using this lysis composition.

[0085] According to one embodiment, the dissolution composition is prepared by combining a dissolution solution with a protease. If necessary, as disclosed herein, additional compounds, particularly additional reactive compounds, are added to prepare the dissolution composition. Furthermore, preparing the dissolution composition may further include adding water.

[0086] According to one embodiment, the protease combined with the lysis solution in step (a) is provided by a solution containing the protease. Suitable concentrations are disclosed elsewhere. The protease may be included in the lysis solution.

[0087] As disclosed herein, the lysis composition may be prepared by combining the lysis solution with a proteolytic enzyme, and optionally water or a dilution buffer.

[0088] According to one embodiment, the lysis solution comprises a salt, a surfactant, and a buffering agent. Furthermore, the lysis solution particularly comprises a reactive compound. Details of said compounds have already been disclosed above and are referred to in the reactivity disclosure. Suitable concentration ranges of the reactive compound, surfactant, and salt are disclosed herein for the lysis mixture and lysis composition. The lysis solution comprises the reactive compound, surfactant, and salt at concentrations suitable to establish the concentrations disclosed herein for the lysis mixture and / or lysis composition.

[0089] According to a preferred embodiment, the dissolution solution comprises a reactive compound. The reactive compound is disclosed and referred to herein. According to a specific embodiment, the dissolution solution comprises a reactive compound comprising one or more primary amine groups, optionally one primary amine group and one or more hydroxyl groups, preferably three hydroxyl groups. According to a specific embodiment, the dissolution solution comprises 2-amino-2-(hydroxymethyl)propane-1,3-diol or a derivative thereof.

[0090] According to certain embodiments, the reactive compound is present in the lysis solution at a concentration of at least 5 mM, particularly at least 10 mM, at least 20 mM, or at least 30 mM. The lysis solution may contain 5 to 500 mM of the reactive compound, particularly 10 to 250 mM, 20 to 100 mM, or 30 to 75 mM, for example, about 53 mM. Particularly suitable reactive compounds having these concentrations in the lysis solution are reactive compounds containing one or more primary amine groups, optionally one primary amine group and one or more hydroxyl groups, preferably three hydroxyl groups, such as 2-amino-2-(hydroxymethyl)propane-1,3-diol or a derivative thereof. According to a preferred embodiment, the reactive compound is a buffering agent.

[0091] According to one embodiment, the reactive compound contains two primary amine groups and preferably one secondary amine group, such as spermidine; optionally, the reactive compound is present in the lysis solution sufficient to establish a concentration of at least 0.25 mM, particularly at least 0.5 mM, at least 1 mM, at least 1.25 mM, or preferably at least 1.5 mM in the lysis composition and / or lysis mixture. According to one embodiment, the lysis solution contains a reactive compound disclosed herein, particularly a reactive compound that is also a buffering agent; optionally, the reactive compound contains one or more primary amine groups, preferably one primary amine group, and one, two, or three hydroxyl groups, preferably three hydroxyl groups, such as 2-amino-2-(hydroxymethyl)propane-1,3-diol; optionally, the reactive compound is present in the lysis solution at a concentration of at least 5 mM, particularly at least 10 mM, at least 20 mM, or at least 30 mM. Alternatively, the lysis solution contains a buffering agent at the above concentrations, but the buffering agent is not the reactive compound. An additional reactive compound containing two primary amine groups and preferably one secondary amine group may be added to the lysis composition or included in the lysis solution, whereby the reactive compound is present in sufficient lysis solution to establish a concentration of at least 0.25 mM, particularly at least 0.5 mM, at least 1 mM, at least 1.25 mM, or preferably at least 1.5 mM in the lysis composition and / or lysis mixture. Reactive compounds containing two primary amine groups and preferably one secondary amine group at the concentrations disclosed above have been found to be advantageous for lysing fixed biological samples and obtaining nucleic acids with large fragment sizes, as demonstrated in the examples.

[0092] According to a preferred embodiment, the lysis solution contains a salt, particularly a salt disclosed herein. In particular, the salt may be a chloride salt, such as sodium chloride, potassium chloride, lithium chloride, or cesium chloride, preferably sodium chloride. According to one embodiment, the salt is present in the lysis solution at a concentration of at least 50 mM, for example, at least 75 mM, at least 100 mM, at least 200 mM, at least 300 mM, or at least 500 mM. According to one embodiment, the lysis solution contains 75 to 2000 mM of salt, particularly 100 to 1500 mM, 200 to 1200 mM, 300 to 1000 mM, or 400 to 800 mM, for example, about 500 to 700 mM. The advantages associated with the salt have been disclosed above, and are referred to here.

[0093] According to a preferred embodiment, the lysis solution contains a surfactant, particularly a surfactant disclosed herein. In particular, the surfactant may be selected from nonionic or ionic surfactants, preferably anionic surfactants. In particular, the surfactant may be an anionic surfactant, such as a sulfate or sulfonate salt of a fatty alcohol, such as sodium dodecyl sulfate, sodium dodecyl sulfonate, or dodecylbenzenesulfonic acid, preferably sodium dodecyl sulfate. According to one embodiment, the surfactant has a concentration of at least 0.01%, particularly at least 0.03%, at least 0.05%, at least 0.07%, or at least 0.1%, in the lysis solution. According to one embodiment, the lysis solution contains 0.01-3% surfactant, particularly 0.03-2.5%, 0.05-2%, 0.07-1%, or 0.1-0.5% surfactant. The advantages associated with the surfactant have been disclosed above, and are further referenced here.

[0094] According to certain embodiments, the lysis solution comprises: - a concentration of at least 5 mM, in particular at least 10 mM, at least 20 mM or at least 30 mM, for example 5 to 500 mM, in particular 10 to 250 mM, 20 to 100 mM or 30 to 75 mM, for example about 53 mM of a reactive compound, in particular a reactive compound comprising one or more primary amine groups, optionally one primary amine group and one or more hydroxyl groups, preferably three hydroxyl groups, such as 2-amino-2-(hydroxymethyl)propane-1,3-diol or a derivative thereof; a salt at a concentration of at least 50 mM, optionally at least 75 mM, at least 100 mM, at least 200 mM, at least 300 mM or at least 400 mM, for example 75 to 2000 mM, in particular 100 to 1500 mM, 200 to 1200 mM, 300 to 1000 mM or 400 to 800 mM, for example about 600 mM, optionally a chloride salt, such as sodium chloride, potassium chloride, lithium chloride and caesium chloride, preferably sodium chloride; and - a surfactant, optionally an anionic surfactant, such as a sulfate or sulfonate of a fatty alcohol, for example sodium dodecyl sulfate, sodium dodecyl sulfonate or dodecylbenzenesulfonic acid, in a concentration of at least 0.01%, in particular at least 0.03%, at least 0.05%, at least 0.07% or at least 0.1%, for example 0.01-4%, in particular 0.03-3%, 0.05-2%, 0.07-1% or at least 0.1-0.5%, for example 0.2%, in the dissolution solution, preferably sodium dodecyl sulfate Includes.

[0095] Such an embodiment is advantageous for the lysis of fixed biological samples, for which corresponding lysis solutions are applied, as demonstrated in the examples.

[0096] Optionally, the lysis solution further comprises a chelating agent. According to one embodiment, the lysis solution further comprises a chelating agent, particularly EDTA, with the chelating agent having a concentration of at least 0.25 mM, particularly at least 0.5 mM, at least 1 mM, or at least 1.5 mM in the lysis solution. According to one embodiment, the chelating agent has a concentration of 0.25-25 mM, particularly 0.5-15 mM, 1-10 mM, or 1.5-5 mM, for example, 2.7 mM in the lysis solution. The chelating agent advantageously assists in the inactivation of nucleases disclosed herein, such as DNases.

[0097] Step (b) Step (b) involves heating the dissolved sample to reverse the cross-linking.

[0098] The heating step (b) is advantageous because it allows the reversal of fixative-induced crosslinks, such as formaldehyde-induced crosslinks. These crosslinks are typically present between proteins, between proteins and nucleic acids, and between nucleic acids. The reversal of crosslinks is temperature-dependent, with higher temperatures resulting in faster reversal (Kennedy-Darling et al., Anal. Chem., 2014, Vol. 86 (12), pp: 5678-5681, "Measuring the Formaldehyde Protein-DNA Cross-Link Reversal A temperature of at least 80°C is particularly suitable for reversing cross-links induced by many fixatives, such as formaldehyde. Furthermore, it is described in the art that heating after the proteolytic digestion step can remove chemical modifications of nucleic acids remaining after the proteolytic digestion in step (a). For example, it is described that methylol groups of nucleic acid bases that may be present after the proteolytic digestion step can be removed by increasing the temperature (see, for example, Masuda et al., Nucleic Acid Research, 1999, Vol. 27 (22), pp: 4436-4443; "Analysis of (See “Chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples”).

[0099] The heating step (b) can also be used to inactivate proteolytic enzymes present in the lysis mixture. Inactivating proteolytic enzymes in the heating step (b) can be advantageous when it is desirable to perform one or more enzymatic treatments different from the proteolytic enzyme digestion step between steps (b) and (c). In one embodiment, the heating step (b) inactivates the proteolytic enzymes present in the lysis mixture. Suitable inactivation conditions, such as minimum inactivation temperatures and incubation times, are described in the art for various proteases and are therefore readily available to those skilled in the art.

[0100] Suitable incubation temperatures and times for reversing fixative-induced crosslinks are also described in the art. According to one embodiment, step (b) comprises heating the lysed sample to a temperature of at least 80°C, e.g., at least 85°C or at least 90°C. According to one embodiment, step (b) comprises heating the lysed sample for at least 15 minutes, at least 20 minutes, or at least 25 minutes to reverse the crosslinks. In a preferred embodiment, step (b) comprises heating the lysed sample for at least 30 minutes, at least 45 minutes, or at least 50 minutes. According to one embodiment, step (b) comprises heating the lysed sample at a temperature suitable for reversing fixative-induced crosslinks for up to 120 minutes, optionally up to 100 minutes. In an embodiment, step (b) comprises heating for up to 80 minutes or up to 70 minutes.

[0101] According to a preferred embodiment, the dissolved sample is heated at a temperature in the range of 80 to 120°C, for example, 80 to 110°C or 85 to 100°C, for 30 to 120 minutes, for example, 45 to 90 minutes or 50 to 70 minutes. Thus, step (b) may comprise heating the dissolved sample at a temperature in the range of 80 to 110°C, for example, 85 to 100°C, for 30 to 120 minutes. Step (b) may further comprise heating the dissolved sample at a temperature in the range of 80 to 110°C, for example, 85 to 100°C, for 45 to 90 minutes. Step (b) may further comprise heating the dissolved sample at a temperature in the range of 80 to 110°C, for example, 85 to 100°C, for 50 to 70 minutes.

[0102] In embodiments, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% of the fixative-induced crosslinks initially present in the fixed biological sample are reversed upon completion of step (b).

[0103] As disclosed herein, one or more additional processing steps may be performed between steps as needed, and in a preferred embodiment are performed between steps (b) and (c).

[0104] Step (c) Step (c) comprises adding a proteolytic enzyme and carrying out proteolytic digestion. Thus, the method according to the first aspect comprises carrying out at least one additional proteolytic digestion step after the decrosslinking step (b). Thus, the method comprises first lysing the fixed biological sample, where the lysis comprises digestion with a proteolytic enzyme (step (a)), followed by heating the lysed sample to reverse crosslinks (step (b)), followed by adding a proteolytic enzyme and carrying out proteolytic digestion (step (c)). This sequential order of steps has been found to be particularly effective in lysing fixed biological samples to release high-quality nucleic acids in good yield. As disclosed herein, one or more additional processing steps may be performed between steps (b) and (c) as needed.

[0105] As demonstrated in the examples, performing step (c) significantly improves the release of nucleic acids during the entire lysis procedure. The released nucleic acids, such as DNA and (or RNA), are of high quality and, after purification, are particularly suitable for amplification reactions. Surprisingly, performing additional proteolytic digestion in step (c) improved nucleic acid yield, as demonstrated in the examples based on DNA. Without wishing to be bound by theory, it is hypothesized that crosslinks generated in fixed biological samples (e.g., by formaldehyde or other aldehyde-based fixatives) may cause particularly persistent protein associations or steric effects with nucleic acids, which can shield proteins from the proteolytic digestion performed in step (a). After the heating step (b), which reverses the crosslinks, these associations are sufficiently weakened that the additional proteolytic digestion performed in step (c) can efficiently remove them. As shown in the examples, performing step (c) surprisingly increases the yield and further enables the release and thus the obtainment of nucleic acids, such as DNA, with longer average sizes. This improvement in quality is also beneficial for subsequent analysis of the released nucleic acids, as it results in better PCR results, especially for large amplicons (500 bp). The results of the examples show that when an additional proteolytic digestion step (c) is performed after the decrosslinking step (b), larger amounts of previously inaccessible long DNA strands become accessible to subsequent PCR reactions. These improvements in DNA yield and accessibility, demonstrated in the examples, also result in improved performance of the released DNA in downstream analytical processes, such as amplification and sequencing. The efficient removal of crosslinks and proteins achieved by the method according to the first aspect, through the sequential implementation of steps (a), (b), and (c), is highly advantageous and also results in improved NGS performance.

[0106] Thus, carrying out step (c) as taught by the method according to the first aspect leads to significant and surprising improvements in the quality and yield of released nucleic acids, such as DNA in particular.

[0107] The protease used in step (c) is preferably a protease, such as proteinase K. Suitable proteases that can be used in step (c) have already been disclosed in connection with step (a), and reference is made to the respective disclosures that also apply here. According to one embodiment, step (c) comprises adding a protease suitable for degrading proteins and / or peptides associated with nucleic acids and / or degrading crosslinks induced by fixatives, particularly crosslinks between nucleic acids and proteins or peptides. According to one embodiment, the added protease is a protease. Suitable proteases are disclosed herein for step (a), and the same proteases may be used in step (c). As mentioned above, preferably a serine protease, particularly proteinase K, is added in step (c).

[0108] The proteolytic digestion in step (c) of the disclosed method is preferably carried out at a temperature suitable for the proteolytic enzyme to degrade proteins and peptides. Preferably, step (c) includes heating the dissolved sample to support proteolytic digestion. In embodiments, the heating in step (c) is carried out at a temperature within a range of 35-75°C, e.g., 40-70°C or 50-70°C. In embodiments, the temperature is within a range of 55-68°C, e.g., 60°C or 65°C. As is known in the art, the heating temperature of the heating device used may be set to a higher temperature, which reaches a preferred incubation temperature in the proteolytic reaction mixture during the heating process (gradient), thereby supporting proteolytic digestion before the final higher temperature is reached in the proteolytic reaction mixture. Such a higher temperature may then inactivate the proteolytic enzyme, as proteolytic digestion is completed during the heating process before reaching this inactivation temperature.

[0109] To enable efficient proteolytic digestion in step (c), the lysed sample may be incubated in the presence of a proteolytic enzyme. The incubation is preferably carried out for at least 5 minutes, e.g., at least 10 minutes or at least 15 minutes. As disclosed herein, the incubation for proteolytic digestion may be carried out at an elevated temperature between 35 and 75°C. The sample may be agitated during incubation. According to one embodiment, step (c) comprises heating to a temperature of at least 35°C, e.g., at least 40°C, at least 45°C, at least 50°C, or at least 55°C, for at least 5 minutes. Agitation may be carried out by any method, e.g., shaking, rotating, inverting, etc.

[0110] According to one embodiment, step (c) comprises heating the lysis composition to a temperature selected from the range of 45°C to 75°C, e.g., 50°C to 75°C or 55°C to 70°C, for 5 to 60 minutes, e.g., 10 to 45 minutes, 10 to 30 minutes, or 10 to 25 minutes. According to one embodiment, step (c) is completed in 30 minutes or less, optionally 20 minutes or less. As demonstrated by the examples, short incubation times, such as 15 minutes (e.g., at 65°C), are feasible for step (c), allowing for significant improvements in the yield and quality of nucleic acids contained in the fixed biological sample. Furthermore, such short incubation times allow for rapid completion of step (c), collectively demonstrating that the method of the first aspect is not only highly efficient but also rapid.

[0111] According to one embodiment, the incubation period for proteolytic digestion in step (c), and therefore for carrying out step (c), is shorter than the incubation period in step (a), and therefore for carrying out step (a). Furthermore, the temperature used in step (c) to support proteolytic digestion is higher in step (c) than in step (a).

[0112] The amount of protease to be added and the concentration of the protease in the proteolysis reaction mixture in step (c) can be determined and selected by those skilled in the art. In one embodiment, the concentration of the protease in the proteolysis reaction mixture is at least 0.5 mg / mL, preferably at least 1 mg / mL. Suitable concentration ranges include, but are not limited to, 0.5 to 10 mg / mL, 0.75 to 7.5 mg / mL, 1 to 5 mg / mL, or 1 to 3 mg / mL. Such concentrations are applied in the Examples. According to one embodiment, the protease is added in step (c) in the form of a solution. The solution may contain the protease at a concentration from 1 mg / mL to its solubility limit, for example, 5 to 40 mg / mL, 7.5 to 35 mg / mL, or 10 to 30 mg / mL, for example, 20 mg / mL.

[0113] The examples show that step (c) advantageously aids in the lysis of fixed biological samples, in particular nucleic acids obtained in higher yields and of higher quality, which are highly suitable for nucleic acid analysis methods such as PCR or NGS as disclosed herein.

[0114] Additional processing steps, if required, between steps (b) and (c) If necessary, one or more additional processing steps may be performed between steps (b) and (c). Additional processing steps may be performed to further improve the method, for example, to remove undesired molecules. Non-limiting examples include removing RNA (e.g., by performing RNase digestion) if DNA is the target nucleic acid of interest, or removing DNA (e.g., by performing DNase digestion) if RNA is the target nucleic acid of interest. Furthermore, lipase treatment may be performed. This may be advantageous, for example, when a fatty biological sample is being processed. Furthermore, processing steps may be performed to remove artifacts present due to fixation of the biological sample, such as uracil nucleobases. This allows for the provision of nucleic acids, such as DNA, that are particularly suitable for amplification and sequencing.

[0115] According to one embodiment, the method according to the first aspect comprises carrying out at least one enzymatic treatment step different from the proteolytic digestion step between steps (b) and (c), According to one embodiment, the at least one enzymatic treatment step comprises the use of one or more of a glycosylase, a nuclease, a lipase or a combination of the foregoing.

[0116] According to one embodiment, at least one enzyme treatment step includes the use of a DNA glycosylase, such as uracil DNA glycosylase. If such a step is performed, uracil-N-glycosylase treatment is preferably performed between steps (b) and (c). According to one embodiment, the method includes performing the enzyme treatment step by adding a glycosylase to the lysed sample and heating. The use of a glycosylate, such as uracil glycosylase, has important advantages. For example, fixed biological samples, such as formalin-fixed, paraffin-embedded (FFPE) tissue samples, can have irreproducible sequence artifacts in DNA. In particular, the C:G>T:A base substitution has been reported as a major type of sequence artifact in DNA recovered from fixed biological samples. This is based on the deamination of cytosine to uracil and subsequent PCR amplification, resulting in a C:G>T:A base substitution (see, for example, Do et al., Oncotarget, 2012, Vol. 3 (5): pp. 546-558, "Dramatic reduction of sequence artifacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase"). This can be advantageously avoided by treating the dissolved sample with a DNA glycosylase, particularly uracil-DNA glycosylase, after step (b) and before step (c). The enzyme removes the pseudouracil, resulting in the formation of an abasic site, which can then induce strand breaks and / or block DNA polymerase in the subsequent polymerization step. As a result, the potential artifact (C:G>T:A base substitution) is eliminated. This allows for improved quality of the released DNA for subsequent sequencing applications. According to one embodiment, the glycosylase treatment is carried out at an elevated temperature that supports glycosylase activity, for example, a temperature in the range of 45-55°C, such as 50°C, is suitable.In embodiments, the glycosylase treatment step is completed in 30 minutes or less, 20 minutes or less, 15 minutes or less, or 10 minutes or less, and optionally the glycosylase is uracil-N-glycosylase.

[0117] According to one embodiment, when an enzymatic treatment step is performed, the sample comprises a salt concentration suitable for performing an enzymatic treatment step, in particular an enzymatic treatment step comprising a glycosylase, preferably a DNA glycosylase, more preferably a uracil DNA glycosylase, e.g., uracil-N-glycosylase; and / or a nuclease, preferably a ribonuclease, more preferably ribonuclease A.

[0118] According to one embodiment, the method includes a step of diluting the dissolved and decrosslinked sample before performing at least one enzymatic treatment step. The sample can be diluted, for example, by adding water or another diluting solution. Dilution can be advantageous to adjust conditions suitable for performing at least one enzymatic treatment between steps (b) and (c). Dilution can be advantageous, for example, to establish a salt concentration suitable for performing the desired enzymatic treatment step. In one embodiment, the salt concentration in the enzymatic treatment mixture generated for performing the enzymatic treatment step is 500 mM or less, for example, 300 mM or less or 250 mM or less. In embodiments, the salt concentration is 200 mM or less, 150 mM or less, or 100 mM or less. Adjusting the salt concentration to a sufficiently low level, as necessary, by diluting the dissolved and crosslinked sample, ensures that the enzymatic treatment is not negatively affected by the salt. The suitable salt concentration varies depending on the intended enzymatic treatment and can be determined by one skilled in the art. For example, providing a low salt concentration in the enzyme treatment mixture, e.g., less than 150 mM or less than 100 mM, has been found to be particularly suitable for enzyme treatment using uracil DNA glycosylase. In particular, these low-salt conditions have been found to enable a reduction in treatment time. For example, treatment time can be reduced to less than 30 minutes, e.g., less than 25 minutes, less than 20 minutes, and preferably less than 15 minutes or less than 10 minutes. Suitable times include 2-25 minutes, 2-20 minutes, 3-15 minutes, and 5-10 minutes. As demonstrated by the examples, digestion with uracil-N-glycosylase could be completed within 5 minutes. This is advantageous because the method can be performed more quickly, allowing for higher sample throughput.

[0119] According to one embodiment, at least one nuclease treatment step is carried out between step (b) and step (c), optionally the nuclease is a ribonuclease, such as ribonuclease A. The use of a ribonuclease allows for the degradation of RNA, thereby providing released DNA that is free from RNA contamination.

[0120] According to one embodiment, at least one, and preferably both, of the following enzymatic treatment steps are performed: - adding a glycosylase, preferably a DNA glycosylase, more preferably a uracil DNA glycosylase, to the sample; and / or - adding a nuclease to the sample, optionally wherein the nuclease is a ribonuclease, such as ribonuclease A.

[0121] Method according to the second aspect According to a second aspect of the present invention, there is provided a method for obtaining purified nucleic acids from a fixed biological sample, comprising lysing the fixed biological sample according to steps (a) to (c) of the lysis method of the first aspect, and further comprising, after step (c) of the method of the first aspect: (d) purifying nucleic acids from the lysed sample. A method is provided, comprising:

[0122] As disclosed herein, according to certain embodiments, the method according to the first aspect comprises, between steps (b) and (c), one or more further processing steps, such as a nuclease digestion step and / or treatment with uracil-N-glycosylase.

[0123] The method according to the second aspect advantageously provides purified nucleic acids from fixed biological samples, where the nucleic acids are of high yield and quality. The high quality and yield of purified nucleic acids are advantageous because they improve subsequent analysis of the isolated nucleic acids, such as amplification and / or sequencing of the purified nucleic acids. Sequencing may be performed by next-generation sequencing. As demonstrated by the examples, the method according to the second aspect provides pure nucleic acids that are particularly suitable for next-generation sequencing applications. As disclosed herein, the method can be adjusted to control the size of the purified nucleic acids. This can be done as disclosed herein by selecting the lysis buffer used in step (a). This allows the degree of fragmentation to be adjusted and controlled. This allows for the provision of nucleic acids optimized for either short or long PCR systems. The nucleic acids can be DNA or RNA, and in embodiments, the nucleic acids are DNA molecules.

[0124] The features of steps (a), (b) and (c), and one or more optional processing steps between steps (b) and (c), are disclosed above in conjunction with the method according to the first aspect, with reference to the respective disclosures also applying to the method according to the second aspect.

[0125] In one embodiment, at least one intermediate processing step is performed between steps (c) and (d). In another embodiment, no further processing step is performed between steps (c) and (d).

[0126] Step (d) The method according to the second aspect comprises a step (d) of purifying nucleic acids from the lysed sample. Since the lysis method according to the first aspect makes the nucleic acids accessible in the lysis mixture, any suitable purification method can be used in step (d). Suitable techniques for purifying nucleic acids from lysed samples are known in the art and therefore do not require any detailed description. Commercially available purification kits may be used in step (d).

[0127] According to a preferred embodiment, step (d) comprises: - binding nucleic acids contained in the lysed sample to a solid phase; - optionally washing the nucleic acid bound to the solid phase; - eluting the nucleic acid from the solid phase; Includes:

[0128] According to one embodiment, step (d) comprises contacting the dissolved sample containing the released nucleic acids (obtained after step (c)) with a binding composition (e.g., a binding buffer) to establish binding conditions for binding the target nucleic acids to the solid phase. The resulting mixture establishing suitable binding conditions is also referred to herein as a binding mixture. According to one embodiment, the binding composition comprises a chaotropic agent, such as a chaotropic salt. Purification methods using chaotropic agents / salts to promote binding of target nucleic acids to the solid phase are well known in the art and therefore need not be described in detail. Chaotropic salts include, but are not limited to, salts including guanidinium, iodide, perchlorate, and thiocyanate, and the chaotropic salt may be selected from guanidinium hydrochloride, guanidinium thiocyanate (GTC), guanidinium isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, and sodium trifluoroacetate. The binding composition and / or binding mixture may further comprise urea and / or a non-chaotropic salt. The binding composition and / or binding mixture may further comprise a surfactant (e.g., an ionic or non-ionic surfactant); and, optionally, an aliphatic alcohol, such as an alkanol preferably containing 1 to 5 or 2 to 3 carbon atoms. Ethanol or isopropanol is commonly used as an alcohol for nucleic acid purification to promote binding to the solid phase. The mixture of nucleic acid and binding composition may then be applied to the solid phase, or the solid phase may be added to the mixture. The solid phase may have a silica surface. The solid phase may comprise unmodified silicon containing a surface to which the target nucleic acid binds. The term "silica surface," as used herein, includes surfaces comprising or consisting of silicon dioxide and / or other silicon oxides, diatomaceous earth, glass, silica gel, zeolite, bentonite, alkyl silica, aluminum silicate, and borosilicate.Exemplary solid phases that can be used in conjunction with the present invention include solid phases comprising an unmodified silica surface, including, but not limited to, silica particles, silica fibers, glass materials such as glass powder, glass fibers, glass particles, or controlled pore glass, silicon dioxide, glass or silica in particle form such as powder, beads, or frits. According to the present disclosure, the use of column-based solid phases or the use of particles, particularly magnetic particles, is preferred. According to one embodiment, the solid phase is contained within a column. The column preferably comprises a solid phase with a surface containing unmodified silicon, which is used for nucleic acid binding, particularly DNA binding.

[0129] Other nucleic acid purification techniques that can be used in step (d) include binding of the target nucleic acid to a solid phase having an anion exchange surface. Again, the solid phase may be provided in a column or bead format. Such methods are known in the art and therefore need not be described in detail.

[0130] The solid phase with the bound nucleic acids may be separated from the remaining sample, and the bound nucleic acids may be washed. The nucleic acids may also be eluted. Elution solutions are well known to those of skill in the art and need not be further defined here.

[0131] Other nucleic acid purification methods may be used in step (d), such as, for example, precipitation-based purification methods. Such methods are known in the art and may involve the use of alcohol.

[0132] The target nucleic acid obtained by the method according to the second aspect of the present disclosure can then be further processed, for example, used in the nucleic acid analysis method disclosed herein.As disclosed herein, the method according to the second aspect, based on the lysis method according to the first aspect, improves the yield and quality of the target nucleic acid extracted from fixed biological samples such as FFPE tissue.As shown by the examples, the provided purified nucleic acid is more suitable for subsequent PCR and NGS analysis compared to prior art methods.

[0133] The target nucleic acid may be DNA and / or RNA. In one embodiment, the bound target nucleic acid comprises or consists essentially of DNA. As disclosed herein, the method according to the second aspect provides:

[0134] Further embodiments of the method according to the first and second aspects nucleic acid The "nucleic acid(s)" may be DNA and / or RNA. Thus, DNA and RNA may be released from a fixed biological sample in the method according to the first aspect, or may be purified in the method according to the second aspect. Furthermore, the nucleic acid may be DNA or RNA. As disclosed herein, primarily DNA (or RNA) may be obtained by either performing a nuclease treatment (e.g., during the method according to the first aspect) to destroy non-target nucleic acids and / or selective target nucleic acid purification, which may be performed in step (d) of the method according to the second aspect. This makes it possible to obtain a target nucleic acid (e.g., DNA) that is free of or contains only small amounts of non-target (e.g., RNA) contaminants.

[0135] According to a preferred embodiment, the nucleic acid comprises or consists essentially of DNA, which can be very efficiently released from fixed biological samples and subsequently purified, as demonstrated in the examples.

[0136] Fixed biological specimens The term "fixed biological sample" specifically refers to any biological material preserved using a fixative. Fixed biological samples contain crosslinks between nucleic acid molecules and protein molecules due to fixation. The fixative used is a crosslinking fixative. Such fixed biological samples include, but are not limited to, tissues or organs fixed with formaldehyde, tissue samples preserved in liquid cytological preservation media, and samples containing fixed cells (such as cervical or gynecological swabs or cell-containing body fluids) preserved in liquid cytological preservation materials.

[0137] In one embodiment, the cross-linking fixative used to fix the biological sample is an aldehyde-containing fixative, such as formaldehyde and / or paraformaldehyde. Cross-linking fixatives include, but are not limited to, aldehyde compounds (such as formaldehyde, paraformaldehyde, and glutaraldehyde), osmium tetroxide, potassium dichromate, chromic acid, and potassium permanganate. This embodiment also includes fixatives known to release cross-linking compounds, such as formaldehyde, over time. Formaldehyde is a well-known cross-reactive molecule that fixes biological samples by cross-linking amino groups, for example, via methylene bridges. According to one embodiment, the biological molecules were fixed using formaldehyde and / or paraformaldehyde. As is known in the art, formaldehyde can be used as a fixative for both solid and liquid biological samples.

[0138] According to a preferred embodiment, the fixed biological sample is a solid fixed biological sample, particularly a fixed tissue sample. Exemplary fixed biological samples from an individual include, but are not limited to, tissues including liver, spleen, kidney, lung, intestine, thymus, colon, tonsil, testis, skin, brain, heart, muscle, and pancreatic tissue. Fixed biological samples may also be samples containing fixed cells, such as cell-containing body fluids and samples derived therefrom, aspirates, cell cultures, bacteria, microorganisms, viruses, plants, fungi, biopsies, bone marrow samples, swab samples, feces, skin fragments, and organisms.

[0139] According to one embodiment, the fixed biological sample is a fixed tissue sample, wherein the tissue sample is an animal tissue, preferably a mammalian tissue sample, in particular a human tissue sample. The tissue may be obtained from an autopsy, biopsy or surgery.

[0140] According to a preferred embodiment, the fixed biological sample is a liquid fixed biological sample. Exemplary liquid fixed biological samples include, but are not limited to, fixed body fluid samples such as blood, serum, plasma, urine, saliva, tears, sweat, stool, mucus, breast milk, bone marrow, and spino-cerebral fluid.

[0141] In one embodiment, the fixed biological sample is a biological sample in a liquid cytological preservation medium. The liquid cytological preservation medium contains a cross-linking fixative, such as an aldehyde-containing fixative, such as formaldehyde. While liquid cytological preservation media are useful for cytological purposes, this may inhibit the efficient isolation of nucleic acids from the fixed biological sample. Exemplary aldehyde-containing fixatives commonly used in liquid cytological preservation media include, but are not limited to, formaldehyde, glyoxal, glutaraldehyde, glyceraldehyde, acrolein, or other aliphatic aldehydes. One commonly used liquid cytological preservation medium containing a fixative is SUREPATH®, which is one of the most commonly used preservation media in clinical settings (e.g., for preserving swabs). SUREPATH® medium has a formaldehyde content of approximately 37% and also contains methanol, ethanol, and isopropanol. Although high formaldehyde content makes formaldehyde a useful fixative, subsequent extraction of target nucleic acids, such as DNA, from such fixed biological samples and their subsequent analysis presents challenges. The method according to the present invention can be advantageously used with such difficult fixed samples. According to one embodiment, the liquid fixed biological sample is a biological sample in SUREPATH®.

[0142] In one embodiment, the fixed biological sample is a biological sample containing solid cells. The fixed biological sample may be embedded in a non-reactive embedding substance such as paraffin. According to one embodiment, the fixed biological sample is embedded in an embedding material such as paraffin. According to one embodiment, the fixed biological sample is a fixed tissue sample fixed using a cross-linking fixative (such as formaldehyde) and embedded in an embedding material, preferably paraffin (such as an FFPE sample). As disclosed in the art, embedding materials include, but are not limited to, paraffin, mineral oil, water-insoluble wax, celloidin, polyethylene glycol, polyvinyl alcohol, agar, gelatin, or other media.

[0143] According to certain embodiments, the fixed biological sample is an FFPE sample.

[0144] When processing a fixed sample embedded in an embedding material such as paraffin, it is within the scope of the present disclosure to include a step of removing the embedding material prior to step (a). According to one embodiment, prior to the contacting step (a), the fixed biological sample is treated to remove any embedding material (such as paraffin) from the fixed biological sample. Removal of embedding material (such as paraffin) from the biological sample can be carried out by any method known in the art for deparaffinizing biological samples. For example, this can be achieved by contacting the sample with a hydrophobic organic solvent such as xylene to dissolve the embedding material (such as paraffin). Suitable deparaffinization methods are described, for example, in WO2012 / 085261, WO2011 / 104027, WO2011 / 157683 and WO2007 / 068764, as well as in GeneRead™'s DNA FFPE handbook (QIAGEN, March 2014) and in "Purification of genomic DNA from FFPE tissue using the The QIAamp® DNA FFPE Tissue Kit and Deparaffinization Solution is described in the QIAamp® DNA FFPE Tissue Handbook (QIAGEN, June 2012).

[0145] Nucleic acid analysis According to a preferred embodiment, the method according to the second aspect comprises the step of (e) analysing the purified nucleic acid.

[0146] The nucleic acid analysis method may be any chemical and / or biotechnological method that can be used to analyze nucleic acids, for example, to amplify, identify, detect, and / or quantify nucleic acids. Preferably, the nucleic acid analysis method includes a detection reaction that allows the detection of the presence, absence, and / or amount of nucleic acids contained in the concentrated nucleic acids. Preferably, step (e) includes the amplification of at least one target nucleic acid. Respective analytical methods are well known in the prior art and are commonly applied in the fields of medicine, diagnosis, and / or prognosis to analyze specific nucleic acids contained or suspected to be contained in nucleic acids and purified nucleic acids.

[0147] According to a preferred embodiment, the purified nucleic acids obtained in step (d) are used in a nucleic acid analysis method that includes amplification. Such a method preferably includes enzymatic amplification, such as polymerase-based amplification. In a specific embodiment, polymerase chain reaction (PCR) is performed to amplify the concentrated nucleic acids.

[0148] According to certain embodiments, the method further comprises amplifying nucleic acid using large amplicon PCR and / or short amplicon PCR. According to one embodiment, the large amplicon PCR is for nucleic acid molecules having a size of at least 500bp. The short amplicon PCR is for nucleic acid molecules having a size of less than 500bp, for example, preferably less than 300bp, less than 200bp, or less than 150bp. In an embodiment, the short amplicon PCR is less than 100bp.

[0149] In embodiments, a lysis composition containing at least 10 mM, particularly at least 20 mM, at least 40 mM, at least 60 mM, or preferably at least 75 mM of a reactive compound, optionally a reactive compound containing a primary amine, preferably 2-amino-2-(hydroxymethyl)propane-1,3-diol, is used for large amplicon PCR. As disclosed herein, for short amplicon PCR, it may be particularly advantageous to provide a lysis composition containing at least 3 mM, particularly at least 5 mM, at least 7 mM, at least 9 mM, or preferably at least 10 mM of a reactive compound, optionally a reactive compound containing a primary amine, preferably 2-amino-2-(hydroxymethyl)propane-1,3-diol. Thus, by selecting suitable conditions for the lysis composition, particularly by selecting suitable reactive compounds and concentrations of the reactive compounds, the resulting nucleic acids can be tailored to suit a particular nucleic acid analysis method, as described in detail elsewhere herein.

[0150] According to one embodiment, the method further comprises a step of analyzing nucleic acids, which comprises performing a next-generation sequencing method. Next-generation sequencing advantageously allows for the determination of nucleic acid sequences in a high-throughput format. However, nucleic acids from fixed biological samples are often associated with artifacts that are detrimental to next-generation sequencing. The method of the present invention advantageously provides high-quality nucleic acids and is particularly suitable for next-generation sequencing, as demonstrated by the examples. In an embodiment, performing next-generation sequencing according to the present disclosure may comprise: i. binding unique molecular identifier sequences to nucleic acids, each nucleic acid molecule comprising a different unique molecular identifier sequence; ii. amplifying the nucleic acids comprising the bound unique molecular identifier sequences; and iii. sequencing the nucleic acids. Such a method advantageously allows for sequencing of the resulting nucleic acids with low reads / unique molecular identifier sequence values, particularly less than 20, for example less than 15, less than 12, and preferably 10 or fewer.

[0151] Further suitable nucleic acid analysis methods may be selected from or include one or more of the following: amplification reactions, polymerase chain reaction (PCR), isothermal amplification, reverse transcription polymerase chain reaction (RT-PCR), reverse transcription amplification, quantitative real-time polymerase chain reaction (qPCR), DNA or RNA sequencing, reverse transcription, LAMP (loop-mediated isothermal amplification), RPA (recombinase polymerase amplification), tHDA (helicase-dependent amplification), NEAR (nicking enzyme amplification reaction) and other types of amplification.

[0152] Use according to the third aspect A third aspect of the invention relates to the use of a proteolytic enzyme, preferably a protease such as proteinase K, for lysing a fixed biological sample, said sample containing crosslinks between nucleic acid molecules and protein molecules due to fixation, and said prior lysis preferably comprising proteolytic digestion in a method according to the first or second aspect of the invention and heating the lysed sample to reverse the crosslinks. Details of the methods according to the first and second aspects are also disclosed in claims 1 to 22, where they are referred to.

[0153] According to a preferred embodiment, the proteolytic enzyme is a protease, in particular a serine protease such as proteinase K. Suitable proteolytic enzymes and digestion conditions are disclosed above in conjunction with the method of the first aspect, particularly in conjunction with steps (a) and (c) to which reference is made and to which the present disclosure also applies. Furthermore, suitable heating conditions and embodiments for reversing crosslinks are described above in conjunction with the method of the first aspect, particularly in conjunction with step (b), to which reference is made in the respective disclosures to which reference is also made.

[0154] Use according to the fourth aspect A fourth aspect relates to the use of a glycosylase, preferably a DNA glycosylase, more preferably a uracil DNA glycosylase such as uracil-N-glycosylase, to carry out an enzymatic treatment which is completed in 30 minutes or less, 20 minutes or less, 15 minutes or less or 10 minutes or less, preferably within the scope of the method according to the first or second aspect of the invention, more preferably between steps (b) and (c).

[0155] As disclosed herein, such glycosylase treatment allows for the removal of artifacts caused by fixation of the biological sample (e.g., caused by formalin cross-linking). Suitable conditions for carrying out the enzymatic treatment using glycosylases, such as suitable temperatures and incubation times, and suitable conditions within the reaction mixture, as well as suitable glycosylases for carrying out such enzymatic treatment, are disclosed herein in conjunction with the method according to the first aspect, and reference is made to each disclosure. Suitable conditions for carrying out such glycosylase treatments are also known in the art. Furthermore, the present disclosure discloses suitable lysis / digestion compositions compatible with such glycosylate treatments, thereby enabling the in-process use of glycosylases, such as uracil-N-glycosylase, to remove artifacts caused by fixative-induced cross-linking.

[0156] Method according to the fifth aspect According to a fifth aspect of the present invention there is provided a method for processing a biological sample, wherein the immobilized biological sample contains cross-links between nucleic acid molecules and protein molecules due to fixation, said method comprising:

[0157] (a) lysing a fixed biological sample, wherein the lysing comprises digestion with a proteolytic enzyme, the lysing comprising preparing a lysis mixture, the lysis mixture comprising: (i) the fixed biological sample; and (ii) a lysis composition comprising a proteolytic enzyme and preferably a reactive compound, more preferably a reactive compound selected from Tris and spermidine; (b) heating the dissolved sample to reverse the crosslinks; (c) optionally adding a proteolytic enzyme and carrying out proteolytic digestion; (d) purifying nucleic acids from the lysed sample; (e) analyzing the purified nucleic acids, the analysis comprising amplifying nucleic acid molecules having a size of less than 500 nt, e.g., 300 nt or less, 200 nt or less, 150 nt or less, or 100 nt or less; A method is provided, comprising:

[0158] Details regarding steps (a) to (e) have already been disclosed in conjunction with the methods according to the first and second aspects, and reference is made to said disclosure, which also applies here. As disclosed above, if necessary, one or more additional processing steps may be carried out between steps (b) and (c), which may be advantageous to prepare the nucleic acid for analysis step (e).

[0159] The purified nucleic acid may be DNA or RNA. Preferably, RNA is reverse transcribed into cDNA before amplification in step (e). When the nucleic acid is a double-stranded molecule, such as a double-stranded DNA molecule, it is preferred that the above designation of "nt" for size (length) refers to "bp". Thus, when a double-stranded DNA molecule has a size of 150 nt, the double-stranded DNA molecule has a size of 150 bp.

[0160] In particular, small nucleic acids can be purified and amplified by the method of the fifth aspect. The method of the fifth aspect is particularly suitable for analyzing nucleic acid molecules with short sizes in amplification-based analytical methods such as PCR. As demonstrated in the examples, short amplicon PCR of nucleic acids purified using the method of the fifth aspect resulted in low Ct values.

[0161] According to one embodiment, the reactive compound (e.g., a formalin scavenger, preferably selected from Tris and spermidine) is present in the lysis mixture and / or lysis composition used in step (a) at a concentration ranging from 3 to 100 mM, 5 to 50 mM, or 10 to 25 mM. Such low concentrations of the reactive compound (which may be a formalin scavenger, such as Tris or spermidine) in the lysis composition / lysis mixture used in step (a), e.g., 3 to 100 mM, preferably 5 to 50 mM, and more preferably 10 to 20 mM, result in nucleic acid fragments of a size that is particularly suitable for performing short amplicon amplification reactions. This was unexpected, since in the prior art, a high degree of fragmentation is believed to be detrimental to subsequent amplification reactions. However, the examples surprisingly show that fragmentation of the purified DNA significantly improves downstream PCR performance when using short amplicon PCR. Without wishing to be bound by theory, it is hypothesized that this effect is due to the greater accessibility of nucleic acids (such as DNA) when they are strongly fragmented, potentially because these breaks occur at points where crosslinks were present in the fixed biological sample. Such crosslink points are inhibitory to PCR, so removing these points during the lysis procedure applied in the method according to the fifth aspect improves the efficiency of the PCR reaction.

[0162] The dissolution mixture particularly comprises a reactive compound as disclosed herein. Examples are disclosed in conjunction with the method according to the first aspect, and reference is made to the respective disclosures that also apply here. In one embodiment, the reactive compound comprises a primary amine group, and is preferably 2-amino-2-(hydroxymethyl)propane-1,3-diol or a derivative thereof. Other features of step (a) are disclosed above in conjunction with the method according to the first aspect, and reference is made thereto.

[0163] The lysis composition used in step (a) comprises (i) salt; and (ii) surfactants It may further include:

[0164] Suitable salts and surfactants and suitable concentrations for the lysis composition used in step (a) have already been disclosed in conjunction with step (a) of the method according to the first aspect, and reference is made to the respective disclosures.

[0165] The lysis composition and / or lysis mixture used in step (a) of the method according to the fifth aspect may have a pH in the range of 6.0 to 9.5, preferably 7.0 to 8.0.

[0166] The nucleic acids advantageously released by carrying out steps (a) and (b) of the method, and optionally but preferably, step (c), are purified in step (d). Further details and embodiments of steps (b), (c) and (d) are particularly disclosed in conjunction with the methods according to the first and second aspects, and reference is made to the respective disclosures that apply here. Preferably, step (c) is carried out in the method according to the fifth aspect. As disclosed in conjunction with the method according to the first aspect, one or more additional processing steps, for example, enzymatic treatment steps including digestion with a nuclease and / or the use of a glycosylase, such as uracil-N-glycosylase, may be carried out between steps (b) and (c). Details are described above in conjunction with the method according to the first aspect, and reference is made to the respective disclosures that apply here.

[0167] Method according to the sixth aspect According to a sixth aspect of the present invention there is provided a method for processing a biological sample, wherein the immobilized biological sample contains cross-links between nucleic acid molecules and protein molecules due to fixation, said method comprising: (a) lysing a fixed biological sample, wherein the lysing comprises digestion with a proteolytic enzyme, the lysing comprising preparing a lysis mixture, the lysis mixture comprising: (i) the fixed biological sample; and (ii) a lysis composition comprising a proteolytic enzyme and preferably a reactive compound, more preferably a reactive compound selected from Tris and spermidine; (b) heating the dissolved sample to reverse the crosslinks; (c) optionally adding a proteolytic enzyme and carrying out proteolytic digestion; (d) purifying nucleic acids from the lysed sample; (e) analyzing the purified nucleic acids, wherein the analysis comprises amplifying nucleic acid molecules having a size of at least 500 nt and / or a size of less than 500 nt; A method is provided, comprising:

[0168] Details regarding steps (a) to (e) have already been disclosed in conjunction with the methods according to the first and second aspects, and reference is made to said disclosure, which also applies here. As disclosed above, if necessary, one or more additional processing steps may be carried out between steps (b) and (c), which may be advantageous to prepare the nucleic acid for analysis step (e).

[0169] The purified nucleic acid may be DNA or RNA. RNA is preferably reverse transcribed into cDNA before amplification in step (e). When the nucleic acid is a double-stranded molecule, such as a double-stranded DNA molecule, it is preferred that the above designation of "nt" for size (length) refers to "bp". Thus, when a double-stranded DNA molecule has a size of 500 nt, the double-stranded DNA molecule has a size of 500 bp.

[0170] As demonstrated by the examples, the method according to the sixth aspect is particularly suitable for analyzing nucleic acids having a short size, such as less than 500 nt, and / or a size of at least 500 nt in an amplification-based analysis method such as PCR. In particular, nucleic acids having small or large sizes can be obtained and amplified using the method of the sixth aspect. Without being bound by theory, it is believed that a concentration of at least 10 mM, particularly at least 20 mM, at least 40 mM, at least 60 mM, or preferably at least 75 mM of a reactive compound (e.g., Tris or spermidine) is very effective in isolating short and large nucleic acids, thereby obtaining more and / or higher quality nucleic acids having short and large sizes.

[0171] In embodiments, the lysis mixture and / or lysis composition contains 10-1000 mM of the reactive compound, particularly 20-500 mM, 50-300 mM, 75-250 mM, or preferably 85-200 mM, for example, about 100-150 mM. The lysis mixture particularly contains a reactive compound disclosed herein, such as Tris or spermidine. Suitable reactive compounds are disclosed in conjunction with the method according to the first aspect, and the respective disclosures are incorporated herein. The reactive compound may include a primary amine, preferably 2-amino-2-(hydroxymethyl)propane-1,3-diol or a derivative thereof.

[0172] The lysis composition used in step (a) comprises (i) salt; and (ii) surfactants It may further include:

[0173] Suitable salts and surfactants and suitable concentrations for the lysis composition used in step (a) have already been disclosed in conjunction with step (a) of the method according to the first aspect, and reference is made to the respective disclosures.

[0174] This may be further assisted by providing a pH of 6.0 to 9.5, such as 7.0 to 9.0 or 8.0 to 9.0, in the dissolution mixture and / or dissolution composition.

[0175] The nucleic acids advantageously released by carrying out steps (a) and (b) of the method, and optionally but preferably, step (c), are purified in step (d). Further details and embodiments of steps (b), (c) and (d) are disclosed herein, in particular in conjunction with the methods according to the first and second aspects, and reference is made to the respective disclosures that apply here. Preferably, step (c) is carried out in the method according to the sixth aspect. As disclosed in conjunction with the method according to the first aspect, one or more additional processing steps, for example, enzymatic treatment steps including digestion with a nuclease and / or the use of a glycosylase, such as uracil-N-glycosylase, may be carried out between steps (b) and (c). Details are described above in conjunction with the method according to the first aspect, and reference is made to the respective disclosures that apply here.

[0176] In step (e), the nucleic acid is analyzed by a method comprising amplifying the nucleic acid. Such amplification methods are disclosed and referred to herein. In particular, the nucleic acid can be amplified using a polymerase enzyme. According to one embodiment, the nucleic acid is amplified using PCR, in particular PCR in which short nucleic acid molecules are amplified (also referred to as short amplicon PCR) and / or PCR in which large nucleic acids are amplified (also referred to as large amplicon PCR). Advantageously, short and large nucleic acid molecules can be amplified using this method. The fragment size can be controlled by adapting the concentration of reactive compounds (e.g., selected from Tris and spermidine) in the lysis composition / lysis mixture. According to one embodiment, the analysis in step (e) comprises amplifying nucleic acid molecules having a size of at least 500 nt, for example, at least 550 nt, at least 600 nt, at least 650 nt, or at least 700 nt.

[0177] The present invention is not limited by the exemplary methods and materials disclosed herein; any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention. Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects or embodiments of the present invention, but rather should be read with reference to the specification as a whole.

[0178] As used in the subject specification and claims, the singular forms "a," "an," and "the" include plural aspects unless the context clearly dictates otherwise. The terms "include," "have," "comprise," and variations thereof are used synonymously and should be interpreted without limitation. Additional components and steps may be present. Throughout this specification, when a composition is described as comprising a component or material, it is further contemplated that the composition, in embodiments, also consists essentially of, or consists of, any combination of the listed components or materials, unless otherwise stated. References to "the disclosure," "the present invention," and the like include single or multiple aspects, etc., taught herein. The aspects taught herein are encompassed by the term "invention."

[0179] It is preferred to select and combine the preferred embodiments described herein, and specific subject matter arising from each combination of preferred embodiments also belongs to this disclosure. [Example]

[0180] The following examples are for illustrative purposes only and should not be construed as limiting the present invention in any way. They demonstrate that DNA extraction from fixed samples can be advantageously improved by performing an additional proteinase digestion step after the first proteinase digestion and crosslink removal steps. This second proteinase digestion step improves the extraction process and provides high-quality, pure DNA. DNA can be purified with high yields, favorable fragment sizes, and better suitability for downstream PCR amplification (such as short amplicon and large amplicon PCR). The purified DNA is also highly advantageous for next-generation sequencing (NGS), as it can obtain particularly low read / UMI (unique molecular identifier, also known as unique molecular index) values.

[0181] Furthermore, the examples show that the size of DNA fragments can be modified by adapting the lysis composition. Furthermore, when implemented, the processing time required for the uracil-N-glycosylase (UNG) treatment step can be significantly reduced compared to prior art methods.

[0182] Throughout the examples, DNA is extracted from FFPE tissue samples. After deparaffinization of the FFPE tissue samples, the samples are lysed. The sample material in the lysis composition (corresponding to the sample + lysis solution) is subjected to a proteinase K digestion step, followed by a cross-link removal step using heat. The lysed sample may then be subjected to an enzymatic treatment, such as RNase and / or UNG treatment. A (second) proteinase K digestion step is then performed to remove proteins and peptides still cross-linked to the DNA. The DNA is then purified from the digested sample, for example, by binding the DNA to a solid phase, followed by repeated washing cycles and elution of the bound DNA. The purified DNA is then ready for further use and analysis.

[0183] 1. Example 1 Improvement by performing an additional proteinase digestion step 1.1. Improved DNA yield, degree of fragmentation, and impact on downstream PCR performance (a) Materials and Methods FFPE tissue samples In this example, various human FFPE tissue samples were used, including prostate, lung, kidney, spleen, and breast cancer. 10 μm sections were cut from the FFPE blocks using a Leica rotary microtome, with two or three 10 μm sections applied per preparation.

[0184] Extraction Protocol DNA was extracted from FFPE tissues according to the following protocol. 1. Preparation of FFPE Tissue Samples - 400 μL of deparaffinization solution (DPS) was added to the FFPE tissue sample and vortexed. - The samples were incubated at 56°C for 3 minutes.

[0185] 2. Lysis of FFPE Tissue Samples Lysis is assisted by the use of a lysis solution such as a lysis buffer. Preferably, the lysis solution contains a detergent, a salt, and a buffer; such a lysis buffer was used in this example. An anionic detergent such as SDS may be used as the detergent. The salt may be a non-buffering salt such as an alkali metal salt. Chloride salts such as NaCl or KCl may also be used. The pH of the lysis buffer may be within the range of 6.0 to 9.5, for example, 7.0 to 9.0. Tris may be used as a buffer. The lysis solution may contain a chelating agent such as EDTA, as needed, to inhibit nucleases such as DNase. In an embodiment, the lysis solution is a lysis buffer containing at least 0.1% (w / v) detergent, at least 300 mM or at least 500 mM salt and a buffering agent. The lysis buffer used may contain 0.1-0.5% (w / v) detergent (e.g., an anionic detergent such as SDS), 400-800 mM salt (alkali metal halide, e.g., KCl or NaCl), and a buffering agent (e.g., Tris). Such lysis buffers are known in the art. In this example, a commercially available lysis buffer was used (FTB, QIAGEN). In this example, a lysis composition was prepared by mixing the lysis buffer, proteinase K (in a solution containing 20 mg / ml proteinase K), and water. In one embodiment, Tris was added to the lysis composition (referred to as "high Tris"; a lysis composition without added Tris is referred to as "low Tris"). As described herein, Tris can act as a formaldehyde scavenger and may aid in the resolution of crosslinks. - A lysis composition as defined below was prepared and added to the FFPE samples processed according to step 1: [Table 1] - Samples were incubated at 56°C for 1 hour and shaken in a thermoshaker for proteinase digestion (using proteinase K). The samples were then incubated at 90°C for 1 hour without shaking to reverse the cross-links. Such a heating step also inactivates the proteinases.

[0186] 3. Digestion with uracil-N-glycosylase (UNG) - Either remove the blue DPS phase from the top aqueous lysate phase or transfer the bottom clear phase to a new tube. - For digestion with uracil-N-glycosylase (UNG), the sample was further diluted using 115 μL of water and 35 μL of UNG (1 U / μL). - The samples were then incubated at 50°C for 5 minutes without shaking.

[0187] 4. RNase A Digestion - 4 μL of RNase A was added per sample. - The sample was mixed and incubated at room temperature for 2 minutes.

[0188] 5. Additional (Second) Proteinase Digestion Step - 20 μL of proteinase K was added. - The samples were mixed and incubated in a thermoshaker at 65°C and 450 rpm for 15 minutes.

[0189] Nucleic acids, such as DNA, are then purified from the digested sample, where the digestion / pretreatment protocol leaves the nucleic acids sufficiently accessible that any suitable purification method can be used.

[0190] 6. DNA Purification - 250 μL each of Buffer AL (QIAGEN) and 96-100% ethanol was added and the sample was mixed. - The lysate was transferred to a QIAamp® MinElute spin column, then centrifuged and the flow-through discarded. - 500 μL of buffer AW1 (QIAGEN) was added, followed by centrifugation and discarding the flow-through. - 500 μL of buffer AW2 (QIAGEN) was added, followed by centrifugation and discarding the flow-through. - 500 μL of 96-100% ethanol was added, followed by centrifugation, discarding the flow-through, and dry spinning at full speed. - 30 μL or 50 μL of elution buffer ATE (QIAGEN) was applied to the membrane, followed by centrifugation. The extracted and purified DNA was present in the resulting flow-through.

[0191] Control For comparison, the above protocol was carried out, except that the additional proteinase K digestion step (see step 5 above) was omitted.

[0192] b) Analysis of DNA yield In this set of experiments, DNA yields of extracts using the protocol disclosed above were determined using QIAxpert and Qubit instruments. DNA yields obtained using either instrument are shown in Figures 1A-1E (UV-Vis = QIAxpert; dsDNA (Qubit) = Qubit).

[0193] Example 1.1 demonstrates that performing an additional proteinase digestion step after the crosslink removal step at elevated temperature improves DNA yield. Furthermore, DNA fragments with a larger average size are obtained, which is particularly advantageous for obtaining DNA suitable for downstream PCR in large amplicon PCR.

[0194] The second proteinase digestion step resulted in higher yields as measured by UV-Vis and fluorimetric determination (Qubit) compared to a control protocol without an additional proteinase K digestion step (see Figures 1A-1E). Higher DNA yields were obtained for all tested FFPE tissue samples and lysis solutions. It is generally believed in the art that protein digestion in samples is essentially complete after the first proteinase digestion step when performed by conventional FFPE extraction methods (e.g., proteinase K at 56°C for 1 hour). Therefore, it was quite surprising that performing a second proteinase digestion step after the heat-assisted crosslink removal step significantly improved the results. Without wishing to be bound by theory, it is hypothesized that crosslinks present in fixed samples may result in particularly persistent protein association with nucleic acids (e.g., DNA), potentially protecting the proteins during the first proteinase digestion step. Furthermore, steric effects may render proteins inaccessible to proteinases during the first proteinase digestion step. After a cross-link reversal step at elevated temperatures (e.g., at least 85°C or at least 90°C), these associations may weaken and / or the sample may be sufficiently denatured that the remaining proteins become accessible and may be sufficiently removed during the second proteinase digestion step.

[0195] Figures 1A-1E further show that for some FFPE tissue types, the high Tris lysis compositions tested resulted in higher DNA yields than the low Tris lysis compositions (prostate, lung; see Figures 1A and 1B). Meanwhile, for kidney tissue, the low Tris lysis composition resulted in higher DNA yields than the high Tris lysis composition (see Figure 1C), whereas no differences were observed for spleen and breast cancer tissue (see Figures 1D and 1E). Thus, modifying the Tris concentration in the lysis composition can be advantageously used to further improve DNA yields, depending on the type of tissue used.

[0196] An additional proteinase K digestion step is advantageous to increase DNA yield, thus improving DNA extraction from various FFPE tissue sample types.

[0197] (c) Analysis of the degree of fragmentation of extracted DNA In this experiment, the degree of fragmentation was analyzed using gel electrophoresis. DNA extracted from FFPE human kidney and breast cancer tissues was used as the sample material. The results are shown in Figure 2.

[0198] Gel electrophoresis in Figure 2 shows that the additional proteinase digestion step results in an increase in the average size of DNA fragments (the bands are shifted; see the "2.PK lysis + at 65°C for 15 min" sample). This increase was observed for both FFPE tissue types and in both lysis solutions, i.e., high Tris and low Tris. This result was surprising and unexpected. The increased fragment size of extracted DNA can be advantageous for downstream PCR applications, so using an additional proteinase digestion after the heat-assisted crosslink removal step improves DNA extraction.

[0199] (d) Impact on downstream PCR performance In this example, the extracted DNA was analyzed by PCR to determine whether PCR performance was affected by the increased DNA size due to the additional proteinase K step. Quantitative real-time PCR was performed to determine Cq values. The "Cq" value can be used interchangeably with the "Ct" value. The Cq value is inversely proportional to the original relative amount of extracted DNA. Both short amplicon PCR using 66 bp fragments and large amplicon PCR using 500 bp fragments were performed. The results are shown in Figure 3 (dark-shaded columns correspond to the same amount of DNA per reaction mixture, and light-shaded columns correspond to the same volume of diluted eluate per reaction mixture).

[0200] As shown in Figure 3, DNA extracted using an additional proteinase K digestion step reduces Cq values ​​overall, thereby improving PCR performance. This improvement was observed for both tested FFPE tissue types (human breast cancer and kidney tissue) and both lysis solutions (high Tris and low Tris). Therefore, improving the quality of extracted DNA also leads to better PCR results, especially with large amplicons (500 bp). This result indicates that by performing a second proteinase digestion after the decrosslinking step, larger amounts of long DNA strands that were previously inaccessible become accessible to the PCR reaction.

[0201] (e) Further conclusions An additional proteinase digestion step after the crosslink removal step enhances DNA extraction from various FFPE tissue samples. In particular, DNA is extracted with higher yields and larger fragment sizes, as well as higher quality in terms of PCR amplification for short and large amplicons. Therefore, an additional proteinase digestion step is highly advantageous for DNA extraction from fixed biological samples, such as FFPE samples, and even fixed liquid samples. As shown, one or more additional enzyme digestion steps may be performed between the crosslink removal step and the second proteinase step.

[0202] 1.2. Next-generation sequencing (NGS) capabilities will be enhanced Example 1.2 demonstrates the improvement of DNA extraction from FFPE tissue samples by implementing an additional proteinase digestion step according to the present disclosure. As previously mentioned, proteinase K may be used as the proteinase. By using the extracted DNA of Example 1.1 for NGS, very high NGS performance was measured. In particular, low reads / UMI values ​​below 10 were measured for all tested tissue samples.

[0203] (a) Materials and Methods DNA extracted from human FFPE tissue samples including prostate, lung, kidney, and breast cancer described in Example 1.1 was used for NGS performance analysis.

[0204] Sample preparation and sequencing workflow The extracted DNA was used as a template for sequencing libraries. The QIAseq™ Targeted Panel for Illumina instrument protocols was used according to the QIAseq™ Targeted DNA Panel Handbook (QIAGEN, 05 / 2017). QIAGEN Targeted DNA The panel used was the Human Lung Cancer Panel using UMI technology, which is based on the incorporation of unique molecular identifier (UMI) sequences (also called unique molecular indexes) into a single gene-specific, primer-based targeted enrichment process, thereby overcoming biases / artifacts of the DNA polymerase and amplification process: - Sequence reads with different UMIs represent different original molecules. - Sequence reads with the same UMI are the result of PCR replication derived from one original molecule.

[0205] Errors from the PCR amplification and sequencing process may also exist in the final reads, resulting in false-positive variants in the sequencing results. These artifact variants can be greatly reduced by calling variants across all reads within a unique UMI instead of picking up variants at the original read level.

[0206] (b) Performance in NGS As discussed above, each molecule of double-stranded DNA is tagged with a UMI barcode before amplification, which allows for the identification of truly unique molecules detected in NGS from PCR amplicons.

[0207] The reads detected per UMI are plotted relative to the extracted DNA in Figure 4. A reads / UMI value greater than 10 indicates that the same molecule was read more than 10 times, indicating over-amplification / insufficient complexity of the starting material.

[0208] As shown in Figure 4, the extraction method of the present disclosure demonstrates very high NGS performance for all samples when fewer than 10 reads / UMIs are measured. This demonstrates that the method of the present disclosure is advantageous for extracting DNA from various FFPE sample types. Furthermore, an additional / second Proteinase K step improves NGS performance, particularly for prostate, kidney, and in some cases lung and breast cancer tissues (see "GR-High Tris" in Figure 4 for high Tris lysis composition).

[0209] Overall, NGS performance is very high when using the purification methods according to the present disclosure.

[0210] 2. Example 2 Comparison of DNA extraction using the disclosed method with prior art methods Example 2 further demonstrates the improvement of DNA extraction from FFPE tissue samples by using an additional proteinase digestion step according to the present disclosure. Similar to Example 1.1, high DNA yields were obtained. Furthermore, larger fragment sizes and better PCR performance were measured. Importantly, NGS performance was also significantly enhanced compared to prior art methods.

[0211] (a) Materials and Methods FFPE tissue samples Human atrial FFPE tissue was used in this example. 10 μm sections were cut from the FFPE blocks using a Leica rotary microtome, with two 10 μm sections applied per preparation.

[0212] Extraction Protocol In this example, the extraction protocol of Example 1.1 was followed.

[0213] Control Similar to Example 1.1, a comparative control omitted the additional proteinase K digestion step (see step 5 above). Additionally, an additional comparative control was performed by performing the first proteinase K step overnight at 56°C and omitting the additional proteinase K digestion step ("o / n 56°C").

[0214] Reference Protocol As a reference protocol (i.e., prior art method), Maxwell® The RSC DNA FFPE Kit Technical Manual (Promega, Revised 11 / 17) and Maxwell® RSC FFPE Plus DNA Kit Technical Manual (Promega, Revised 12 / 19) were followed. Additionally, the QIAGEN supplemental protocol "Purification of genomic DNA" was used as a reference protocol. from FFPE tissue using the QIAamp® DNA FFPE Tissue Kit and Deparaffinization Solution, according to the QIAamp® DNA FFPE Tissue Handbook (QIAGEN, June 2012).

[0215] b) Analysis of DNA yield In this set of experiments, DNA yields of extracts were determined using QIAxpert and Qubit instruments. DNA yields obtained using either instrument are shown in Figures 5A and 5B for human lung cancer tissue and human atrium, respectively (UV-Vis = QIAxpert; dsDNA (Qubit) = Qubit).

[0216] An additional proteinase K digestion step at 65°C after the cross-linking step resulted in higher yields compared to a control protocol without an additional proteinase K digestion step, as measured by UV-Vis and fluorimetric determination (Qubit) (see Figures 5A and 5B). Higher yields were obtained for both tested FFPE tissue types and both lysis compositions (high Tris and low Tris). Importantly, performing the first proteinase K step for a longer period, as in the overnight control sample at 56°C (see "o / n 56°C"), did not result in higher yields. In fact, performing a second proteinase K step after the de-cross-linking step resulted in higher yields compared to the o / n 56°C control, demonstrating that it is not the protein digestion time itself that is important, but rather the specific sequence of steps performed in the disclosed method, in which the second proteinase digestion step is performed after the de-cross-linking step.

[0217] Figures 5A and 5B further demonstrate that the low-Tris lysis composition resulted in even higher DNA yields for both tissue types tested. Therefore, for these tissue types, a low-Tris lysis composition (e.g., a Tris concentration in the lysis composition less than 50 mM, less than 30 mM, less than 25 mM, or optionally in the range of 1 mM to 20 mM) may be used to further increase yields. Significantly higher yields were obtained than with the reference protocol.

[0218] (c) Analysis of the degree of fragmentation of extracted DNA The degree of fragmentation was analyzed using gel electrophoresis. DNA extracted from FFPE human lung cancer and atrial tissues was used as the sample material. The results are shown in Figures 6A and 6B, respectively.

[0219] Gel electrophoresis in Figures 6A and 6B shows that the additional proteinase digestion step results in an increase in the average size of DNA fragments (the bands are shifted; see the "2.PK lysis + at 65°C for 15 min" sample). This increase was observed for both FFPE tissue types and for both lysis compositions used. As with the discussion of higher yields, this result was surprising and unexpected. Compared to the reference protocol, performing a second proteinase K step according to the present disclosure resulted in an overall larger fragment size. Larger fragment sizes may be advantageous for downstream PCR performance.

[0220] (d) Impact on downstream PCR performance In this example, the extracted DNA was analyzed by PCR to determine whether PCR performance was affected by the increased DNA size due to the additional proteinase K step. Quantitative real-time PCR was performed as before, and Cq values ​​were determined. Both short amplicon PCR using 66-bp fragments and large amplicon PCR using 500-bp fragments were performed. The results are shown in Figures 7A and 7B for the short and large amplicons, respectively (the dark-shaded columns correspond to the same amount of DNA per reaction mixture, and the light-shaded columns correspond to the same volume of diluted eluate per reaction mixture).

[0221] As shown in Figures 7A and 7B, DNA extracted using an additional proteinase digestion step generally reduces Cq values, thereby improving PCR performance, particularly for large-amplicon PCR using 500-bp fragments. The disclosed method using an additional / second proteinase digestion step generally results in lower Cq values, especially for short amplicons, compared to the reference protocol. Furthermore, the disclosed method using an additional / second proteinase digestion step generally outperformed the control sample, which underwent an overnight proteinase K digestion step at 56°C. This result indicates that by performing an additional proteinase digestion after decrosslinking, a larger amount of previously inaccessible long DNA strands became accessible to the PCR reaction. Furthermore, this result indicates that it is the sequence of steps performed in the disclosed method, rather than the total amount of digestion time, that is important for achieving better PCR performance.

[0222] (e) Performance in NGS In this example, we investigated the performance of extracted DNA for NGS analysis. DNA was extracted from FFPE human atrial tissue samples using the extraction protocol described above (see Example 1.1 for the lysis composition used) with and without an additional proteinase digestion step (using proteinase K as the proteinase).

[0223] The extracted DNA was used as a template for sequencing libraries. The sample preparation and sequencing workflow described in Example 1.2 was followed, and the reads / UMI values ​​were determined as above.

[0224] result Each molecule of double-stranded DNA is tagged with a unique molecular identifier (UMI) barcode prior to amplification. This allows for the identification of truly unique molecules detected in NGS from PCR amplicons. In Figure 8, the reads detected per UMI are plotted relative to the extracted DNA. A read / UMI value greater than 10 indicates that the same molecule was read more than 10 times, indicating over-amplification / insufficient complexity of the starting material.

[0225] As shown in Figure 8, the extraction method according to the present disclosure outperforms the QIAamp® FFPE DNA kit ("QA FFPE") and the Maxwell RSC DNA FFPE kit or Maxwell RSC FFPE Plus DNA kit from Promega. All of these kits have UMI values ​​well above 10. DNA extracted by the method according to the present disclosure all have values ​​below 10. An additional proteinase K incubation step resulted in even lower reads / UMI values.

[0226] Overall, NGS performance is significantly improved by the extraction method according to the present disclosure, which can be performed quickly and produces excellent results.

[0227] (e) Conclusion This example demonstrates that performing an additional proteinase digestion step after the first proteinase digestion and decrosslinking step improves the quality of the purified nucleic acid, as shown for DNA. The additional proteinase digestion step, in particular, increases yield and fragment size, improves PCR performance, and, importantly, significantly enhances NGS performance compared to prior art methods. As discussed above, these results are highly surprising and unexpected. Furthermore, by comparing the disclosed method, which includes an additional / second protein digestion step, with the first Proteinase K digestion step performed overnight at 56°C, it was shown that the specific sequence of steps performed in the disclosed method, rather than the overall digestion time, is responsible for the superior performance.

[0228] 3. Example 3 Effect of DNA extraction using diluted lysis composition Example 3 demonstrates that the lysis composition affects DNA extraction from FFPE tissue samples. Dilution of the lysis composition allows for control of DNA fragmentation during the crosslink removal step. Furthermore, this example shows that controlling fragmentation can result in shorter DNA fragments, which improves downstream PCR performance when using short amplicon PCR.

[0229] (a) Materials and Methods FFPE tissue samples To analyze the effect of lysis composition within the DNA extraction protocol, two different FFPE tissue samples were used: heart and lung tissue, both from rats. 10 μm sections were cut from the FFPE blocks using a Leica rotary microtome, with one 10 μm section applied per preparation.

[0230] Extraction Protocol The extraction protocol described in Example 1.1 was used with the following modifications: - The lysis composition in step 2. was prepared by combining 25 μL of lysis buffer (FTB, QIAGEN), 20 μL of proteinase K and 55 μL of water (corresponding to the "low Tris" buffer used above for Example 1.1). - In step 3, UNG was replaced with water and incubation was carried out at 50°C for 60 minutes. - Step 5 was omitted (i.e., no second proteinase K digestion step).

[0231] Reference Protocol The QIAamp® DNA FFPE Tissue Handbook (QIAGEN, June 2012) was used as a reference protocol, using QIAGEN's supplemental protocol "Purification of genomic DNA from FFPE tissue using the QIAamp® DNA FFPE Tissue Kit and Deparaffinization Solution." For lysis, 180 μL of Buffer ATL (QIAGEN) was mixed with 20 μL of Proteinase K.

[0232] (b) The degree of fragmentation can be controlled by the choice of lysis solution in the crosslink removal step. In a first set of experiments, DNA extracted from FFPE tissue samples was analyzed by gel electrophoresis (see Figure 9), which allows the size distribution of the extracted DNA to be visualized and conclusions to be drawn regarding the degree of fragmentation.

[0233] As shown in Figure 9, greater fragmentation of DNA was observed when using the extraction method with diluted lysis composition ("GR std" = diluted lysis composition; "QA std" = reference lysis composition; L1-L3 = see the DNA ladder shown in Figure 9). After a further 2.5-fold dilution, diluted lysis composition (see "GR std") for lysis and crosslink removal was used to allow uracil-N-glycosylase activity within the lysate. This was found to result in a smaller average size of extracted DNA compared to the reference protocol (see "QA std"). The smaller average size of extracted DNA was obtained for both FFPE tissue samples, i.e., rat heart and rat lung.

[0234] Overall, the extraction method using diluted lysis composition results in greater fragmentation of DNA.Therefore, the dilution of compounds present in the lysis solution allows for the control of the degree of fragmentation.These compounds include, in particular, Tris.

[0235] (c) The performance of downstream PCR is affected by fragmentation of extracted DNA. In this set of experiments, the effect of DNA fragmentation on PCR amplification was analyzed. The specific objective was to investigate the quality of the extracted DNA with respect to its suitability for amplification by PCR. The DNA extracted above from FFPE tissue samples was used as the sample material. As before, quantitative real-time PCR was performed and Cq values ​​were determined. The obtained Cq values ​​are plotted in Figures 10 and 11.

[0236] In general, the smaller the molecular weight and, therefore, the average length of the DNA (the more fragmented the DNA), the less reliable the downstream analysis is likely to be. As shown in Figure 10, the performance of PCR using long amplicons (727 bp) was slightly worse when using more fragmented DNA obtained by the extraction method using a diluted lysis composition. This is reflected by the higher Cq value ("Fragmented"; approximately 23.6) compared to the reference protocol ("Standard"; Cq value approximately 23.2). This is due to the fact that when DNA is more fragmented, fewer fragments with the minimum length required to amplify large PCR products are available.

[0237] Surprisingly, however, the more fragmented DNA obtained with the diluted lysis composition significantly improved downstream PCR performance when using short amplicon PCR (78 bp) (see Figure 11). While the reference protocol ("standard") has a Cq value of approximately 23, the DNA sample obtained from the diluted lysis composition ("fragmented") has a reduced Cq value of approximately 21.5. This was unexpected. Without wishing to be bound by theory, it is hypothesized that this effect is due to the greater accessibility of DNA when it is more fragmented, potentially resulting in breaks at points where crosslinks were present. Because such crosslinks are inhibitory to PCR, removing these points is believed to improve the efficiency of the PCR reaction.

[0238] (d) Further conclusions The extraction method using diluted dissolution composition results in higher DNA fragmentation, and therefore the average DNA size is smaller.This may be due to the lower concentration of compounds used to aid dissolution, such as surfactants, salts and, if necessary, chelating agents.Despite the smaller fragment size, the extracted DNA demonstrates improved performance in downstream PCR when using short amplicon PCR.In this respect, obtain higher quality DNA sample.

[0239] 4. Example 4 Effect of additives in lysis solution on the incubation of extracted DNA and UNG Example 4 demonstrates that the addition of certain compounds to the lysis composition can be advantageously used to control the degree of fragmentation of DNA extracted from FFPE tissue samples. Thus, the extraction protocol can be adjusted to optimize for either short or long fragments used in downstream PCR. Furthermore, Example 4 demonstrates that the disclosed method advantageously allows for a reduction in processing time. In particular, the uracil-N-glycosylase (UNG) treatment step can be reduced from 60 minutes to just 5 minutes of incubation.

[0240] FFPE tissue samples In this example, FFPE tissue samples from rat kidney and rat lung were used. 10 μm sections were cut from the FFPE blocks using a Leica rotary microtome, with two 10 μm sections applied per preparation.

[0241] Extraction Protocol The extraction protocol of Example 1.1 above was followed without step 5 (i.e., without the second proteinase K digestion step). For lysis of FFPE tissue samples, the following lysis composition was prepared by mixing with lysis buffer (FTB, QIAGEN): [Table 2]

[0242] Reference Protocol As a reference protocol, the GeneRead™ DNA FFPE Handbook (QIAGEN, March 2014) was followed, including a 60-minute UNG treatment step.

[0243] (a) The degree of fragmentation can be controlled by additives in the dissolution composition Fragmentation was analyzed by gel electrophoresis of the extracted DNA samples, and the results are shown in Figure 12.

[0244] As visible by the band shift in Figure 12, Tris had the strongest effect on fragmentation for rat kidney and lung FFPE tissues (see "GR-High Tris" compared to "GR-Low Tris"). Notably, higher Tris concentrations increased the average size of DNA fragments, while lower Tris concentrations decreased the average size. Furthermore, the addition of spermidine affected the size of DNA fragments. The addition of spermidine to the low Tris lysis composition increased the fragment size (see "GR-Low Tris + Spermidine") compared to the low Tris lysis composition (see "GR std"). As shown in Figure 12, the degree of fragmentation was controlled by the addition of certain compounds (additives) to the system. Compounds that showed a fragmentation effect include Tris and spermidine. DTT, glycine, and spermine did not affect fragmentation in this example in a similar manner. Tris had the strongest effect on fragmentation.

[0245] (b) The incubation time of UNG can be reduced by providing a suitable dissolution solution. UNG activity was tested using a bisulfide DNA test. In bisulfide DNA, the cytosine nucleobase of DNA is exchanged with a uracil nucleobase. The uracil nucleobase is cleaved by UNG. As a result, the DNA is partially degraded, reducing the amount of recoverable DNA.

[0246] Workflow A lysis composition containing bisulfide DNA was prepared as described above, except that 2.5 μL of water was replaced with 2.5 μL of bis.gDNA having a concentration of 830 ng / μL. The mixture was then diluted with 115 μL of water, and 35 μL of UNG was added. The sample was then incubated at 50° C. for 5 minutes. The DNA was then purified as described in Example 1.1 (see point 6. DNA purification).

[0247] Control As a control, samples were incubated for 60 minutes at 50° C., as performed in the GeneRead™ DNA FFPE workflow. As a further control, no UNG digestion was performed (“w / o UNG”).

[0248] result The results of the UNG activity test are shown in Figure 13. Specifically, 5 ng (dark-shaded column) or 10 ng (light-shaded column) of extracted DNA was analyzed by quantitative real-time PCR using an amplicon size of 110 bp and wobble bases in the primer sequences to ensure primer annealing. Ct values ​​were determined as before. Because UNG digests bisulfide DNA, small amounts of DNA are recovered, and therefore, higher Ct values ​​are desirable.

[0249] As shown in Figure 13, overall, good results were obtained with regard to UNG digestion, except for the sample with a higher Tris concentration and additional glycine, and the sample with a lower Tris concentration and additional DTT. These had relatively slightly lower Ct values. However, they still had significantly higher Ct values ​​than the control without UNG digestion. Importantly, the lysis solution samples were only incubated for 5 minutes compared to the control sample ("GR FFPE std"), which was incubated for 60 minutes. However, comparable Ct values ​​were measured, demonstrating similar UNG activity. Therefore, by providing the lysis composition according to the present disclosure, the UNG digestion time can be successfully reduced to 5 minutes. A significant reduction in processing time is achieved.

[0250] (c) Conclusion As noted above, by starting with the diluted low-salt lysis composition of Example 3 and adding additives such as Tris or spermidine as needed, extraction protocols can be optimized for either short or long fragments used in downstream PCR. Additionally, processing times can be reduced by providing the lysis composition of the present disclosure, as the digestion time for UNG is reduced from 60 minutes to 5 minutes without loss of activity.

[0251] 5. Overall Conclusion As demonstrated in the Examples, additional proteinase digestion performed after the decrosslinking step significantly enhances the performance of DNA purification, particularly from fixed biological samples such as FFPE samples. The results indicate that despite the first proteinase digestion step and crosslink removal step, nucleic acids such as DNA likely still contain DNA protein crosslinks or similar protein-associated modifications that interfere with downstream processes (e.g., PCR amplification or NGS). Performing an additional proteinase digestion step after decrosslinking removes these, improving DNA quality and enabling enhanced downstream processing and analysis. Improvements were verified with various reference protocols, demonstrating that the disclosed method offers significant improvements over prior art methods. Furthermore, comparing the performance of an extended first proteinase digestion (overnight) with the disclosed method, which uses a second proteinase digestion step after decrosslinking, demonstrated that the specific sequence of steps, rather than the overall digestion time, is critical. Significant improvements are achieved by performing a first proteinase digestion step, followed by a heating step to remove crosslinks, followed by an additional / second proteinase digestion step. This sequence of steps allows for the production of high-quality nucleic acids (e.g., DNA) while allowing the workflow to be completed within a short time frame, making the method very rapid and at the same time very effective in producing high-quality DNA.

[0252] The improvement in DNA yield and accessibility is also reflected in improved NGS performance. Moreover, the method of the present disclosure with adjustable size modification works well for all variants. Furthermore, it is possible to reduce the incubation time of UNG from 60 minutes to 5 minutes, which demonstrates that processing time can be significantly reduced. In all cases, the method according to the present invention shows advantages compared to prior art methods, including traditional QIAamp® FFPE kits. In comparison, the method according to the present disclosure has a significantly faster and simpler workflow, and also provides better UMI values.

[0253] 6. Example 5 Isolation of DNA from various FFPE tissue samples using the EZ1 Advanced XL instrument Approximately 2 mm was measured using the EZ1 Advanced XL instrument for the bind / wash / elute steps, respectively. 3 DNA was isolated from a variety of FFPE tissues using the EZ1 Advanced XL instrument, a robotic workstation for automated nucleic acid purification.

[0254] Before processing the samples on the robotic instrument, they were deparaffinized, lysed, and decrosslinked as follows: >300 μl deparaffinization solution (DPS), vortex, spin >56°C for 3 minutes > Cool to room temperature Master mix: 55 μl RNase-free water, 25 μl Buffer FTB (QIAGEN), and 20 μl Proteinase K per sample. >Vortex and spin the sample >56°C for 1 hour, 1000 rpm >90°C for 1 hour >Remove the top layer >115 μl Rnase-free water >35 μl Rnase-free water >50°C for 5 minutes >Spin >2 μl RNase A, 2 min at RT

[0255] The performance of this protocol was compared to a modified protocol according to the invention which included a second proteinase K step before binding. >20 μl PK, vortex, 65°C, 450 rpm for 15 min

[0256] Yields were determined by UV VIS and Qubit dsDNA BR measurements for human kidney and human breast samples. Figure 14 shows the results. Performance of the isolated DNA in qPCR was determined by adding equal volumes, adjusted for the actual elution volume, to each reaction. 66-bp and 500-bp human 18S rRNA genes were amplified from eluates obtained after extraction using either protocol option. Figure 15 shows the results.

[0257] Approximately 2 mm 3 The experiment was repeated for 100 human heart samples. Yields were again determined by UV VIS and Qubit dsDNA BR measurements, and Figure 16 shows the results. The performance of the isolated DNA in qPCR was again determined by adding the same volume, adjusted for the actual elution volume, to each reaction. 66 bp and 500 bp human 18S rRNA genes were amplified from the eluates obtained after extraction using either protocol option, and Figure 17 shows the results.

[0258] Example 5 demonstrates, based on UV VIS and Qubit dsDNA measurements, and by qPCR, that including a second proteolytic digestion according to the teachings of the present invention improves nucleic acid yield, thereby improving performance in qPCR. The present invention provides, for example, the following items. (Item 1) 1. A method for lysing a fixed biological sample, wherein the fixed biological sample contains crosslinks between nucleic acid molecules and protein molecules resulting from fixation, the method comprising: (a) lysing the fixed biological sample, wherein lysis comprises digestion with a proteolytic enzyme; (b) heating the dissolved sample to reverse the crosslinks; (c) adding a proteolytic enzyme and performing proteolytic digestion; Including, Optionally, one or more additional processing steps are performed between steps (b) and (c). (Item 2) 1. A method for obtaining purified nucleic acids from a fixed biological sample, comprising lysing the fixed biological sample according to the lysis method of item 1, wherein the method further comprises, after step (c): (d) purifying nucleic acids from the lysed sample. A method comprising: (Item 3) The dissolution step (a) comprises preparing a dissolution mixture, the dissolution mixture comprising: (i) the fixed biological sample, and (ii) a dissolution composition containing the protease 3. The method according to item 1 or 2, comprising: (Item 4) Step (a) has the following characteristics: (i) the proteolytic enzyme is a protease, optionally a serine protease, such as proteinase K; (ii) the lysis mixture is heated to aid digestion by the proteolytic enzyme, optionally heating at a temperature in the range of 35 to 75°C, optionally 40 to 70°C; (iii) the lysis mixture is incubated for at least 30 minutes, e.g., at least 45 minutes or at least 50 minutes, and optionally the incubation is performed at an elevated temperature between 35 and 75°C, and / or the sample is agitated during incubation; and / or (iv) step (a) is completed in 120 minutes or less, optionally 100 minutes or less, 90 minutes or less, or 70 minutes or less; Item 4. The method of item 3, comprising one or more of: (Item 5) Step (b) has the following characteristics: (i) step (b) comprises heating the lysed sample to a temperature of at least 80°C, optionally at least 85°C or at least 90°C; (ii) heating the dissolved sample to reverse crosslinks for at least 30 minutes, at least 45 minutes, or at least 50 minutes; and / or (iii) Heating the dissolved sample at a temperature in the range of 80 to 110°C, for example, 85 to 100°C, for 30 to 120 minutes, for example, 45 to 90 minutes or 50 to 70 minutes. 5. The method according to one or more of items 1 to 4, comprising one or more of: (Item 6) Step (c) has the following characteristics: (i) the proteolytic enzyme is a protease, optionally a serine protease, such as proteinase K; (ii) step (c) includes heating to aid digestion by the proteolytic enzyme, and optionally the heating is carried out at a temperature in the range of 35 to 75°C, for example, 40 to 70°C or 50 to 70°C; (iii) step (c) comprises incubation for at least 5 minutes, e.g., at least 10 minutes or at least 15 minutes, optionally wherein the incubation is carried out at an elevated temperature between 35 and 75°C, and / or the sample is agitated during incubation; (iv) the incubation in step (c) is shorter than the incubation in step (a) and / or the incubation temperature in step (c) is higher than that in step (a); and / or (v) step (c) is completed in 30 minutes or less, optionally 20 minutes or less; 6. The method according to one or more of items 1 to 5, comprising one or more of: (Item 7) 7. The method according to one or more of items 1 to 6, comprising carrying out at least one enzymatic treatment step different from the proteolytic digestion step between step (b) and step (c). (Item 8) 8. The method of claim 7, wherein the at least one enzymatic treatment step comprises the use of one or more of a glycosylase, a nuclease, a lipase, or a combination thereof. (Item 9) 9. The method according to item 7 or 8, wherein the at least one enzymatic treatment step comprises the use of a DNA glycosylase, such as a uracil DNA glycosylase, preferably a uracil-N-glycosylase. (Item 10) 10. The method of claim 9, wherein the glycosylase treatment step is performed at an elevated temperature and / or the glycosylase treatment step is completed in 30 minutes or less, 20 minutes or less, 15 minutes or less, or 10 minutes or less, and optionally the glycosylase is uracil-N-glycosylase. (Item 11) 11. The method according to one or more of items 7 to 10, comprising a step of diluting the sample obtained from step (b) in order to perform the at least one enzymatic treatment step before step (c). (Item 12) During the enzyme treatment step, the salt concentration in the enzyme treatment mixture is adjusted to have the following characteristics: (i) the salt concentration is 500 mM or less, and optionally selected from 300 mM or less, 250 mM or less, 200 mM or less, 150 mM or less, and 100 mM or less; (ii) the salt concentration is in the range of 10 mM to 500 mM, for example, in the range of 15 mM to 300 mM, 15 mM to 200 mM, or 15 to 150 mM; and Optionally, the at least one enzymatic treatment step comprises the use of a DNA glycosylase, e.g., uracil DNA glycosylase. 12. The method according to any one of items 7 to 11. (Item 13) 13. The method according to one or more of items 3 to 12, wherein the dissolution composition in step (a) has a pH in the range of 6.0 to 9.5, preferably 6.5 to 9.0 or 7.0 to 9.0. (Item 14) The dissolved composition in step (a) comprises the following compounds: (i) salt; (ii) surfactants; (iii) buffering agent 14. The method according to one or more of items 3 to 13, further comprising one or more, preferably all, of: (Item 15) The dissolution composition comprises a reactive compound, the reactive compound having the following characteristics: (i) the reactive compound reacts with a fixative or chemical moiety released in heating step (b) and / or crosslinks induced by the fixative, optionally wherein the fixative is an aldehyde-containing fixative, such as formaldehyde; (ii) the reactive compound contains a nucleophilic group, preferably an amine group; (iii) the reactive compound is one or more primary amine groups, optionally one primary amine group and one or more hydroxyl groups, preferably three hydroxyl groups; or - Two primary amine groups and optionally one secondary amine group and / or (iv) The reactive compound is 2-amino-2-(hydroxymethyl)propane-1,3-diol or a derivative thereof, or spermidine or a derivative thereof, or a combination thereof. 15. The method according to one or more of items 1 to 14, comprising one or more of: (Item 16) The following features: (i) the reactive compound is present in the lysis composition and / or the lysis mixture at a concentration in the range of 1 mM to 500 mM, for example, 5 mM to 500 mM; (ii) the concentration of the reactive compound, preferably selected from Tris or spermidine, is selected to control the length of the released nucleic acid molecules, wherein a lower concentration of the reactive compound in the lysis composition and / or lysis mixture results in a higher degree of fragmentation of the released nucleic acid molecules. 16. The method of claim 14 or 15, wherein the method comprises one or more of the following: (Item 17) The lysed composition has the following characteristics: (a) the salt has the following characteristics: (i) the salt is a monovalent or divalent salt; (ii) the salt is a chaotropic salt or a non-chaotropic salt; (iii) the salt is a non-buffering salt; (iv) the salt is an alkali metal salt, optionally an alkali metal halide; and / or (v) the salt is a chloride salt, optionally selected from sodium chloride, potassium chloride, lithium chloride and cesium chloride, preferably the salt is sodium chloride. having one or more of the following; (b) the surfactant has the following characteristics: (i) the surfactant is an ionic or nonionic surfactant; (ii) the surfactant is an anionic surfactant, and / or (iii) the surfactant is a sulfate or sulfonate of a fatty alcohol, such as sodium dodecyl sulfate, sodium dodecyl sulfonate, or dodecylbenzenesulfonic acid, preferably sodium dodecyl sulfate; having one or more of the following; (c) The dissolution composition comprises the reactive compound according to item 15, which is a buffering agent, or the dissolution composition comprises a buffering agent, optionally the buffering agent has a pKa value in the range of 5.0 to 10.5, optionally the pKa value is selected from 5.5 to 10.0, 6.0 to 10.0, 6.5 to 10.0, 7.0 to 9.8, or 7.2 to 9.8, and optionally the buffering agent is Tris; and / or (d) Optionally, the dissolution composition comprises a chelating agent, preferably the chelating agent is an aminopolycarboxylic acid, more preferably ethylenedinitrilotetraacetic acid (EDTA). 17. The method according to one or more of items 14 to 16, comprising one or more of: (Item 18) In the dissolution composition and / or the dissolution mixture, (a) the salt is present at a concentration of at least 15 mM, at least 50 mM, at least 75 mM or at least 100 mM, optionally with the concentration of the salt in the range of 15 mM to 500 mM, e.g., 50 mM to 350 mM, 75 mM to 300 mM, 100 mM to 250 mM or 125 mM to 200 mM; and / or (b) The method according to item 17, wherein the surfactant is present in the lysis composition and / or lysis mixture at a concentration of at least 0.01%, at least 0.02%, at least 0.03%, or at least 0.04%, and optionally the concentration of the surfactant is in the range of 0.01 to 3.0%, for example, 0.02 to 2.75%, 0.03 to 2.5%, or 0.04 to 2.0%. (Item 19) 19. The method according to one or more of items 3 to 18, wherein the lysis composition is prepared by combining a lysis solution with the proteolytic enzyme, and optionally preparing the lysis composition further comprises adding water or a dilution buffer. (Item 20) The following features: (i) the dissolution solution contains a reactive compound, and optionally - the reactive compound is a reactive compound as defined in item 15, and / or - the reactive compound is present in the lysis solution at a concentration of at least 5 mM, in particular at least 10 mM, at least 20 mM or at least 30 mM; (ii) the lysis solution comprises a reactive compound that is a buffering agent, or the lysis solution further comprises a buffering agent; (iii) the dissolution solution comprises a reactive compound as defined in item 15, and optionally - the reactive compound comprises one or more primary amine groups, preferably one primary amine group, and one, two or three hydroxyl groups, preferably three hydroxyl groups, and optionally the reactive compound is present in the lysis solution at a concentration of at least 10 mM, such as at least 20 mM, at least 40 mM, at least 60 mM or at least 75 mM; or - the reactive compound comprises two primary amine groups and preferably one secondary amine group, and optionally the reactive compound is present in the lysis solution at a concentration of at least 0.25 mM, in particular at least 0.5 mM, at least 1 mM, at least 1.25 mM or preferably at least 1.5 mM; (iv) the lysis solution contains a salt, and optionally - the salt is as defined under item 17 a); - the salt is present in the lysis solution at a concentration of at least 50 mM, optionally at least 75 mM, at least 100 mM, at least 150 mM, at least 200 mM or at least 250 mM; (v) the dissolution solution contains a surfactant, and optionally - the surfactant is a surfactant as defined under item 17 b); - the surfactant is an ionic or non-ionic surfactant, preferably an anionic surfactant; - the surfactant is present in the lysis solution at a concentration of at least 0.01%, at least 0.01%, at least 0.02%, at least 0.03%, or at least 0.04%, optionally with a concentration of the surfactant in the range of 0.01-3.0%, for example 0.02-2.75%, 0.03-2.5%, or 0.04-2.0%; 20. The method of claim 19, wherein the method comprises one or more of the following: (Item 21) The following features: (i) the nucleic acid comprises or consists essentially of DNA; (ii) the fixed biological sample is a sample containing cells; (iii) the fixed biological sample is a solid fixed biological sample, in particular a fixed tissue sample; (iv) the fixed biological sample is a liquid fixed biological sample; (v) the fixed biological sample is a sample fixed using a cross-linking fixative, optionally an aldehyde-containing fixative, such as formaldehyde and / or paraformaldehyde; and / or (vi) the fixed biological sample is an FFPE sample; 21. The method according to one or more of items 1 to 20, comprising one or more of: (Item 22) step (d) comprising: binding the nucleic acid to a solid phase; optionally washing the nucleic acid bound to the solid phase; and eluting the nucleic acid from the solid phase; and / or The method further comprises (e) analyzing the purified nucleic acid, wherein the analyzing step optionally comprises: (i) amplifying said nucleic acid, preferably using a polymerase enzyme; (ii) amplifying the nucleic acid using large amplicon PCR and / or short amplicon PCR, optionally wherein the large amplicon PCR is for nucleic acid molecules having a size of at least 500 nt and the short amplicon PCR is for nucleic acid molecules having a size of less than 500 nt, preferably 300 nt or less, 200 nt or less, or 150 nt or less; and / or (iii) performing a next-generation sequencing method, said method optionally comprising: (aa) attaching unique molecular identifier sequences to said nucleic acids, wherein each nucleic acid molecule comprises a different unique molecular identifier sequence; (bb) amplifying the nucleic acid containing the attached unique molecular identifier sequence; and (cc) sequencing said nucleic acid. to carry out, including 22. The method according to one or more of items 2 to 21, including one or more of: (Item 23) (a) lysing the fixed biological sample, wherein the lysing comprises digestion with a proteolytic enzyme, and the lysing step (a) comprises preparing a lysis mixture, The lysis mixture is (i) the fixed biological sample, and (ii) a dissolution composition containing the protease Including, Incubating the lysis mixture at an elevated temperature between 35 and 75°C for at least 30 minutes, and optionally completing step (a) in 120 minutes or less; (b) heating the dissolved sample to reverse crosslinking, wherein the dissolved sample is heated at a temperature in the range of 80-110°C, e.g., 85-100°C, for 30-120 minutes, e.g., 45-90 minutes or 50-70 minutes; (c) adding a proteolytic enzyme and carrying out proteolytic digestion, wherein step (c) comprises incubation at an elevated temperature between 35 and 75°C, such as between 40 and 70°C or between 50 and 70°C, for at least 5 minutes, such as at least 10 minutes or at least 15 minutes; The incubation in step (c) is shorter than the incubation in step (a) and / or the incubation temperature in step (c) is higher than that in step (a). 23. The method according to one or more of items 1 to 22, comprising: (Item 24) 24. The method according to item 23, wherein the fixed biological sample is a solid fixed biological sample, optionally an FFPE sample, and the method comprises carrying out at least one enzymatic treatment step different from a proteolytic digestion step between step (b) and step (c), wherein the at least one enzymatic treatment step comprises the use of one or more of a glycosylase, a nuclease, a lipase or a combination thereof. (Item 25) 25. The method according to item 24, wherein the at least one enzyme treatment step comprises the use of a DNA glycosylase, such as a uracil DNA glycosylase, preferably a uracil-N-glycosylase, and the DNA glycosylase treatment step is carried out at an elevated temperature and is completed in 30 minutes or less, 20 minutes or less, 15 minutes or less, or 10 minutes or less, and optionally, the method comprises the step of diluting the sample obtained from step (b) in order to perform the DNA glycosylase treatment step prior to step (c). (Item 26) 26. The method according to item 24 or 25, wherein at least one nuclease treatment step is carried out between step (b) and step (c), and the nuclease is a ribonuclease, such as ribonuclease A. (Item 27) said enzymatic treatment step, which is different from the proteolytic digestion step carried out between step (b) and step (c), - adding a DNA glycosylase, more preferably a uracil DNA glycosylase; - adding a ribonuclease, e.g., ribonuclease A; 27. The method according to any one of items 24 to 26, comprising: (Item 28) - step (a) is characterized in that the protease is a protease, for example proteinase K, step (a) is completed in 120 minutes or less, optionally 100 minutes or less, 90 minutes or less, or 70 minutes or less, and heating is carried out at a temperature in the range of 35 to 75°C, optionally 40 to 70°C; - step (c) is characterized in that the protease is a protease, for example proteinase K, step (c) is completed in 30 minutes or less, optionally 20 minutes or less, and heating is carried out at a temperature in the range of 35 to 75°C, optionally 40 to 70°C or 50 to 70°C; 28. The method according to one or more of items 23 to 27, wherein the incubation in step (c) is shorter than the incubation in step (a) and the incubation temperature in step (c) is higher than that in step (a). (Item 29) 29. The method of claim 28, wherein the sample is agitated during incubation in steps (a) and (c). (Item 30) lysing the fixed biological sample according to a lysis method as defined in one or more of items 23 to 29, preferably as defined in one or more of items 26 to 29, After step (c), the method comprises: (d) purifying DNA from the lysed sample, wherein step (d) comprises: - binding the DNA to a solid phase; - optionally washing the DNA bound to the solid phase; - eluting the DNA from the solid phase; Steps including and said method comprises: (e) analyzing the purified DNA, said analyzing step comprising: (i) amplifying the DNA, preferably using a polymerase enzyme; (ii) amplifying the DNA using large amplicon PCR and / or short amplicon PCR, optionally wherein the large amplicon PCR is for DNA molecules having a size of at least 500 nt and the short amplicon PCR is for DNA molecules having a size of less than 500 nt, preferably 300 nt or less, 200 nt or less, or 150 nt or less; and / or (iii) performing a next-generation sequencing method; the step including one or more of 30. The method according to one or more of items 2 to 29, further comprising: (Item 31) Step (e) comprises performing a next generation sequencing method, said sequencing method comprising: (aa) attaching unique molecular identifier sequences to said DNA molecules, wherein each DNA molecule comprises a different unique molecular identifier sequence; (bb) amplifying the DNA molecules containing the attached unique molecular identifier sequences; and (cc) sequencing said DNA molecule. Item 31. The method according to Item 30, comprising:

Claims

[Claim 1] The invention described in this specification.

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