Patent Application
20230140574
The present invention pertains to methods for lysing a fixed biological sample and/or obtaining purified nucleic acids from a fixed biological sample.
Fixation of biological samples allows long-term preservation and archiving of the samples. Fixation is regularly achieved by protein-precipitating or protein-crosslinking compounds such as acids, alcohols, ketones or other organic substances 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 percent by weight aqueous solution referred to as “formalin”), which can be followed by embedding of the fixed material in paraffin (called “formalin-fixed, paraffin-embedded” (FFPE) material) is a widely used fixation technology. Formaldehyde-based fixation has the advantage that tissue structures are attained comparatively well in the fixation. Fixation involving cross-linking fixatives are also used for fixing and thus preserving liquid samples. A commonly used preservative that comprises ethanol and formaldehyde is SurePath®.
Fixed biological samples (e.g. FFPE samples) are a valuable resource for investigating diseases. However, such samples are primarily used for histopathology, and are poorly suited for nucleic acid extraction and analysis, due to extensive damage and crosslinking of DNA resulting from the exposure to formaldehyde and storage. The most critical quality issue with DNA extracted from fixed biological samples is the crosslinking of the nucleic acids to protein and to each other. This crosslinking makes the DNA inaccessible to enzymes during reactions such as PCR, leading to very poor performance, and potentially false results in tests. Consequently, the efficient release and purification of nucleic acids (DNA or RNA) fixed biological samples (solid or liquid) is difficult. For numerous investigations on a molecular level, in particular for clinical or diagnostic applications, however, analysis of the nucleic acids is of great importance.
Methods to extract nucleic acids such as DNA from such fixed biological samples must reverse the cross-links that were introduced due to the fixation while preventing any further damage to the nucleic acids. A standard method of de-crosslinking DNA is to treat the crude lysate with heat e.g. for 1 hour at 90° C. or 4 hours at 80° C. and such de-crosslinking steps have all been used to dissolve the cross-links present in the fixed biological samples. Numerous commercial kits and methods for the extraction of FFPE are available in the art. All kits use a combination of heat, enzymes, and chemical lysis in order to remove the tissue from paraffin, digest the tissue, and purify the DNA. Further methods for isolate/release nucleic acids from fixed samples have been described in WO 2007/068764, WO 2014/072366, WO2005/075642, WO 2001/46402 and US 2005/0014203. Most of the available kits and methods have to make tradeoffs in terms of total yield and/or quality of DNA that is extracted (i.e. a higher DNA yield is often accompanied by fragmentation, while largely intact, high molecular weight DNA is often not completely de-crosslinked and is of worse quality in PCR applications). Further Important indicators for assessing the quality of extracted nucleic acid are the performance in PCR and/or NGS applications.
It is an object to provide a method for lysing a fixed biological sample that effectively releases contained nucleic acids, such as DNA and/or RNA, in particular DNA. It is furthermore an object of the present invention to provide a method for purifying nucleic acids, such as DNA and/or RNA from a fixed biological sample. In embodiments, it is an object of the present invention to avoid a drawback from the prior art methods. In embodiments, the methods of the present invention provide an improvement in at least one criterion, e.g. yield, fragmentation and/or performance in a subsequent nucleic acid analysis method, such as PCR and/or next generation sequencing.
The present invention provides an improved method for lysing a fixed biological sample, wherein the fixed biological sample comprises crosslinks between nucleic acid molecules and protein molecules. These crosslinks are present because of the used fixation (e.g. formaldehyde and/or paraformaldehyde based fixation). It was found that the lysis and digestion of fixed biological samples can be significantly improved by a step-wise sequential lysis process, wherein the fixed biological sample is digested with a proteolytic enzyme, followed by heating to reverse crosslinks, followed by adding a proteolytic enzyme to perform an additional proteolytic digestion. The examples show that performing a second proteolytic digestion after performing a crosslink reversal step results in important and unexpected improvements.
According to a first aspect of the invention, a method is provided for lysing a fixed biological sample, wherein the fixed biological sample comprises crosslinks between nucleic acid molecules and protein molecules due to the fixation, the method comprising
optionally wherein one or more additional treatment steps are performed between steps (b) and (c).
As is 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 fixed cell-containing liquid sample).
According to a second aspect of the invention, a method is provided for obtaining purified nucleic acids from a fixed biological sample, wherein the fixed biological sample comprises crosslinks between nucleic acid molecules and protein molecules due to the fixation, said method comprising lysing the fixed biological sample according to the lysis method of the first aspect, steps (a) to (c), wherein subsequent to step (c) of the method of the first aspect the method comprises
As is disclosed herein, one or more additional treatment steps (e.g. at least one further enzymatic treatment step different from a proteolytic digestion step) may be optionally performed between steps (b) and (c) of the method according to the first aspect. The step-wise lysis/digestion procedure according to the first aspects 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 from the digested sample in step (d) of the method according to the second aspect with high quality and/or high yield. Thereby, an improved method is provided for purifying nucleic acids, such as DNA, from a fixed biological sample.
A third aspect of the invention pertains to the use of a proteolytic enzyme, such as proteinase K, for performing a proteolytic digestion after lysing a fixed biological sample, wherein such prior lysis involves digestion of the fixed biological sample with a proteolytic enzyme and heating the lysed sample to reverse crosslinks, preferably in a method according to the first or second aspect of the present invention. As disclosed herein, the fixed biological sample comprises crosslinks between nucleic acid molecules and protein molecules due to the fixation. According to one embodiment, fixation involved the use of formaldehyde or paraformaldehyde.
A fourth aspect of the present invention pertains to the use of a glycosylase, such as a DNA glycosylase, preferably a uracil DNA glycosylase, for performing an enzymatic treatment, wherein the enzymatic treatment is completed in 30 min or less, 20 min or less, 15 min or less or 10 min or less. Such use may be performed in a method according to the first or second aspect of the present invention. In a preferred embodiment, the uracil glycosylase is a uracil-N-glycosylase.
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 application, are given by way of illustration only.
The present invention provides an improved method for lysing a fixed biological sample, wherein the fixed biological sample comprises crosslinks between nucleic acid molecules and protein molecules due to the fixation. Furthermore, improved methods are described for obtaining purified nucleic acids such as DNA and/or RNA from a fixed biological sample.
The invention provides improvements in yield and/or quality of nucleic acids extracted from a fixed biological sample, such as FFPE tissue, enabling better analysis of the material, for instance using PCR and NGS sequencing methods. It was in particular found that an additional round of proteolytic digestion subsequent to a decrosslinking step (that is performed after a first proteolytic digestion step) provides important improvements.
In addition, the present disclosure provides methods that allow to control core parameters that are relevant for the analysis of fixed biological samples. In particular, it was found that by adjusting the lysis conditions in the first step it is possible to control the size of the released and this obtained nucleic acids. This important finding allows to optimize the methods for either short or long amplicon PCR systems. Furthermore, adjusting the lysis conditions as disclosed herein also improves the performance of enzymatic steps during the extraction, thereby enabling e.g. the in-process use of Uracil-n-glycolysase to remove artifacts caused by formalin cross-linking.
The different aspects and embodiments of the invention disclosed herein therefore make important contributions to the art.
According to a first aspect of the invention, a method is provided for lysing a fixed biological sample, wherein the fixed biological sample comprises crosslinks between nucleic acid molecules and protein molecules due to the fixation, the method comprising
optionally wherein one or more additional treatment steps are performed between steps (b) and (c).
The individual steps and preferred embodiments will now be described in detail.
The method according to the first aspect comprises in step (a) lysing the fixed biological sample, wherein lysis involves digestion with a proteolytic enzyme. During lysis step (a), the fixed biological sample is degraded such that nucleic acids are released.
Step (a) can be performed using lysis and proteolytic digestion conditions known in the art. Assisting the lysis of the fixed biological sample by digestion with a proteolytic enzyme is highly advantageous, as it allows degrading protein and peptide crosslinked to the nucleic acid. As disclosed herein and known in the art, such cross-links in the fixed biological sample are induced due to the used fixation, e.g. when using a crosslinking fixative. For instance, using a crosslinking fixative such as an aldehyde containing fixative, e.g. formaldehyde, leads to crosslinking between protein and nucleic acid, as well as between nucleic acids. By performing lysis step (a), nucleic acid-crosslinked protein can be degraded, as well as partially crosslinks between nucleic acids.
According to one embodiment, lysis step (a) comprises preparing a lysis mixture, wherein the lysis mixture comprises (i) the fixed biological sample, and (ii) a lysis composition comprising the proteolytic enzyme. Any contacting order of the fixed biological sample and the components of the lysis composition is encompassed by this embodiment for preparing the lysis mixture in step (a). E.g. the lysis composition may be prepared separate and the prepared lysis composition may then be contacted with the fixed biological sample or vice versa. Thus, the lysis composition can be first adjusted without affecting the fixed biological sample. It is furthermore within the scope of this embodiment that the lysis mixture is prepared by contacting the fixed biological sample in any order with the proteolytic enzyme and/or the further components of the lysis composition to prepare the lysis mixture.
The proteolytic enzyme supports the digestion of the fixed biological sample and improves the release of the comprised nucleic acids, such as DNA and/or RNA. In embodiments, the proteolytic enzyme is a protease, such as proteinase K. Proteases may either qualify as endopeptidases, which cleave peptide bonds within the protein, and/or are exopeptidases, which cleave off amino acids from the ends of a protein chain. Typically, proteases are further categorized by mechanism, such as serine proteases (e.g., chymotrypsin, trypsin, elastase, subtilisin, and proteinase K); cysteine (thiol) proteases (e.g., bromelain, papain, cathepsins, parasitic proteases, and bacterial virulence factors); aspartic proteases (e.g., pepsin, cathepsins, rerun, fungal and viral proteases); and metalloproteases (e.g., thermolysin). Such proteases may be used in the context of the present invention. In a core embodiment, the proteolytic enzyme used in step (a) is a serine protease. According to one embodiment, the proteolytic enzyme 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 the digestion of fixed biological samples that comprise crosslinks because of the applied fixation. Proteinase K advantageously remains active even at higher temperatures and in the presence of a detergent, denaturing agents such as urea and chaotropic agents and salt. Proteinase K was also used in the present examples. All disclosures described herein for a proteolytic enzyme or protease in general specifically apply and particularly refer to the preferred embodiment proteinase K. As is apparent from the listed examples, the proteolytic enzyme may be a heat-stable protease. This allows to support the lysis and digestion in step (a) by heating the lysis mixture as is also described below. Examples of suitable heat-stable proteases are proteinase K, trypsin, chymotrypsin, papain, pepsin, pronase and endoproteinase Lys-C. The heat-stable protease may be inactivated at or above an inactivation temperature, which may lie in a range of 85° C.-120° C. Suitable inactivation temperatures for different proteases are described in the art for different proteases.
According to one embodiment, step (a) comprises heating to assist the digestion by the proteolytic enzyme. The prepared lysis mixture may thus be heated to a suitable elevated temperature that supports the lysis/digestion of the fixed biological sample. The lysis/digestion temperature is chosen such that the proteolytic enzyme is active. In embodiments, heating in step (a) is performed at a temperature in the range of 35-75° C., such as 40-70° C. or 45-65° C. As is known in the art, heating the lysis may enhance the activity of the proteolytic enzyme, thereby allowing for a fast and efficient sample lysis and digestion in step (a). According to one embodiment, digestion with a proteolytic enzyme in step (a) comprises heating to a temperature of at least 30° C., in particular at least 35° C., at least 40° C., at least 45° C., preferably at least 50° C.
According to one embodiment, the lysis mixture is incubated in step (a) for at least 15 min, such as at least 20 min, at least 25 or at least 30 min. In embodiments, the lysis mixture is incubated in step (a) for at least 45 min or at least 50 min. Advantageously, step (a) may be performed within a short timeframe. According to a one embodiment, step (a) is completed in 120 min or less. Step (a) may be completed in 100 min or less, 90 min or less or 70 min or less, such as about 60 min. Incubation may occur at an elevated temperature as described above. Furthermore, incubation, preferably assisted by heating as disclosed herein, may be supported by agitation. Therefore, the lysis mixture may be agitated during incubation in step (a). According to one embodiment, lysis and digestion with a proteolytic enzyme in step (a) comprises agitating and heating the lysis mixture at a temperature in the range of 35-75° C., such as 40-70° C. or 45-65° C. for 15 to 120 min, such as 20 to 100 min, 30 to 90 min or 45 to 75 min. Agitation may be performed by any method, such as shaking, rolling, inverting, etc.
The person skilled in the art can chose a suitable concentration of the proteolytic enzyme in the lysis composition and lysis mixture and suitable concentrations are known in the art. According to one embodiment, the proteolytic enzyme of step (a) is present in the lysis mixture and/or lysis composition at a concentration of at least 0.5 mg/mL, such as 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 proteolytic enzyme used in step (a) is a serine protease, such as proteinase K, present in the lysis mixture and/or lysis composition at a concentration selected from 1-10 mg/mL, such as 1.5-7.5 mg/mL, 2-7 mg/mL, 3-6 mg/mL or 3.5-5 mg/mL. Such concentrations may be used when the fixed biological sample is a solid fixed biological sample, such as a fixed tissue sample. Such concentrations may also be used in the lysis mixture when processing fixed liquid biological samples. The lysis composition is in such case adapted to take into account any dilution effects because of the fixed liquid biological sample. For example, a dilution of the proteolytic enzyme by the liquid fixed biological sample may be compensated to providing a higher concentration of proteolytic enzyme in the lysis composition. Suitable concentrations can be readily calculated by the skilled person considering the suitable concentrations of proteolytic enzyme in the lysis mixture of step (a).
As disclosed above, step (a) comprises in a core embodiment preparing a lysis mixture, wherein the lysis mixture comprises (i) the fixed biological sample, and (ii) a lysis composition comprising the proteolytic enzyme.
In embodiments, the lysis mixture comprises the components of the lysis composition in the same or similar concentration as the lysis composition (e.g. allowing a deviation of up to 50%, such as up to 40%, up to 30%, or up to 20% or up to 10%). This is in particular when providing a fixed biological sample that is a solid sample, e.g. a fixed tissue sample. For instance, a solid fixed biological sample does not result in dilution of the compounds present in the lysis composition, i.e. the concentration of the compounds present in the lysis composition is the same or about the same as in the lysis mixture (with the solid fixed biological sample). In case the sample is a liquid fixed biological sample, the liquid sample dilutes the compounds of the lysis composition, so that typically a higher concentration of compounds is used in the lysis composition to take into account such dilution effect. For instance, a higher concentration of compounds can be provided in the lysis composition and/or a higher volume ratio of lysis composition can be provided in order to establish suitable conditions for lysis in the lysis mixture of step (a).
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. An according pH range has been found to be particularly suitable for lysis step (a) according to the method of the present disclosure as is also demonstrated in the examples. In embodiments the pH is in a range of such as 7.0-8.0. In further embodiments, the pH is in the range of 8.0 to 9.0, such as 8.2 to 8.8. In particular, applying such alkaline pH in the lysis composition and thus in the lysis mixture led to less fragmentation as compared to more acidic conditions.
According to a preferred embodiment, the lysis composition in step (a) further comprises one or more, preferably all of the following compounds:
In a particular embodiment, the lysis composition comprises a salt, a detergent and a buffering agent. Optionally, the lysis composition further comprises a chelating agent, such as EDTA. The lysis composition has been found to be suitable for the method of the present disclosure, as is demonstrated in the examples. Individual embodiments of the compounds of the lysis composition are disclosed below.
According to a preferred embodiment, the lysis composition comprises at least one reactive compound which may act as a formaldehyde scavenger. A reactive compound is in particular capable of reacting with the fixative and/or reacting with crosslinks induced by the fixative. Preferably, one or two reactive compounds are comprised in the lysis composition of step (a). However, a reactive compound can also be added separately to the lysis mixture, e.g. in form of a solution or solid comprising the reactive compound.
According to a preferred embodiment, the reactive compound reacts with the fixative or a chemical moiety released in heating step (b) and/or with a crosslink induced by the fixative, such as a crosslink induced by an aldehyde-containing fixative, such as formaldehyde. For the biological sample may be fixed with a fixative, such as an aldehyde-containing fixative, e.g. formaldehyde or a formaldehyde derivative, which induces crosslinks between nucleic acid molecules and protein molecules or in-between protein or nucleic acid molecules. The reactive compound of the present invention can advantageously react with the fixative, e.g. formaldehyde. The fixative or chemical moiety derived therefrom that reacts with the reactive compound may 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, which greatly favors de-crosslinking (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, scavenging the fixative or chemical moiety derived therefrom that is released in decrosslinking step (b). Alternatively or additionally, the reactive compound may reacts with a crosslink that is induced by the fixative. In particular, the reactive compound may directly break down fixative-induced crosslinks comprised in the fixed biological sample, preferably in step (a) and/or step (b). Reaction of the reactive compound with crosslinks induced by the fixative preferably takes place during step (b) of the method of the present disclosure. Furthermore, the reactive compound can react with a fixative-induced crosslink that has formed throughout the reversible crosslinking reaction. For instance, an aldehyde-containing fixative, such as formaldehyde, can lead to a formation of an aminal group between two biological molecules, e.g. protein and DNA. The aminal group can reversibly form an imine group under release of a protonated amine group of one of the biological molecules. The imine group can then react with a reactive compound according to the present disclosure and thereby react with a crosslink induced by the fixative. Further conversion can then lead to imine base formation at the reactive compound under release of the second biomolecule. Hence, the crosslink induced by the fixative is reversed and the fixative is bound to the reactive compound. Advantageously the biomolecules, in particular nucleic acid, is released.
According to a preferred embodiment, the reactive compound comprises a nucleophilic group, preferably an amine group. The reactive compound may be selected from a nucleophilic agent as described in WO 2007/068764 A1. A reactive compound comprising a nucleophilic group was found particularly suitable to react with the fixative or chemical moiety released in heating step (b) and/or with a crosslink induced by the fixative, in particular a crosslink induced by an aldehyde-containing fixative, such as formaldehyde.
According to a preferred embodiment, the reactive compound comprises one or more primary amine groups, optionally one primary amine group and one or more hydroxyl groups, preferably three hydroxyl groups. The reactive compound may furthermore comprise two primary amine groups and optionally one secondary amine group. Such reactive compounds have been found to be particularly suitable for the present invention as demonstrated in the Examples. In particular, nucleic acid crosslinks were efficiently removed by the method of the present disclosure using such a reactive compound, leading to high quality which is particularly suitable for analysis methods, e.g. for PCR or NGS. Exemplary reactive compounds, which can be advantageously used are 2-amino-2-(hydroxymethyl)propane-1,3-diol or a derivative thereof or 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.
According to a preferred embodiment, the reactive compound comprises at least two nucleophilic groups, preferably nucleophilic groups having different nucleophile strength. Such an embodiment has been found advantageous in the Examples, in particular resulting in nucleic acids that are of high quality which is particularly suitable for analysis methods, e.g. PCR or NGS. A first nucleophilic group of the reactive compound may be stronger than a second nucleophilic group. In an according reactive compound one or more types of nucleophilic groups can be present. For instance, one or two first nucleophilic groups and one, two or three second nucleophilic groups. According to one embodiment, the reactive compound comprises a first nucleophilic group which is a primary amine group and a second nucleophilic group which is a hydroxyl group or a secondary amine group. According to a particular embodiment, the reactive compound comprises a primary amine group and a hydroxyl group, preferably three hydroxyl groups. According to another embodiment, the reactive compound comprises 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.
According to one embodiment, the reactive compound comprises a polyamine, preferably a natural polyamine, such as spermidine. Such a reactive compound has been found advantageous to modify the nucleic acid fragmentation, in particular by increasing the fragment size as demonstrated in the Examples.
A preferred reactive compound of the present disclosure is 2-amino-2-(hydroxymethyl)propane-1,3-diol, also referred to as Tris. Kawashima et al., 2014 describe that Tris acts as a formaldehyde scavenger by producing a Schiff base, cyclic hemiaminal and cyclic acetal adducts. Moreover, Tris may be directly involved in breaking down crosslinks, as a kind of transamination catalyst. Furthermore, Tris has the advantage that it can form a cyclic compound with the aldehyde-containing fixative, such as formaldehyde, and thus one molecule of Tris scavenges one molecule for aldehyde-containing fixative. As demonstrated in the Examples, 2-amino-2-(hydroxymethyl)propane-1,3-diol also allows to modify the nucleic acid fragmentation and also suitability for subsequent nucleic acid analysis methods. For instance, using 2-amino-2-(hydroxymethyl)propane-1,3-diol resulted in high PCR and NGS performance.
According to one embodiment, the lysis composition comprises more than one reactive compound. It may comprise two reactive compounds, wherein optionally 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.
According to one embodiment, the lysis mixture comprises a reactive compound in a concentration suitable for reacting with at least a portion of the fixative, in particular in heating step (b). According to one embodiment, the lysis mixture comprises one or more reactive compounds in a concentration suitable for reacting with at least a portion of the fixative, in particular in heating step (b). In embodiments, the portion of the fixative corresponds to at least 15% of the fixative present in the fixed biological sample, in particular at least 25%, at least 35%, at least 45%, at least 55%, at least 65%, at least 75% of the fixative present in the fixed biological sample.
According to a one embodiment, the reactive compound is present in the lysis composition and/or the lysis mixture in a concentration in a range of 1 mM to 500 mM or 5-500 mM in the lysis composition and optionally also in the lysis mixture.
According to one embodiment, the lysis mixture and/or the lysis composition comprises a reactive compound, which comprises two primary amine groups and preferably one secondary amine group, such as spermidine. Said reactive compound (e.g. spermidine) may be comprised in a concentration of at least 0.5 mM, such as at least 1 mM, at least 1.5 mM or at least 2 mM. The concentration may be selected from 0.25-25 mM, such as 0.5-20 mM, 1-15 mM, 1.25-10 mM or 1.5-7 mM, such as about 2.5 mM. Such a concentration has been found particularly advantageous for sample lysis, resulting in nucleic acids of high fragment size. Furthermore, by varying said concentration, the fragment size can be flexibly controlled. The lysis composition may comprise a buffering agent or a reactive compound which is also a buffering agent.
According to one embodiment, the lysis mixture and/or lysis composition comprise a reactive compound which 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. In embodiments, it is comprised in the lysis mixture and/or lysis composition in a concentration of at least 3 mM, such as at least 5 mM, at least 7 mM, or at least 10 mM. A suitable concentration may be selected from 3 mM-100 mM, in particular 5 mM-50 mM, 7 mM-30 mM, 9 mM-25 mM or preferably 10 mM-20 mM, such as 10 mM-15 mM. Such concentrations are advantageous for sample lysis, resulting in nucleic acids of small fragment size and high quality, in particularly for a nucleic acid analysis method involving amplification of small fragments, e.g. a short amplicon PCR, as disclosed herein. Furthermore, the high nucleic acid quality leads to enhanced NGS performance. Such applications are also disclosed in conjunction with the method according to the fifth aspect.
According to one embodiment, the lysis mixture and/or lysis composition comprise a reactive compound which 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. In embodiments, it is comprised in the lysis mixture and/or lysis composition in 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 10 mM-750 mM, such as 20 mM-500 mM, 30 mM-300 mM, 50 mM-250 mM or preferably 75 mM-200 mM, such as about 100 mM to 150 mM. This concentration has been found advantageous for sample lysis, resulting in nucleic acids having a high quality, particularly for a nucleic acid analysis method involving amplification of large and/or small fragments, in particular large amplicon PCR, as disclosed herein. It is referred to the method according to the sixth aspect. Furthermore, the high nucleic acid quality leads to enhanced NGS performance.
According to one embodiment, the reactive compound comprised in the lysis composition is additionally a buffering agent. In particular, both functions are advantageously fulfilled by such a compound, in particular buffering the lysis mixture and providing a reactive compound to react with the fixative or chemical moiety released in heating step (b) and/or with a crosslink induced by the fixative, in particular a crosslink induced by an aldehyde-containing fixative, such as formaldehyde. Such a compound may preferably comprise 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. According to one embodiment, the lysis mixture and/or the lysis composition comprises a compound, which is a reactive compound and also buffering agent at a concentration selected from the range of 3-500 mM. Suitable concentration ranges were described above. As disclosed herein, the choice of the concentration of said reactive compound allows to control the fragment size of the nucleic acid molecules released during lysis.
Optionally, the lysis composition comprises two reactive compounds. In such an embodiment, preferably the first reactive compound is selected from a reactive compound, which comprises two primary amine groups and preferably one secondary amine group, such as spermidine, and the second reactive compound is selected from a reactive compound which 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 sample lysis and obtaining nucleic acids having an increased size or less fragmentation.
According to one embodiment, the lysis composition comprises the reactive compound in a concentration leading to a concentration in the lysis mixture suitable for reacting with at least a portion of the fixative, in particular in heating step (b). According to one embodiment, the above disclosed concentrations of the reactive compound in the lysis mixture correspond to the concentrations of the reactive compound in the lysis composition, in particular in case the fixed biological sample is a solid fixed biological sample, such as a fixed tissue sample. According to one embodiment, the lysis composition comprises the reactive compound in a concentration selected from the range of 0.25-500 mM, wherein the fixed biological sample is a solid fixed biological sample, in particular a fixed tissue sample. As demonstrated in the Examples, such a concentration of reactive compound, e.g. 2-amino-2-(hydroxymethyl)propane-1,3-diol and/or sperimidine, has been found advantageous for lysis of the fixed tissue samples, in particular by allowing to modify the nucleic acid size/fragmentation and obtaining nucleic acids of high quality, in particular suitable for a nucleic acid analysis method, e.g. PCR, NGS.
In case the fixed biological sample is a liquid fixed biological sample, the concentration of the reactive compound in the lysis composition in adapted to establish an above disclosed concentration in the lysis mixture. Alternatively or additionally, a higher volume ratio of lysis composition may be added to a liquid fixed biological sample in order to establish a concentration of the reactive compound in the lysis mixture as disclosed above. According to one embodiment, the lysis composition comprises the reactive compound in a concentration suitable to be in the lysis mixture present at a concentration selected from the range of 0.25-500 mM, wherein the fixed biological sample is a liquid fixed biological sample. According adjustment of the concentration of the reactive compound is well within the abilities of the skilled person.
According to one embodiment, the lysis composition and/or lysis mixture has a pH selected from the range of 6.0 to 9.5 and the lysis composition further comprises a reactive compound, in particular reacting with a fixative and/or reacting with a crosslink induced by the fixative, wherein the reactive compound comprises a nucleophilic group, in particular a primary amine group. Exemplary reactive agents 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 comprised in the lysis mixture and/or lysis composition in a concentration of 5-50 mM preferably 10-20 mM and the lysis composition has a pH in the range of 6.0 to 9.5, preferably 7.0 to 8.0. According to another embodiment, the reactive compound is comprised in the lysis mixture and/or lysis composition in a concentration of 60-250 mM, preferably 75-200 mM and the lysis composition has a pH in the range of 7.0 to 9.5, preferably 8.0 to 9.0. These embodiments are particularly advantageous, as they allows for improved release of a nucleic acid, e.g. DNA, from the fixed biological sample. By providing a neutral or slightly alkaline pH and such a reactive compound, the fixative of the fixed biological sample is advantageously removed by the reaction equilibrium by reacting with the reactive compound, allowing for releasing the nucleic acid with less fragmentation than at acidic conditions.
According to a preferred embodiment, the lysis composition comprises a salt. Salt supports the lysis. The salt is preferably provided to the lysis mixture by the lysis composition. The salt may preferably be a mono- or divalent salt. Preferably, the salt is a chaotropic or non-chaotropic salt. According to a preferred embodiment, the salt is a non-buffering salt. Also a mixture of salts can be used. A particular salt may be selected from an alkali metal salt, optionally an alkali metal halide. According to a preferred embodiment, the salt is a chloride salt, optionally selected from sodium chloride, potassium chloride, lithium chloride and cesium chloride, wherein preferably the salt is sodium chloride. Such salts have been found suitable as is demonstrated by the Examples.
The concentration of the salt in the lysis composition depends on the type of fixed biological sample from which the nucleic acid is released and can be flexibly adjusted by the skilled person. According to a preferred embodiment, the salt is present in the lysis composition and/or the lysis mixture in a concentration of at least 15 mM, in particular at least 30 mM, at least 50 mM, preferably at least 100 mM. Suitable concentration ranges of salt in the lysis composition and/or the lysis mixture may be selected from 15-500 mM, in particular 30-440 mM, 50-300 mM or preferably 100-250 mM, such as about 150 mM. According to one embodiment, the salt is present in the lysis composition and/or the lysis mixture in a concentration of less than 500 mM, in particular less than 400 mM, less than 300 mM or preferably less than 250 mM, such as less than 200 mM. Such salt concentrations have been found advantageous as demonstrated in the Examples for sample lysis. For instance, by applying such a salt concentration the digestion using a DNA glycosylase, such as a uracil DNA glycosylase, preferably a uracil-N-glycosylase, is advantageously completed in 30 min or less, 20 min or less, 15 min or less or 10 min or less. As disclosed above, when providing a solid fixed biological sample, the salt concentration in the lysis mixture corresponds to or is about the salt concentration in the lysis composition, while a liquid fixed biological sample dilutes the concentration such that it is lower in the lysis mixture than in the lysis composition. Accordingly, for a liquid fixed biological sample, the salt concentration can be adapted to establish the above disclosed concentration in the lysis mixture, in particular by providing a higher salt concentration in the lysis composition and/or providing a higher volume ratio of the lysis composition.
According to a preferred embodiment, the lysis composition comprises a detergent. Detergents support the lysis of the sample and dissolve protein aggregates. The detergent is preferably provided to the lysis mixture by the lysis composition.
According to a preferred embodiment, the detergent is a ionic or non-ionic detergent. Exemplary detergents are known by the skilled person. According to one embodiment, the detergent is an ionic detergent, preferably an anionic detergent. This is in particular suitable when the fixed biological sample is a solid fixed biological sample, such as a fixed tissue sample. A detergent in particular can be a sulfate or sulfonate of a fatty alcohol, such as sodium dodecyl sulfate, sodium dodecyl sulfonate or dodecylbenzenesulfonic acid, preferably the detergent is sodium dodecyl sulfate (SDS). Also a mixture of detergents can be used.
According to a preferred embodiment, the detergent is present in the lysis composition and/or the lysis mixture in a concentration of at least 0.01%, at least 0.02%, preferably at least 0.03%. Suitable concentration ranges of detergent in the lysis composition and/or the lysis mixture may be selected from 0.01-3.0%, 0.02-2.75%, preferably 0.03-2.5% or 0.04% to 2.0%. In embodiments, the concentration is in a range of 0.03-1%. As disclosed above, when providing a solid fixed biological sample, the detergent concentration in the lysis mixture corresponds to the concentration in the lysis composition, while a liquid fixed biological sample dilutes the concentration such that it is lower in the lysis mixture than in the lysis composition. Accordingly, for a liquid fixed biological sample, the detergent concentration can be adapted to establish the above disclosed concentration in the lysis mixture, in particular by providing a higher detergent concentration in the lysis composition and/or providing a higher volume ratio of the lysis composition.
According to one embodiment, the lysis composition comprises a buffering agent. Preferably, the buffering agent is selected from the group consisting of 2-amino (hydroxymethyl)propane-1,3-diol (also referred to as Tris), MOPS, HEPES, phosphate and borate, preferably selected from Tris. The buffering agent is preferably provided to the lysis mixture by the lysis composition. The buffering agent advantageously facilitates maintenance of the pH. As disclosed above, providing a suitable pH is particularly advantageous for the method according to the present disclosure and suitable pH ranges were disclosed above. Also a mixture of buffering agents can be used.
According to a preferred embodiment, the reactive compound is a buffering agent or the lysis composition or the lysis mixture comprises a buffering agent, optionally wherein the buffering agent has a pKa value which is 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.
According to one embodiment, the buffering agent is additionally a reactive compound, which is capable of reacting with a fixative and/or reacting with a crosslink induced by the fixative, optionally, it comprises a nucleophilic group, such as a primary amine group. For instance, the buffering agent may be 2-amino-2-(hydroxymethyl)propane-1,3-diol or a derivative thereof. In such an embodiment, the concentration disclosed above for the reactive compound corresponds to the concentration of the buffering agent.
According to one embodiment, the lysis composition comprises 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 comprises a chelating agent, optionally wherein the chelating agent is 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), ethylene glycol tetraacetic acid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA). According to a preferred embodiment, EDTA is used. As used herein, the term “EDTA” indicates inter alia the EDTA portion of an EDTA compound such as, for example, K2EDTA, K3EDTA or Na2EDTA. Using a chelating agent such as EDTA has the advantageous effect that nucleases such as DNases and RNases are inhibited.
The chelating agent may be employed in a concentration of 0.05 mM to 5 mM, such as 0.075 mM to 2 mM, 0.1 mM to 1.5 mMin the lysis mixture and/or the lysis composition. A suitable concentration of the chelating agent in the lysis composition may depends on the applied sample type (solid or liquid fixed biological sample) and can be readily found by the skilled person considering suitable concentrations disclosed above for the lysis mixture.
According to one embodiment, the lysis composition is prepared by adding a reactive compound, wherein the reactive compound is selected from:
This embodiment is advantageous for flexibly modifying the type and concentration of the reactive compound independently from further compounds of the lysis composition. As demonstrated in the Example, by adding at least one of said reactive compounds, the nucleic acid size/fragmentation can be modified, e.g. for optimizing the nucleic acids for a nucleic analysis method. For instance, fragment size of nucleic acids have been increased, rendering the nucleic acids highly suitable for amplification of large nucleic acids as disclosed here, e.g. large amplicon PCR.
According to one embodiment, the lysis composition is aqueous and comprises:
optionally the lysis composition further comprises
According to one embodiment, the lysis composition is aqueous and comprises:
(i) a reactive compound comprising a primary amine, preferably 2-amino-2-(hydroxymethyl)propane-1,3-diol, wherein said reactive compound is comprised in the lysis mixture and/or lysis composition in 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, e.g. 3-100 mM, in particular 5-50 mM, 7-30 mM, 9-25 mM or preferably 10-20 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, e.g. 10-1000 mM, in particular 20-500 mM, 40-300 mM, 60-250 mM or preferably 75-200 mM, such as about 100 mM to 150 mM;
or
(ii) a reactive compound comprising two primary amine groups and preferably one secondary amine group, wherein said reactive compound is comprised in the lysis mixture and/or lysis composition in 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, e.g. 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, such as about 2.5 mM;
and
(iii) a protease enzyme, optionally a serine protease, preferably proteinase K;
(iv) a detergent, preferably an anionic detergent, more preferably the detergent 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.
The combination of features of this lysis composition has been found advantageous for sample lysis as demonstrated in the Examples and has been disclosed herein.
According to one embodiment, the lysis composition comprises a reactive compound which is 2-amino-2-(hydroxymethyl)propane-1,3-diol, wherein said reactive compound is comprised in the lysis mixture and/or lysis composition in a concentration of 5-50 mM preferably 10-20 mM; wherein 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 advantageous as demonstrated in the Examples. In particular, high quality nucleic acids were obtained using this lysis composition, which are suitable for a nucleic acid analysis method involving amplification of small nucleic acids. Accordingly, in one embodiment using the lysis composition of this embodiment, nucleic acids are analyzed by amplifying nucleic acids smaller than 500 nt, which may be referred to as short amplicon PCR. Details are also disclosed elsewhere herein and it is referred to the respective disclosure.
According to one embodiment, the lysis composition comprises a reactive compound which is 2-amino-2-(hydroxymethyl)propane-1,3-diol, wherein said reactive compound is comprised in the lysis mixture and/or lysis composition in a concentration of 60-250 mM, preferably 75-200 mM; optionally wherein the lysis composition has a pH in the range of 7.0 to 9.5, such as 8.0 to 9.0. Also this embodiment has been found highly advantageous as demonstrated in the Examples. In particular, high quality nucleic acids were obtained using this lysis composition, which are suitable for a nucleic acid analysis method involving amplification of small and/or large nucleic acid molecules. Details of short and long amplicon PCR are disclosed elsewhere.
According to one embodiment, the lysis composition comprises a reactive compound which is spermidine, wherein said reactive compound is comprised in the lysis mixture and/or lysis composition in a concentration of 0.5-12.5 mM preferably 1.5-5 mM; and wherein the lysis composition further comprises 2-amino-2-(hydroxymethyl)propane-1,3-diol which is comprised in the lysis mixture and/or lysis composition in a concentration of 5-50 mM preferably 10-20 mM. This embodiment has been found highly advantageous as demonstrated in the Examples. In particular, high quality nucleic acids were obtained using this lysis composition, which are large in size and show less fragmentation.
According to one embodiment, the lysis composition is prepared by combining a lysis solution and the proteolytic enzyme. Optionally, further compounds are added to prepare the lysis composition, in particular additional reactive compound, as disclosed herein. Moreover, preparing the lysis composition may further comprise adding water.
According to one embodiment, the proteolytic enzyme combined with the lysis solution in step (a) is provided by a solution comprising the proteolytic enzyme. Suitable concentrations are disclosed elsewhere. The proteolytic enzyme can also be comprised in the lysis solution.
As disclosed herein, the lysis composition may be prepared by combining a lysis solution and the proteolytic enzyme, and optionally water or a dilution buffer.
According to one embodiment, the lysis solution comprises a salt, a detergent and a buffering agent. Moreover, the lysis solution in particular comprises a reactive compound. Details of said compounds were already disclosed above and it is referred to the reactive disclosure. Suitable concentration ranges of the reactive compound, detergent and salt have been disclosed herein for the lysis mixture and lysis composition. The lysis solution comprises the reactive compound, detergent and salt at a concentration suitable to establish a concentration as disclosed herein for the lysis mixture and/or lysis composition.
According to a preferred embodiment, the lysis solution comprises a reactive compound. A reactive compound has been disclosed herein and it is referred thereto. According to a particular embodiment, the lysis 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 particular embodiment, the lysis solution comprises 2-amino-2-(hydroxymethyl)propane-1,3-diol or a derivative thereof.
According to a particular embodiment, the reactive compound is present in the lysis solution in a concentration of at least 5 mM, in particular at least 10 mM, at least 20 mM or at least 30 mM. The lysis solution may comprise 5-500 mM reactive compound, in particular 10-250 mM, 20-100 mM or 30-75 mM, such as about 53 mM. A particularly suitable reactive compound having said concentration in the lysis solution is 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. According to a preferred embodiment, the reactive compound is a buffering agent.
According to one embodiment, the reactive compound comprises two primary amine groups and preferably one secondary amine group, such as spermidine, optionally wherein the reactive compound is present in the lysis solution sufficient for establishing a concentration of at least 0.25 mM in the lysis composition and/or lysis mixture, in particular at least 0.5 mM, at least 1 mM, at least 1.25 mM or preferably at least 1.5 mM. According to one embodiment, the lysis solution comprises a reactive compound as disclosed herein, in particular a reactive compound which is also a buffering agent, optionally wherein 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, such as 2-amino-2-(hydroxymethyl)propane-1,3-diol, optionally wherein the reactive compound is present in the lysis solution in a concentration of at least 5 mM, in particular at least 10 mM, at least 20 mM or at least 30 mM. Alternatively, the lysis solution comprises a buffering agent in said concentrations, wherein the buffering agent is not a reactive compound. Further reactive compound comprising two primary amine groups and preferably one secondary amine group can be added to the lysis composition or are comprised in the lysis solution, optionally wherein this reactive compound is present in the lysis solution sufficient for establishing a concentration of at least 0.25 mM in the lysis composition and/or lysis mixture, in particular at least 0.5 mM, at least 1 mM, at least 1.25 mM or preferably at least 1.5 mM. A reactive compound comprising two primary amine groups and preferably one secondary amine group in the above disclosed concentrations has been found advantageous for lysing the fixed biological sample and obtain nucleic acids of large fragment size, as is demonstrated in the Examples.
According to a preferred embodiment, the lysis solution comprises a salt, in particular a salt as disclosed herein. In particular the salt may be a chloride salt, such as sodium chloride, potassium chloride, lithium chloride and cesium chloride, preferably sodium chloride. According to one embodiment, the salt is present in the lysis solution in a concentration of at least 50 mM, such as 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 comprises 75-2000 mM salt, in particular 100-1500 mM, 200-1200 mM, 300-1000 mM or 400-800 mM, such as about 500 to 700 mM. Advantages associated with said salt have been disclosed above and it is here referred thereto.
According to a preferred embodiment, the lysis solution comprises a detergent, in particular a detergent as disclosed herein. In particular, the detergent may be selected from a non-ionic or ionic detergent, preferably an anionic. In particular the detergent may be an anionic detergent, such as sulfate or sulfonate of a fatty alcohol, such as sodium dodecyl sulfate, sodium dodecyl sulfonate or dodecylbenzenesulfonic acid, preferably sodium dodecyl sulfate. According to one embodiment, the detergent has a concentration of at least 0.01% in the lysis solution, in particular at least 0.03%, at least 0.05%, at least 0.07% or at least 0.1%. According to one embodiment, the lysis solution comprises 0.01-3% detergent, in particular 0.03-2.5%, 0.05-2%, 0.07-1% or at least 0.1-0.5. Advantages associated with said detergent have been disclosed above and it is here referred thereto.
According to a particular embodiment, the lysis solution comprises
Such an embodiment is advantageous for the lysis of the fixed biological sample as has been demonstrated in the examples, wherein an according lysis solution has been applied.
Optionally, the lysis solution further comprises a chelating agent. According to one embodiment, the lysis solution further comprises a chelating agent, in particular EDTA, wherein the chelating agent has a concentration of at least 0.25 mM in the lysis solution, in particular at least 0.5 mM, at least 1 mM or at least 1.5 mM. According to one embodiment, the chelating agent has a concentration of 0.25-25 mM in the lysis solution, in particular 0.5-15 mM, 1-10 mM or 1.5-5 mM, such as 2.7 mM. The chelating agent advantageously supports inactivation of nucleases, such as DNases, as disclosed herein.
Step (b) comprises heating the lysed sample to reverse crosslinks.
Performing a heating step (b) is advantageous as it allows to reverse the crosslinks induced by the fixative, such as e.g. formaldehyde induced crosslinks. These crosslinks are usually present between proteins, proteins and nucleic acid, as well as between nucleic acids. The crosslink reversal reaction is temperature dependent and a high temperature leads to a quicker reversal reaction (see Kennedy-Darling et al., Anal. Chem., 2014, Vol. 86 (12), pp: 5678-5681, “Measuring the Formaldehyde Protein—DNA Cross-Link Reversal Rate”). A temperature of at least 80° C. is particularly suitable for reversing the crosslinks induced by numerous fixatives, such as e.g. formaldehyde. Moreover, it is described in the art that heating after a proteolytic digestion step can remove chemical modifications of nucleic acids, which remain after the proteolytic digestion of step (a). For instance, it is described that methylol groups of nucleobases which can be present after a proteolytic digestion step can be removed by elevating the temperature (see e.g. Masuda et al., Nucleic Acid Research, 1999, Vol. 27 (22), pp: 4436-4443; “Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples”).
Heating step (b) can also be employed to inactivate the proteolytic enzyme that is present in the lysis mixture. Inactivating the proteolytic enzyme in heating step (b) may be advantageous in case it is desired to perform one or more enzymatic treatments different than a proteolytic enzyme digestion step between step (b) and step (c). In one embodiment, heating step (b) inactivates the proteolytic enzyme that is present in the lysis mixture. Suitable inactivation conditions such as minimum inactivation temperatures and incubation times are described in the art for different proteases and thus are readily available to the skilled person.
Incubation temperatures and incubation times suitable to reverse 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., such as at least 85° C. or at least 90° C. According to one embodiment, step (b) comprises heating the lysed sample for at least 15 min, at least 20 min or at least 25 min in order to reverse crosslinks. In a preferred embodiment, step (b) comprises heating the lysed sample for at least 30 min, at least 45 min or at least 50 min. According to one embodiment, step (b) comprises heating the lysed sample at a temperature suitable for reversing crosslinks induced by the fixative up to 120 min, optionally up to 100 min. In embodiments, step (b) comprises heating up to 80 min or up to 70 min.
According to a preferred embodiment, the lysed sample is heated at a temperature in a range of 80-120° C., such as 80° C. to 110° C. or 85-100° C., for 30-120 min, such as for 45-90 min or 50-70 min. Step (b) may thus comprise heating the lysed sample at a temperature in the range of 80-110° C., such as 85-100° C., for 30-120 min. Step (b) may furthermore comprise heating the lysed sample at a temperature in the range of 80-110° C., such as 85-100° C., for 45-90 min. Step (b) may furthermore comprise heating the lysed sample at a temperature in the range of 80-110° C., such as 85-100° C., for 50-70 min.
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).
As is disclosed herein, one or more additional treatment steps may optionally be performed between the steps, and are in preferred embodiments performed between step (b) and step (c).
Step (c) comprises adding a proteolytic enzyme and performing a proteolytic digestion. Therefore, the method according to the first aspect comprises performing at least one additional proteolytic digestion step subsequent to decrosslinking step (b). Accordingly, the method comprises first lysing the fixed biological sample, wherein lysis involves 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 performing a proteolytic digestion (step (c)). This sequential order of steps was found particularly effective in lysing the fixed biological sample to release high quality nucleic acids with good yield. As is disclosed herein, one or more additional treatment steps may optionally be performed between steps (b) and (c).
As is demonstrated by the examples, performing step (c) significantly improves the release of nucleic acids during the overall lysis procedure. The released nucleic acids such as DNA and (or RNA) are of high quality and following purification, particularly suitable for amplification reactions. Surprisingly, performing an additional proteolytic digestion in step (c) improved the nucleic acid yield, as is demonstrated based on DNA in the examples. Without wishing to be bound in theory, it is assumed that the cross-links generated in the fixed biological sample (e.g. due to formaldehyde or other aldehyde-based fixatives) can cause particularly persistent protein associations with the nucleic acids or steric effects that may shield the protein from the proteolytic digestion performed in step (a). After heating step (b) which reverses crosslinks, these associations are weakened sufficiently that the additional proteolytic digestion step performed in step (c) is able to efficiently remove them. As is shown in the examples, performing step (c) surprisingly increases the yield and furthermore allows to release and thus obtain nucleic acids, such as DNA, of longer average size. This improvement in quality is beneficial for subsequent analyses of the released nucleic acids, as it also leads to better PCR results, in particular with large amplicons (500 bp). The results of the examples indicate that a larger amount of previously inaccessible long DNA strands have been made accessible to the subsequent PCR reaction when performing the additional proteolytic digestion step (c) after de-crosslinking step (b). These improvements that are demonstrated in the examples with respect to DNA yield and accessibility also result in an improved performance of the released DNA in downstream analytic processes, such as amplification and sequencing. The efficient removal of crosslinks and proteins that is achieved by the method according of the first aspect due to the sequential performance of steps (a), (b) and (c) is highly advantageous and also results in an improved NGS performance.
Therefore, performing step (c) as is taught by the method according to the first aspect leads to important and surprising improvements regarding the quality and yield of the released nucleic acid, such as particularly DNA.
The proteolytic enzyme used in step (c) is preferably a protease, such as proteinase K. Suitable proteases that can be used in step (c) were already disclosed in conjunction with step (a) and it is referred to the respective disclosure which also applies here. According to one embodiment, step (c) comprises adding proteolytic enzyme suitable for degrading protein and/or peptide associated with the nucleic acid and/or degrading crosslinks induced by the fixative, in particular crosslinks between the nucleic acid and a protein or peptide. According to one embodiment, the added proteolytic enzyme is a protease. Suitable proteases have been disclosed herein for step (a), and the same proteases may be used in step (c). As described above, preferably a serine protease is added in step (c), in particular proteinase K.
The proteolytic digestion in step (c) of the method of the present disclosure is preferably performed at a temperature suitable for degrading proteins and peptides by the proteolytic enzyme. Preferably, step (c) comprises heating the lysed sample to assist the digestion by the proteolytic enzyme. In embodiments, heating in step (c) is performed at a temperature in the range of 35-75° C., such as 40-70° C. or 50-70° C. In embodiments, the temperature is in the range of 55-68° C., such as 60° C. or 65° C. As is known in the art, the heating temperature of the used heating device may also be set to a higher temperature, wherein the preferred incubation temperature is reached in the proteolytic reaction mixture during the heating process (ramping) thereby assisting the proteolytic enzyme digestion before the higher temperature is then finally reached in the proteolytic reaction mixture. Such higher temperature may then also inactivate the proteolytic enzyme because the proteolytic digestion is completed before this inactivation temperature is reached in the course of the heating process.
To allow an efficient proteolytic digestion in step (c), the lysed sample may be incubated in the presence of the proteolytic enzyme. Incubation is preferably performed for at least 5 min, such as at least 10 min or at least 15 min. As disclosed herein, incubation for the proteolytic digestion may occur at an elevated temperature between 35-75° C. The sample may be agitated during incubation. According to one embodiment, step (c) comprises heating at a temperature of at least in 35° C., such as at least 40° C., at least 45° C., at least 50° C. or at least 55° C. for at least 5 min. Agitation may be performed by any method, such as shaking, rolling, inverting, etc.
According to one embodiment, step (c) comprises heating the lysis composition to a temperature selected from the range of 45° C. to 75° C., such 50° C. to 75° C. or 55° C. to 70° C. for 5-60 min, such as 10-45 min, 10-30 min, or 10-25 min. According to one embodiment, step (c) is completed in 30 min or less, optionally 20 min or less. As is demonstrated by the examples, a short incubation time such as 15 min (e.g. 65° C.) is feasible in step (c) and allows for significant improvements of yield and quality of nucleic acids comprised in the fixed biological sample. Moreover, such short incubation time enable the rapid completion of step (c), overall supporting that the method of the first aspect is not only highly efficient but also fast.
According to one embodiment, the incubation for the proteolytic digestion in step (c) and thus the time period for performing step (c) is shorter than the incubation in step (a) and thus the time period for performing step (a). Furthermore, the temperature used in step (c) to assist the proteolytic digestion may be higher in step (c) than in step (a).
The amount of the proteolytic enzyme added and the concentration of the proteolytic enzyme in the proteolytic reaction mixture of step (c) can be determined and chosen by the person skilled in the art. In one embodiment, the concentration of the proteolytic enzyme in the proteolytic reaction mixture is at least 0.5 mg/mL, preferably at least 1 mg/mL. Suitable concentration ranges include but are not limited to 0.5-10 mg/mL, 0.75-7.5 mg/mL, 1-5 mg/mL, or 1 to 3 mg/ml. Such concentrations have been applied in the examples. According to one embodiment, the proteolytic enzyme is added in step (c) is in form of a solution. The solution may comprise the proteolytic enzyme in a concentration of 1 mg/mL up to the solubility limit, e.g. in a concentration of 5-40 mg/mL, 7.5-35 mg/mL or 10-30 mg/mL, such as 20 mg/mL.
The examples show that step (c) advantageously supports the lysis of the fixed biological sample. In particular, nucleic acids are obtained with a higher yield and greater quality, which are highly suitable for a nucleic acid analysis method, such as PCR or NGS as disclosed herein.
Optionally, one or more additional treatment steps may be performed between steps (b) and (c). The additional treatments steps can be performed to further improve the method, e.g. in order to remove unwanted molecules. Non-limiting examples are the removal of RNA in case DNA is the target nucleic acid of interest (e.g. by performing a RNase digestion) or the removal of DNA in case RNA is the target nucleic acid of interest (e.g. by performing a DNase digestion). Furthermore, a treatment with a lipase may be performed. This can be advantageous, e.g. in case a fatty biological sample is processed. Furthermore, a treatment step can be performed to remove artifacts that are present due to the fixing of the biological sample, such as e.g. uracil nucleobases. This allows to provide nucleic acids such as DNA particularly suitable for amplification and sequencing.
According to one embodiment, the method according to the first aspect comprises performing at least one enzymatic treatment step different from a proteolytic digestion step between step (b) and step (c). According to one embodiment, the at least one enzymatic treatment step involves the use of one or more of a glycosylase, a nuclease, a lipase or a combination of the foregoing.
According to one embodiment, the at least one enzymatic treatment step involves the use of a DNA glycosylase, such as a uracil DNA glycosylase. If such step is performed, preferably an uracil-N-glycosylase treatment is performed between step (b) and step (c). According to one embodiment, the method comprises performing an enzymatic treatment step by adding a glycosylase to the lysed sample and heating. Using a glycosylate, such as a uracil glycosylase has important advantages. Fixed biological samples, such as for instance formalin-fixed and paraffin-embedded (FFPE) tissue samples, can have non-reproducible sequence artifacts in the DNA. Especially, C:G>T:A base substitution has been reported as the predominant type of sequence artifact in DNA recovered from fixed biological samples. This is based on cytosine deamination to uracil and sequential PCR amplification, leading to a C:G>T:A base substitution (see e.g. 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 lysed sample after step (b) and prior to step (c) with a DNA glycosylase, in particular a uracil-DNA glycosylase. The enzyme removes the false uracil leading to formation of an abasic site. This in turn can induce breakage of the strand and/or block the DNA polymerase in a subsequent polymerization step. As a result, a potential artifact (C:G>T:A base substitution) is eliminated. This allows to improve the quality of the released DNA for subsequent sequencing applications. According to one embodiment, the glycosylase treatment is performed at an elevated temperature assisting the glycosylase activity. Suitable are e.g. a temperature in the range of 45-55° C., such as 50° C. In embodiments, the glycosylase treatment step is completed in 30 min or less, 20 min or less, 15 min or less or 10 min or less, optionally wherein the glycosylase is a uracil-N-glycosylase.
According to one embodiment, when performing the enzymatic treatment step the sample comprises a salt concentration suitable for performing the enzymatic treatment step, in particular an enzymatic treatment step comprising a glycosylase, preferably a DNA glycosylase, more preferably a uracil DNA glycosylase, such as a uracil-N-glycosylase; and/or a nuclease, preferably a ribonuclease, more preferably a ribonuclease A.
According to one embodiment, the method comprises diluting the lysed and decrosslinked sample prior to performing the at least one enzymatic treatment step. The sample can be diluted, e.g. by adding water or other dilution solution. Dilution can be advantageous to adjust suitable conditions for performing at least one enzymatic treatment between step (b) and step (c). Dilution can be e.g. advantageous 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, such ≤300 mM or ≤250 mM. In embodiments, the salt concentration is ≤200 mM, ≤150 mM or ≤100 mM. Adjusting a sufficiently low salt concentration, optionally achieved by dilution of the lysed and cross-linked sample, ensures that the enzymatic treatment is not negatively affected by the salt. The suitable salt concentration depends on the intended enzymatic treatment and can be determined by the skilled person. For instance, providing a low salt concentration in the enzymatic treatment mixture, e.g. of less than 150 mM or less than 100 mM, was found particularly suitable for the enzymatic treatment using a uracil DNA glycosylase. In particular, it was found that these low salt conditions allow to reduce the treatment time. For instance, the treatment time can be reduced to less than 30 min, such as less than 25 min, less than 20 min, preferably less than 15 min or less than 10 min. Suitable time periods include 2-25 min, 2-20 min, 3-15 min and 5-10 min. As is demonstrated by the examples, an uracil-N-glycosylase digestion could be completed within 5 min. This is advantageous, as the method can be performed faster allowing a higher sample throughput.
According to one embodiment, at least a nuclease treatment step is performed in-between step (b) and step (c), optionally wherein the nuclease is a ribonuclease, such as ribonuclease A. The use of a ribonuclease allows to degrade RNA, thereby allowing to provide released DNA, devoid of RNA contaminations.
According to one embodiment, at least one, preferably both of the following enzymatic treatment steps are performed:
According to a second aspect of the invention, a method is provided for obtaining purified nucleic acids from a fixed biological sample, comprising lysing the fixed biological sample according to the lysis method of the first aspect, steps (a) to (c), wherein subsequent to step
As disclosed herein, according to certain embodiment the method according to the first aspect comprises performing one or more further treatment steps in-between steps (b) and (c), such as a nuclease digestion step and/or a treatment with a uracil-N-glycosylase.
The method according to the second aspect advantageously provides purified nucleic acids from a fixed biological sample, wherein the nucleic acids are of high yield and quality. The high quality and yield of purified nucleic acids is advantageous, as it improves the subsequent analyses of the isolated nucleic acids, such as the amplification and/or sequencing of the purified nucleic acids. Sequencing may be performed by next generation sequencing. As is demonstrated in 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 choice of the lysis buffer that is used in step (a). Thereby, the degree of fragmentation can be adjusted and controlled. This allows to provide nucleic acids that are either optimized for short or long PCR systems.
The nucleic acids may be DNA or RNA and in embodiments the nucleic acids are DNA molecules.
Features of step (a), (b) and (c), as well as the one or more optional treatment steps between step (b) and step (c) have been disclosed above in conjunction with the method according to the first aspect and it is referred to the respective disclosure which also applies to the method according to the second aspect.
In one embodiment, at least one intermediate treatment step is performed between step (c) and step (d). In another embodiment, no further treatment step is performed between step (c) and step (d).
The method according to the second aspect comprises step (d) purifying nucleic acids from the lysed sample. In step (d), any suitable purification method can be used, as the lysis method according to the first aspect renders the nucleic acids accessible in the lysis mixture. Suitable techniques for purifying nucleic acids from a lysed sample are known in the art and therefore, do not need any detailed description. Commercially available purification kits may be used in step (d).
According to a preferred embodiment, step (d) comprises
According to one embodiment step (d) comprises contacting the lysed sample comprising the released nucleic acids (obtained after step (c)) with a binding composition (e.g. a binding buffer) in order to establish binding conditions for binding the target nucleic acids to the solid phase. The resulting mixture that establishes suitable binding conditions is also referred to as binding mixture herein. According to one embodiment, the binding composition comprises a chaotropic agent such as a chaotropic salt. Purification methods employing a chaotropic agent/chaotropic salt in order to promote binding of target nucleic acids to a solid phase are well-known in the art and therefore, do not need to be described in detail. Chaotropic salts include but are not limited to salts comprising 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, sodium trifluroacetate. The binding composition and/or the binding mixture may furthermore comprise urea and/or a non-chaotropic salt. The binding composition and/or the binding mixture may furthermore comprise a detergent (e.g. an ionic or non-ionic detergent); and optionally an aliphatic alcohol, such as an alkanol, preferably comprising 1-5 or 2-3 carbon atoms. Ethanol or isopropanol are commonly used as alcohol for nucleic acid purification to promote binding to a solid phase. The mixture of the nucleic acids and the binding composition can then be applied to a solid phase or a solid phase may be added to said mixture. The solid phase may have a silica surface. The solid phase may comprise an unmodified silicon containing surface to which the target nucleic acids bind. 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, zeolithe, bentonite, alkylsilica, aluminum silicate and borosilicate. Exemplary solid phases that can be used in conjunction with the present invention include, but are not limited to, solid phases comprising an unmodified silica surface, including but not limited to, silica particles, silica fibres, glass materials such as e.g. glass powder, glass fibres, glass particles or controlled pore glass, silicon dioxide, glass or silica in particulate form such as powder, beads or frits. According to the present disclosure, the use of a column based solid phase or the use of particles, in particular magnetic particles, is preferred. According to one embodiment, the solid phase is comprised in a column. The column preferably comprises the solid phase having an unmodified silicon containing surface that is used for nucleic acid binding, particularly for DNA binding.
Other nucleic acid purification technologies that can be used in step (d) involve 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, do not need to be described in detail.
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 furthermore be eluted. Elution solutions are well known by the skilled person and do not need to be further defined here.
Other nucleic acid purification methods can also be used in step (d), such as e.g. precipitation based purification methods. Such methods are known in the art and may involve the use of alcohols.
The target nucleic acids obtained according to the method according to the second aspect of the present disclosure may then be further processed and e.g. used for a nucleic analysis method as disclosed herein. As disclosed herein, the method according to the second aspects which is based on the lysis method according to the first aspect provides improvements in yield and quality of target nucleic acids extracted from fixed biological samples such as FFPE tissue. As is shown by the examples, the provided purified nucleic acids are better suitable for subsequent PCR and NGS analysis compared to prior art methods.
The target nucleic acids may be DNA and/or RNA. In one embodiment, the target nucleic acids to be bound comprise or substantially consist of DNA. As disclosed herein, the method according to the second aspect provides
The “nucleic acid” or “nucleic acids” may be DNA and/or RNA. Therefore, DNA and RNA may be released from the fixed biological sample in the method according to the first aspect, or purified in the method according to the second aspect. Furthermore, the nucleic acid may be DNA or RNA. As disclosed herein, predominantly DNA (or RNA) can be obtained by either performing a nuclease treatment (e.g. during the method according of the first aspect) in order to destroy the non-target nucleic acid and/or a target nucleic acid selective purification may be performed in step (d) of the method according to the second aspect. This allows to obtain the target nucleic acid (e.g. DNA) with no or a low amount of non-target (e.g. RNA) contamination.
According to a preferred embodiment, the nucleic acids comprise or substantially consist of DNA. As is demonstrated in the examples, DNA can be very efficiently released from the fixed biological sample and subsequently purified.
The term “fixed biological sample” in particular refers to any biological material that has been preserved with a fixative. The fixed biological sample comprises crosslinks between nucleic acid molecules and protein molecules due to the fixation. The used fixative is a cross-linking fixative. Such fixed biological samples include but not limited to formaldehyde-fixed tissues or organs, tissue samples stored in liquid cytological preservation media, and fixed cell-containing samples, stored in liquid cytological preservation material (such as cervical or gynecological swabs or cell-containing body fluids).
In one embodiment, the cross-linking fixative used for fixing 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. Also included in this embodiment are fixatives known to release cross-linking compounds, such as formaldehyde over time. Formaldehyde is a well-known cross-reactive molecule that fixes the biological sample by cross-linking e.g. amino groups by methylene bridges. According to one embodiment, the biological sample was fixed using formaldehyde and/or paraformaldehyde. As is known in the art, formaldehyde can be used as fixative for solid and liquid biological samples.
According to a preferred embodiment, the fixed biological sample is a solid fixed biological sample, in particular a fixed tissue sample. Exemplary solid fixed biological samples include, but are not limited to tissues, including but not limited to, liver, spleen, kidney, lung, intestine, thymus, colon, tonsil, testis, skin, brain, heart, muscle and pancreas tissue. The fixed biological sample may furthermore be a fixed cell containing sample, such as a cell-containing body fluid and samples derived therefrom, aspirates, cell culture, bacteria, microorganisms, viruses, plants, fungi, biopsies, bone marrow samples, swab samples, faeces, skin fragments and organisms.
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 autopsy, biopsy or from surgery.
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, feces, mucous, breast milk, bone marrow, and spino-cerebral fluid.
In one embodiment, the fixed biological sample is a biological sample in a liquid cytological preservative medium. The liquid cytological preservative medium comprises a cross-linking fixative, such as an aldehyde-containing fixative, such as formaldehyde. Although the liquid cytological preservative medium is useful for cytology purposes, it can inhibit the efficient isolation of nucleic acids from the fixed biological samples. Exemplary aldehyde-containing fixatives commonly used in liquid cytology preservative medium include, but is not limited to, formaldehyde, glyoxal, glutaraldehyde, glyceraldehyde, acrolein, or other aliphatic aldehydes. One commonly used liquid cytological preservative medium that comprises a fixative is SUREPATH®, which is one of the most commonly used preservative media in clinical settings (e.g. to preserve swabs). SUREPATH® medium has a nearly 37% formaldehyde content and also contains methanol, ethanol, and isopropanol. The high formaldehyde content makes it a useful fixative, but poses challenges for subsequently extracting the target nucleic acids such as DNA from such fixed biological samples and using them for subsequent analysis. The methods according to the present invention provide can be advantageously used for such difficult fixed samples. According to one embodiment, the liquid fixed biological sample is a biological sample in SUREPATH®.
In one embodiment, the fixed biological sample is a solid cell-containing biological sample. 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 that has been fixed using a cross-linking fixative (such as formaldehyde), and embedded in an embedding material, preferably in paraffin (such as a FFPE sample). As disclosed in the art, embedding materials include, but are not limited to, paraffin, mineral oil, non-water soluble waxes, celloidin, polyethylene glycols, polyvinyl alcohol, agar, gelatin or other media.
According to a particular embodiment, the fixed biological sample is an FFPE sample.
When processing fixed samples that have been embedded in an embedding material such as paraffin, it is within the scope of the present disclosure to include a step to remove said embedding material prior to step (a). According to one embodiment, prior to contacting step (a) the fixed biological sample is processed in order to remove any embedding material (such as paraffin) from the fixed biological sample. The removal of the embedding material (such as paraffin) from the biological sample can take place by any method known in the art for deparaffinization of biological samples. E.g. this may be achieved by contacting the sample with a hydrophobic organic solvent, such as xylene, in order to dissolve out the embedding material such as paraffin. Suitable deparaffinization methods are e.g. described in WO 2012/085261, WO 2011/104027, WO 2011/157683 and WO 2007/068764, as well as in the GeneRead™ DNA FFPE handbook (QIAGEN, March 2014) and the QIAamp® DNA FFPE Tissue Handbook (QIAGEN, June 2012) using the QIAGEN supplementary protocol on “Purification of genomic DNA from FFPE tissue using the QIAamp® DNA FFPE Tissue Kit and Deparaffinization Solution”.
According to a preferred embodiment, the method according to the second aspect comprises (e) analyzing the purified nucleic acids.
The nucleic acid analysis method may be any chemical and/or biotechnological method that can be used to analyze nucleic acids e.g. in order to amplify, identify, detect and/or quantify a nucleic acid. Preferably, the nucleic acid analysis method comprises a detection reaction which allows to detect the presence, absence and/or quantity of nucleic acids comprised in the enriched nucleic acid. Preferably, said step (e) comprises the amplification of at least one target nucleic acid. Respective analytical methods are well-known in the prior art and are also commonly applied in the medical, diagnostic and/or prognostic field in order to analyze nucleic acids or a specific nucleic acid comprised in or suspected to be comprised in the purified nucleic acids.
According to a preferred embodiment, the purified nucleic acid obtained in step (d) is used in a nucleic acid analysis method that comprises amplification. Such a method preferably involves enzymatic amplification, such as a polymerase-based amplification. In a particular embodiment, a polymerase chain (PCR) reaction is performed to amplify the enriched nucleic acid.
According to a particular embodiment, the method further comprises amplifying the nucleic acids using a large amplicon PCR and/or a short amplicon PCR. According to one embodiment, the large amplicon PCR is for nucleic acid molecules having a size of at least 500 bp. The short amplicon PCR is for nucleic acid molecules having a size of less than 500 bp, such as preferably 300 bp, 200 bp or 150. In embodiments, the short amplicon PCR is less than 100 bp.
In embodiments, a lysis composition comprising at least 10 mM, in particular at least 20 mM, at least 40 mM, at least 60 mM or preferably at least 75 mM reactive compound, optionally a reactive compound comprising a primary amine, preferably 2-amino-2-(hydroxymethyl)propane-1,3-diol for a large amplicon PCR. As disclosed herein, may be particularly advantageous to provide a lysis composition comprising at least 3 mM, in particular at least 5 mM, at least 7 mM, at least 9 mM or preferably at least 10 mM reactive compound, optionally a reactive compound comprising a primary amine, preferably 2-amino-2-(hydroxymethyl)propane-1,3-diol for a short amplicon PCR. Thus, by selecting suitable conditions of the lysis composition, in particular by selecting a suitable reactive compound and concentration of the reactive compound, the obtained nucleic acids can be adjusted to be suitable for certain nucleic acid analysis methods. details are described elsewhere herein.
According to one embodiment, the method further comprises analyzing the nucleic acids, wherein analyzing includes performing a next generation sequencing method. Next generation sequencing advantageously allows to determine the sequence of nucleic acids in a high throughput format. However, nucleic acids from fixed biological samples have often been associated with artifacts that are detrimental in next generation sequencing. The methods of the present invention advantageously provide high quality nucleic acids, which are particularly suitable for next generation sequencing as demonstrated by the examples. In embodiments, performing next generation sequencing according to the present disclosure may involve i. attaching a unique molecular identifier sequence to the nucleic acids, wherein each nucleic acid molecule comprises a different unique molecular identifier sequence; ii. amplifying the nucleic acids with the attached unique molecular identifier sequence; and iii. sequencing the nucleic acids. Such a method advantageously allows sequencing of the obtained nucleic acids having a low reads-per-unique molecular identifier sequence value, in particular less than 20, such as less than 15, less than 12, preferably 10 or less.
Further suitable nucleic acid analysis methods may be selected from or involve one or more of an amplification reaction, a 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 amplifications.
The third aspect of the invention pertains to the use of a proteolytic enzyme, preferably a protease, such as proteinase K, for performing a proteolytic digestion after lysing a fixed biological sample, said sample comprising crosslinks between nucleic acid molecules and protein molecules due to the fixation, wherein said prior lysis involves digestion with a proteolytic enzyme and heating the lysed sample to reverse crosslinks, preferably in a method according to the first or second aspect of the present invention. Details of the method according to the first and second aspect are also disclosed in claims 1 to 22 to which it is referred.
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 have been disclosed above in conjunction with the method of the first aspect, in particular in conjunction with step (a) and step (c) to which it is referred and the disclosure also applies here. Furthermore, suitable heating conditions and embodiments for reversing crosslinks have been described above in conjunction with the method according to the first aspect, in particular in conjunction with step (b). It is referred to the respective disclosure which also applies here.
The fourth aspect pertains to the use of a glycosylase, preferably a DNA glycosylase, more preferably a uracil DNA glycosylase, such as a uracil-N-glycosylase, for performing an enzymatic treatment, wherein the enzymatic treatment is completed in 30 min or less, 20 min or less, 15 min or less or 10 min or less. Preferably such use is within the method according to the first or second aspect of the present invention, more preferably between step (b) and step (c).
As is disclosed herein, such glycosylase treatment allows to remove artifacts caused by the fixation of the biological sample (such as caused by formalin crosslinking). Suitable glycosylases for performing such enzymatic treatment has been disclosed herein in conjunction with the method according to the first aspect, as well as suitable conditions for performing the enzymatic treatment using a glycosylase, such as suitable temperatures and incubation times, as well as suitable conditions within the reaction mixture. It is referred to the respective disclosure. Suitable conditions for performing such glycosylase treatment are also known in the art. Furthermore, the present disclosure discloses suitable lysis/digestion compositions that are compatible with such glycosylate treatment thereby allowing the in-process use of a glycosylase, such as uracil-N-glycosylase, to remove artefacts caused by the fixative induced crosslinking.
According to a fifth aspect of the invention, a method is provided for processing a biological sample, wherein the fixed biological sample comprises crosslinks between nucleic acid molecules and protein molecules due to the fixation, said method comprising
Details with respect to steps (a) to (e) were already disclosed in conjunction with the methods according to the first and second aspect and it is referred to the above disclosure which also applied here. As disclosed above, optionally one or more additional treatment steps may be performed between steps (b) and (c) and this may be advantageous in order to prepare the nucleic acids for analysis step (e).
The purified nucleic acid may be DNA or RNA. RNA is preferably reverse-transcribed to cDNA prior to amplification in step (e). If the nucleic acid is a double-stranded molecule, such as a double stranded DNA molecule, what is preferred, the above indications with respect to the size (length) in “nt” refers to “bp”. Thus, if a double-stranded DNA molecule has a size of 150 nt, said double-stranded DNA molecule has a size of 150 bp.
In particular, nucleic acids having a small size can be purified and amplified by the method of the fifth aspect. The method according to the fifth aspect is in particular suitable for analyzing nucleic acid molecules having a short size in an amplification-based analysis method such as a PCR. As is demonstrated in the examples a short amplicon PCR of nucleic acids purified using the method according to the fifth aspect led to low Ct-values.
According to one embodiment, a 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) in a concentration in the range of 3-100 mM, 5-50 mM or 10-25 mM. Such lower concentration of reactive compound (which may be a formalin scavenger such as Tris or spermidine) in the lysis composition/lysis mixture used in step (a) of e.g. 3-100 mM, preferably 5-50 mM, more preferably 10-20 mM, provides nucleic acid fragments of a size that is particularly suitable for performing a short amplicon amplification reaction. This was unexpected as the prior art considers a higher degree of fragmentation as disadvantageous for subsequent amplification reactions. However, the examples surprisingly show that the fragmentation of the purified DNA improves the performance of a downstream PCR when using a short amplicon PCR, by a very significant amount. Without wishing to be bound in theory, it is assumed that this effect is due to a greater accessibility of the nucleic acid (such as DNA) when it is fragmented strongly, potentially because these breaks occur at points where a crosslink was present in the fixed biological sample. As such crosslink points are inhibitory to the PCR, removing those points during the lysis procedure as applied in the method according to the fifth aspect improves the efficiency of the PCR reaction.
The lysis mixture in particular comprises a reactive compound as disclosed herein. Examples are disclosed in conjunction with the method according to the first aspect and it is referred to the respective disclosure which also applied here. In one embodiment, the reactive compound comprises a primary amine, and preferably is 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 it is referred thereto.
The lysis composition used in step (a) may furthermore comprise
Suitable salts and detergents for the lysis composition used in step (a) as well as suitable concentrations were already disclosed in conjunction with step (a) of the method according to the first aspect and it is referred to the respective disclosure.
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.
The nucleic acids, which are advantageously released by performing method steps (a) and (b) 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 aspect, and it is referred to the respective disclosure which also applies here. Preferably, step (c) is performed in the method according to the fifth aspect. As disclosed in conjunction with the method according to the first aspect, one or more additional treatment steps may be performed between step (b) and step (c), such as a nuclease digestion and/or an enzymatic treatment step involving the use of a glycosylase, such as uracil-N-glycosylase. Details were described above in conjunction with the method according to the first aspect and it is referred to respective disclosure which also applies here.
According to a sixth aspect of the invention, a method is provided for processing a biological sample, wherein the fixed biological sample comprises crosslinks between nucleic acid molecules and protein molecules due to the fixation, said method comprising
Details with respect to steps (a) to (e) were already disclosed in conjunction with the methods according to the first and second aspect and it is referred to the above disclosure which also applied here. As disclosed above, optionally one or more additional treatment steps may be performed between steps (b) and (c) and this may be advantageous in order to prepare the nucleic acids for analysis step (e).
The purified nucleic acid may be DNA or RNA. RNA is preferably reverse-transcribed to cDNA prior to amplification in step (e). If the nucleic acid is a double-stranded molecule, such as a double stranded DNA molecule, what is preferred, the above indications with respect to the size (length) in “nt” refers to “bp”. Thus, if a double-stranded DNA molecule has a size of 500 nt, said double-stranded DNA molecule has a size of 500 bp.
The method according to the sixth aspect is in particular suitable for analyzing nucleic acids have 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, as is demonstrated by the Examples. In particular, nucleic acids having a small or large size can be obtained and amplified using the method of the sixth aspect. Without being bound to theory, it is believed that a concentration of reactive compound (e.g. Tris or spermidine) of at least 10 mM, in particular at least 20 mM, at least 40 mM, at least 60 mM or preferably at least 75 mM is highly effective in isolating nucleic acids having short sizes and large sizes and thus obtain more nucleic acids and/or nucleic acids of higher quality having a short size and large size.
In embodiments, the lysis mixture and/or lysis composition comprise 10-1000 mM reactive compound, in particular 20-500 mM, 50-300 mM, 75-250 mM or preferably 85-200 mM, such as about 100-150 mM. The lysis mixture in particular comprises a reactive compound as disclosed herein, e.g. Tris or spermidine. Suitable reactive compounds were disclosed in conjunction with the method according to the first aspect and it is referred to the respective disclosure which also applies here. The reactive compound may comprise a primary amine and preferably is 2-amino-2-(hydroxymethyl)propane-1,3-diol or a derivative thereof.
The lysis composition used in step (a) may furthermore comprise
Suitable salts and detergents for the lysis composition used in step (a) as well as suitable concentrations were already disclosed in conjunction with step (a) of the method according to the first aspect and it is referred to the respective disclosure.
This may be further supported 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 lysis mixture and/or lysis composition.
The nucleic acids, which are advantageously released by performing method steps (a) and (b) 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 aspect, and it is referred to the respective disclosure which also applies here. Preferably, step (c) is performed in the method according to the sixth aspect. As disclosed in conjunction with the method according to the first aspect, one or more additional treatment steps may be performed between step (b) and step (c), such as a nuclease digestion and/or an enzymatic treatment step involving the use of a glycosylase, such as uracil-N-glycosylase. Details were described above in conjunction with the method according to the first aspect and it is referred to respective disclosure which also applies here.
In step (e), the nucleic acids are analyzed by a method comprising amplifying nucleic acids. Such amplification methods have been disclosed herein and it is referred thereto. In particular, nucleic acids may be amplified using a polymerase enzyme. According to one embodiment, the nucleic acids are amplified using a PCR, in particular a PCR wherein short nucleic acid molecules are amplified (also referred to as short amplicon PCR) and/or a PCR wherein large nucleic acid amplified are amplified (also referred to as large amplicon PCR). Advantageously, short and large nucleic acid molecules can be amplified using this method. By adapting the concentration of the reactive compound (e.g. selected from Tris and spermidine) in the lysis composition/lysis mixture, one can control the fragment size. According to one embodiment, step (e) analysis comprises amplifying nucleic acid molecules having a size of at least 500 nt, such as at least 550 nt, at least 600 nt, at least 650 nt or at least 700 nt.
This invention is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this invention. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects or embodiments of this invention which can be read by reference to the specification as a whole.
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 their variants are used synonymously and are to be construed as non-limiting. Further components and steps may be present. Throughout the specification, where compositions are described as comprising components or materials, it is additionally contemplated that the compositions can in embodiments also consist essentially of, or consist of, any combination of the recited components or materials, unless described otherwise. Reference to “the disclosure” and “the invention” and the like includes single or multiple aspects taught herein; and so forth. Aspects taught herein are encompassed by the term “invention”.
It is preferred to select and combine preferred embodiments described herein and the specific subject-matter arising from a respective combination of preferred embodiments also belongs to the present disclosure.
The following examples are for illustrative purpose only and are not to be construed as limiting this invention in any manner. They demonstrate that the DNA extraction from fixed samples can be advantageously improved by performing an additional proteinase digestion step after a first proteinase digestion step and a crosslink removal step. This second proteinase digestion step improves the extraction process and provides pure DNA of high quality. The DNA can be purified with high yield, good fragment size and better suitability for downstream PCR amplification (such short and large amplicon PCR). The purified DNA is also highly advantageous for Next Generation Sequencing (NGS) applications, as a particular low read-per-UMI (=Unique Molecular Identifier, also referred to as Unique Molecular Index) value can be obtained.
Moreover, the examples show that the size of the DNA fragments can be modified by adaption of the lysis composition. In addition, the processing time required for a uracil-N-glycosylase (UNG) treatment step, if performed, can be significantly reduced compared to prior art methods.
Throughout the examples, DNA is extracted from FFPE tissue samples. After deparaffinizing the FFPE tissue sample, the sample is lysed. The sample material in the lysis composition (corresponding to sample+lysis solution) is subjected to a proteinase K digestion step, followed by a crosslink removal step applying heating. Afterwards, the lysed sample can be subjected to an enzymatic treatment such as an RNase and/or UNG treatment. Then, a (second) proteinase K digestion step is performed, which removes protein and peptides still crosslinked to the DNA. Afterwards, the DNA is purified from the digested sample, e.g. by binding the DNA to a solid phase, followed by wash cycles and elution of the bound DNA. The purified DNA is then ready for further use and analysis.
(a) Materials and Methods
FFPE Tissue Samples
Various human FFPE tissue samples were used in the present example, including prostate, lung, kidney, spleen and breast cancer. 10 μm sections were cut from FFPE blocks using a Leica rotary microtome and two or three 10 μm sections were applied per preparation.
Extraction Protocol
DNA was extracted from FFPE tissue according to the following protocol:
Control
For comparison, the protocol above was performed except for the additional proteinase K digestion step (see above step 5.), which was omitted.
b) Analysis of the DNA Yield
In this set of experiments, the DNA yield of the extract using the above disclosed protocol was determined using the QIAxpert and Qubit instruments. The obtained DNA yields using either instrument are shown in
Example 1.1 demonstrates that performing an additional proteinase digestion step after the crosslink removal step at high temperatures improves the DNA yield. Moreover, DNA fragments having a larger average size are obtained, which is particularly advantageous for obtaining DNA suitable for downstream PCR within a large amplicon PCR.
The second proteinase digestion step resulted in higher yields as measured with UV-Vis and by fluorometric determination (Qubit) compared to the control protocol without the additional proteinase K digestion step (see
The additional proteinase K digestion step advantageously increases the DNA yield and thus improves the extraction of DNA from various FFPE tissue sample types.
(c) Analysis of the Degree of Fragmentation of the Extracted DNA
In this experiment, the degree of fragmentation was analyzed using gel electrophoresis. As sample material, the above extracted DNA was used from human kidney and breast cancer FFPE tissue. The obtained results are shown in
The gel electrophoresis in
(d) Impact on Downstream PCR Performance
In this example, the extracted DNA was analyzed by PCR to determine, whether the PCR performance is affected by the increase in size of DNA by the additional proteinase K step. A quantitative Real Time PCR was performed and the Cq values were determined. “Cq” values can be interchangeably used with “Ct” values. Cq values are inversely proportional to the original relative amount of the extracted DNA. Both short amplicon PCR with 66 bp fragments and large amplicon PCR with 500 bp fragments were performed. The obtained results are shown in
As shown in
(e) Further Conclusions
An additional proteinase digestion step after the crosslink removal step enhances the DNA extraction from various FFPE tissue samples. In particular, DNA is extracted with a higher yield and with a larger fragment size, as well as a higher quality in respect to PCR amplification for short and large amplicons. Therefore, the additional proteinase digestion step is highly advantageous for DNA extraction from fixed biological samples, such as FFPE samples but also fixed liquid samples. As shown, one or more further enzymatic digestion steps may be performed in-between the de-crosslinking step and the second proteinase step.
1.2. Next Generation Sequencing (NGS) Performance is Enhanced
Example 1.2 demonstrates the improvements of DNA extraction from FFPE tissue samples by performing an additional proteinase digestion step according to the present disclosure. As noted, Proteinase K may be used as proteinase. By using the extracted DNA of Example 1.1 for NGS, very high NGS performance was measured. In particular, a low reads-per-UMI value of below 10 was measured for all tested tissue samples.
(a) Materials and Methods
DNA extracted from human FFPE tissue samples, including prostate, lung, kidney, and breast cancer as described in Example 1.1 were used for NGS performance analysis.
Sample Preparation and Sequencing Workflow
The extracted DNA was used as a template for a sequencing library. The QIAseg™ Targeted DNA Panel Handbook (QIAGEN, 05/2017) was followed using the QIAseg™ Targeted Panel for Illumina Instruments protocol. As QIAGEN targeted DNA Panel the Human Lung Cancer Panel with UMI technology was used. The UMI technology is based on integration of unique molecular identifier (UMI) sequences (also referred to as Unique Molecular Index) into a single gene-specific, primer-based targeted enrichment process, which overcomes biases/artifacts of the DNA polymerase and amplification processes:
Errors from PCR amplification and from the sequencing process may also be present in final reads that lead to false positive variants in 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.
(b) Performance in NGS
As discussed above, each molecule of double stranded DNA is tagged with an UMI barcode prior to amplification. This allows to distinguish truly unique molecules that were detected in NGS from PCR amplicons.
In
As shown in
Overall, the NGS performance is very high when using the purification method according to the present disclosure.
Example 2 further demonstrates the improvements of DNA extraction from FFPE tissue samples by using an additional proteinase K digestion step according to the present disclosure. In line with Example 1.1, a high DNA yield was obtained. Moreover, larger fragment size and better PCR performance was measured. Importantly, also the NGS performance was significantly enhanced compared to prior art methods.
(a) Materials and Methods
FFPE Tissue Samples
Human atrium FFPE tissue was used in the present example. 10 μm sections were cut from FFPE blocks using a Leica rotary microtome and two 10 μm sections were applied per preparation.
Extraction Protocol
The extraction protocol of Example 1.1 was followed in the present Example.
Control
In line with Example 1.1, the additional proteinase K digestion step (see above step 5.) was omitted in the control for comparison. Moreover, an additional control was performed for comparison by performing the first proteinase K step over night at 56° C. and omitting the additional proteinase K digestion step (“o/n 56° C.”).
Reference Protocols
As reference protocols (i.e. prior art methods), the Maxwell® RSC DNA FFPE Kit Technical Manual (Promega, Revised 11/17) was followed, as well as the Maxwell® RSC FFPE Plus DNA Kit Technical Manual (Promega, Revised 12/19). Moreover, as reference protocol, the QIAamp® DNA FFPE Tissue Handbook (QIAGEN, June 2012) was followed using the QIAGEN supplementary protocol on “Purification of genomic DNA from FFPE tissue using the QIAamp® DNA FFPE Tissue Kit and Deparaffinization Solution”.
b) Analysis of the DNA Yield
In this set of experiments, the DNA yield of the extract was determined using the QIAxpert and Qubit instruments. The obtained DNA yields using either instrument are shown in
The additional proteinase K digestion step at 65° C. after the cross-linking step resulted in higher yields as measured with UV-Vis and by fluorometric determination (Qubit) compared to the control protocol without the additional proteinase K digestion step (see
(c) Analysis of the Degree of Fragmentation of the Extracted DNA
The degree of fragmentation was analyzed using gel electrophoresis. As sample material, the above extracted DNA was used from human lung cancer and atrium FFPE tissue. The obtained results are shown in
The gel electrophoresis in
(d) Impact on Downstream PCR Performance
In this example, the extracted DNA was analyzed by PCR to determine, whether the PCR performance is affected by the increase in size of DNA by the additional proteinase K step. As before, a quantitative Real Time PCR was performed and the Cq values were determined. Both short amplicon PCR with 66 bp fragments and large amplicon PCR with 500 bp fragments were performed. The obtained results are shown in
As shown in
(e) Performance in NGS
In this example, the performance of the extracted DNA for NGS analysis was investigated. DNA was extracted from human atrium FFPE tissue samples using the above described extraction protocol (see Example 1.1. for used lysis compositions), with/without the additional proteinase digestion step (using proteinase K as proteinase).
The extracted DNA was used as a template for a sequencing library. The sample preparation and sequencing workflow described in Example 1.2 was followed and the reads-per-UMI values were determined as above.
Results
Each molecule of double stranded DNA is tagged with a unique molecular identifier (UMI) barcode prior to amplification. This allows to distinguish truly unique molecules that were detected in NGS from PCR amplicons. In
As shown in
Overall, the NGS performance is significantly improved by the extraction method according to the present disclosure, which can be performed fast and provides excellent result.
(e) Conclusions
This example demonstrates that performing an additional proteinase digestion step after a first proteinase digestion step and de-crosslinking step improves the quality of the purified nucleic acid, as shown based on DNA. The additional proteinase digestion step in particular increases the yield and fragment size, improves PCR performance and importantly, significantly enhances the NGS performance compared to the prior art methods. As discussed above, these results are highly surprising and unexpected. Moreover, by comparing the method according to the present disclosure comprising the additional/second proteinase digestion step with a first overnight 56° C. proteinase K digestion step, it was shown that not the overall digestion time is the cause of the better performance but the particular sequence of steps as performed in the method according to the present disclosure.
Example 3 demonstrates that the lysis composition affects the DNA extraction from FFPE tissue samples. Dilution of the lysis composition allows to control the fragmentation of DNA in the crosslink removal step. Moreover, the example shows that by controlling the fragmentation shorter DNA fragments can be obtained that improve the performance of downstream PCR when using short amplicon PCR.
(a) Materials and Methods
FFPE Tissue Samples
To analyse the impact of the lysis composition in frame of a DNA extraction protocol, two different FFPE tissue samples were used: heart tissue and lung tissue, both from rat. 10 μm sections were cut from FFPE blocks using a Leica rotary microtome and one 10 μm section was applied per preparation.
Extraction Protocol
An extraction protocol as described in Example 1.1 was used, with the following modifications:
Reference Protocol
As reference protocol, the QIAamp® DNA FFPE Tissue Handbook (QIAGEN, June 2012) was followed using the QIAGEN supplementary protocol on “Purification of genomic DNA from FFPE tissue using the QIAamp® DNA FFPE Tissue Kit and Deparaffinization Solution”. For lysis, 180 μL buffer ATL (QIAGEN) was mixed with 20 μL proteinase K.
(b) The Degree of Fragmentation May be Controlled by the Choice of the Lysis Solution in the Crosslink Removal Step
In the first set of experiments, the DNA extracted from the FFPE tissue samples was analyzed by gel electrophoresis (see
As shown in
Overall, a greater fragmentation of DNA was observed resulting from the extraction method with the diluted lysis composition. Therefore, a dilution of compounds present in the lysis solution allows to control the degree of fragmentation. These compounds in particular include Tris.
(c) The Performance of Downstream PCR is Affected by Fragmentation of the Extracted DNA
In this set of experiments, the influence of the DNA fragmentation on PCR amplification was analyzed. It was a particular object to investigate the quality of the extracted DNA in respect to the suitability to be amplified by PCR. As sample material, the DNA extracted above from the FFPE tissue samples was used. As before, a quantitative Real Time PCR was performed and Cq values were determined. The obtained Cq-values are plotted in
It is generally believed that a lower molecular weight and thus average length of DNA (a more fragmented DNA) is less reliable for downstream analyses. As shown in
However, surprisingly the more fragmented DNA obtained by the diluted lysis composition improved the performance of downstream PCRs when using short amplicon PCR (78 bp), by a very significant amount (see
(d) Further Conclusions
Using an extraction method with the diluted lysis composition led to higher DNA fragmentation and thus smaller average DNA size. This could be due to the lower concentrations of the compounds used to assist the lysis, such as in particular the detergent, salt and optionally the chelating agent. Despite the smaller fragment size, the extracted DNA demonstrated an improved performance in downstream PCR when using short amplicon PCR. In this respect, a higher quality DNA sample was obtained.
Example 4 demonstrates that the addition of certain compounds to the lysis composition can advantageously be used to control the degree of fragmentation of DNA extracted from FFPE tissue samples. Therefore, the extraction protocol can be tuned to optimize it for either short or long fragments used in the downstream PCR. Moreover, Example 4 demonstrates that the method of the present disclosure advantageously allows to reduce the processing time. In particular, the uracil-N-glycosylase (UNG) treatment step can be reduced in incubation from 60 min to only 5 min.
FFPE Tissue Samples
In the present example, FFPE tissue samples from rat kidney and rat lung were used. 10 μm sections were cut from FFPE blocks using a Leica rotary microtome and two 10 μm section were applied per preparation.
Extraction Protocol
The extraction protocol of Example 1.1 above was followed without step 5 (i.e. no second proteinase K digestion step). For lysis of the FFPE tissue sample, following lysis compositions were prepared by mixing with the lysis buffer (FTB, QIAGEN):
Reference Protocols
As reference protocol, the GeneRead™ DNA FFPE Handbook (QIAGEN, March 2014) was followed, comprising a 60 min UNG treatment step.
(a) The Degree of Fragmentation can be Controlled by Additives in the Lysis Composition
The fragmentation was analysed by gel electrophoresis of the extracted DNA samples. The results are shown in
As visible by the band shift in
(b) The Incubation Time of UNG can be Reduced by Providing a Suitable Lysis Solution
The UNG activity was tested using a bisulfide DNA test. In bisulfide DNA the cytosine nucleobases of the DNA are exchanged with uracil nucleobases. The uracil nucleobases are cleaved by the UNG. As a result DNA is partially degraded, reducing the amount of recoverable DNA.
Workflow
The lysis compositions containing the bisulfide DNA were prepared as disclosed above with the exception that 2.5 μL of water were substituted with 2.5 μL of bis.gDNA having a concentration of 830 ng/μL. Then, the mixture was diluted with 115 μL water and 35 μL UNG were added. Samples were then incubated for 5 min at 50° C. Afterwards, the DNA was purified as explained in Example 1.1 (see point 6. Purification of DNA).
Control
As control a sample was incubated for 60 min at 50° C., as is performed in the GeneRead™ DNA FFPE workflow. As further controls no UNG digestion was performed (“w/o UNG”).
Results
The results of the UNG activity test are shown in
As shown in
(c) Conclusions
By starting with a diluted low salt lysis composition of Example 3, as described above, and optionally adding an additive such as Tris or spermidine, the extraction protocol can be optimized for either short or long fragments used in the downstream PCR. Moreover, processing time can be reduced by providing a lysis composition of the present disclosure, as the UNG digestion time is reduced from 60 min down to 5 min without a loss in activity.
5. Overall Conclusions
As demonstrated in the examples, the additional proteinase digestion performed after the de-crosslinking step significantly enhances the performance of the DNA purification from fixed biological samples, such as in particular FFPE samples. The results indicate that despite a first proteinase digestion step and a crosslink removal step, the nucleic acids such as DNA appear to still contain protein cross-links or similar protein related modifications of the DNA, which hamper downstream processes (e.g. PCR amplification or NGS). By performing the additional proteinase digestion step after de-crosslinking, these are removed, which improves the quality of the DNA, allowing for enhanced downstream processing and analysis. The improvements were verified for various reference protocols demonstrating that the method of the present disclosure leads to significant improvements over prior art methods. Moreover, by comparing performance of an extended first proteinase digestion (overnight) to the method of the present disclosure using the second proteinase digestion step after de-crosslinking, it was shown that not the overall digestion time is important but the particular sequence of steps. By performing a first proteinase digestion step, followed by a heating step to remove crosslinks, followed by an additional/second proteinase digestion step important improvements are achieved. This sequence of steps allows obtaining high quality nucleic acids (such as DNA), while allowing completing the workflow within a short time period. Thus, the method is very fast and at the same time highly effective in obtaining a high quality DNA.
The improvements in DNA yield and accessibility are also reflected in the improved NGS performance. Moreover, the method of the present disclosure with the tunable size modifications works well in all variants. In addition, it was possible to reduce the UNG incubation time from 60 min to 5 min, which demonstrates that the processing time can be significantly reduced. In all cases, the method according to the present invention shows a benefit compared to prior art methods, including the legacy QIAamp® FFPE kit. The method according to the present disclosure is a much faster and more convenient workflow in comparison, and still provides better UMI values.
DNA was isolated from about 2 mm3 of different FFPE tissues, respectively, using the EZ1 Advanced XL instrument for bind wash/elute/steps. The EZ1 Advanced XL instrument is a robotic workstation for automated purification of nucleic acids.
Deparaffinization, lysis and cross-link removal was carried out as follows prior to processing the sample with the robotic instrument:
Performance of this protocol was compared to a modified protocol according to the present invention that includes a second Proteinase K step prior to binding:
The yield was determined by UV VIS and Qubit dsDNA BR measurement for a human kidney and a human breast sample.
The experiment was repeated with about 2 mm3 of a human heart sample. The yield was again determined by UV VIS and Qubit dsDNA BR measurement and
Example 5 demonstrates based on UV VIS and Qubit dsDNA measurement as well as by qPCR that the inclusion of a 2nd proteolytic digest according to the teachings of the present invention leads to an improvement in nucleic acid yield and thereby improves the performance in the qPCR.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20167445.4 | Mar 2020 | WO | international |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/058514 | 3/31/2021 | WO |
