Method for treatment of a biological sample

Abstract

The present disclosure relates to methods and kits for treatment of biological samples comprising non-sperm cells and sperm cells. The present disclosure relates more particularly to methods and kits for treatment of forensic samples comprising non-sperm cells and sperm cells.

Description

METHOD FOR TREATMENT OF A BIOLOGICAL SAMPLEFIELD

[0001] The present disclosure relates to methods and kits for treatment of biological samples comprising non-sperm cells and sperm cells. The present disclosure relates more particularly to methods and kits for treatment of forensic samples comprising non-sperm cells and sperm cells.BACKGROUND

[0002] Extraction of DNA (deoxyribonucleic acid) is important in forensic genetics because the patterns of short tandem repeats (STR) differ between individuals and therefore DNA is used to identify individuals. In sexual assault cases it is therefore important to clearly identify all persons involved in the crime and the victim using STR profiles. Most commonly, the male perpetrator leaves sperm cells as evidence. Sperm cells are usually mixed with non-sperm cells of the victim. In order to identify the male perpetrator, it is therefore important to separate the DNA of non-sperm cells from the DNA from sperm cells.

[0003] In sexual assault cases, for example, it is paramount to clearly identify all persons involved in the crime as well as the victim. Most commonly, the male perpetrator leaves sperm cells as evidence. In sexual assault samples, sperm cells of the perpetrator are usually mixed with non-sperm cells of the victim. For STR profiling and identification it is therefore important to separate the DNA of non-sperm cells from the DNA of sperm cells so that the STR profile of the sperm cells shows a clear male profile (from the “perpetrator”) and not a mixed profile (from both the “perpetrator” and the “victim”).

[0004] Sperm cells carry haploid genomes, while non-sperm cells predominantly carry diploid genomes. As a result, the amount of DNA that is obtained from sperm cells is noticeably less than the amount of DNA obtained from non-sperm cells. Furthermore, the amount of DNA extracted from sperm decreases with the length of time the semen remains in a victim's body. The longer the time since intercourse, the less sperm DNA can be obtained and the less favorable the mixing ratio of non-sperm cells (usuallymostly epithelial cells) to sperm cells becomes in living organisms. When the amount of non-sperm cell DNA like epithelial cell DNA in a forensic sample is very high in comparison to the amount of sperm cell DNA, it is often difficult and sometimes impossible to remove enough of said epithelial DNA so that a clean sperm DNA profile is obtained. In these cases, the result oftentimes is an undesirable mixed DNA profile.

[0005] The success of genetic typing or profiling procedures depends largely on the availability of sufficient amounts of target DNA of the appropriate quality and purity.

[0006] Extraction and separation of sperm cell DNA from non-sperm cell DNA is mostly achieved by the so-called differential digestion method developed by Gill, Jeffreys, Werrett ("Forensic application of DNA ‘fingerprints’." Nature 318.6046 (1985): 577-579.). In this method, firstly non-sperm cells like epithelial cells are lysed with SDS and proteinase K, while sperm cells and sperm nuclei remain intact. After removal of the lysed epithelial cells and other non-sperm cellular material the remaining sperm fraction, which contains the nucleus, is cleaned of any residual non- sperm cell DNA by a series of centrifugation and wash steps. Finally, the sperm heads are lysed by addition of SDS, proteinase K, and a reducing agent like dithiothreitol (DTT) which disrupts the disulphide bonds protecting the sperm nucleus.

[0007] Although this is the most common differential extraction method used in forensic laboratories, it is very technique-dependent, and the quality of results can vary between analysts. Particularly in the case of sexual assault samples, the non-sperm cells are usually present in large numbers in relation to the number of sperm cells and it is often difficult and sometimes impossible to remove enough of the non-sperm DNA like the epithelial DNA so that a clean sperm DNA profile is obtained, and the result often is a mixed DNA profile.

[0008] Furthermore, the sperm pellet washing steps of this method can be insufficient at removing soluble DNA from the cell pellet, leading to incomplete separation of sperm and non-sperm fractions, particularly in samples that contain large numbers of the victim's cells relative to sperm cells. Moreover, the several centrifugation and washing steps usually result in significant loss of sperm cells and sperm nuclei. This usually leads to low sperm DNA yields insufficient for subsequent analysis.

[0009] The time-consuming nature of the process often precludes this method as a viable solution in an efficiency-minded and automated laboratory setting.

[0010] In view of the above, new methods and kits for treatment of biological samples, such as forensic samples, comprising sperm cells and non-sperm cells that overcome at least some of the problems in the art are needed. In particular, there is a need in forensic sample preparation for time and cost-effective protocols for the separation of sperm fractions from non-sperm cell fractions.SUMMARY

[0011] The present disclosure is generally directed to methods and kits for treatment of biological samples comprising sperm cells and / or sperm nuclei and non-sperm cells.

[0012] It is an object of the present disclosure to provide an efficient method for treatment of a biological sample comprising sperm cells and / or sperm nuclei and non- sperm cells that allows for the selective isolation of sperm cells and / or sperm nuclei and sperm cell DNA that is essentially free of non-sperm cell DNA.

[0013] It is another object of the present disclosure to provide a method for treatment of a forensic sample, and in particular a sexual assault sample, comprising sperm cells and / or sperm cell nuclei and non-sperm cells that allows for the selective isolation of sperm cell DNA from said sample that is essentially free from non-sperm cell DNA.

[0014] It is a further object of the present disclosure to provide a DNA profiling method that allows for reliable identification of a sperm donor and a non-sperm cell donor, from a biological sample, particularly a forensic sample, including both sperm cells and / or sperm nuclei and non-sperm cells, like epithelial cells.

[0015] Another object of the present disclosure is to provide a kit for the processing of samples, particularly forensic samples, comprising sperm cells and / or sperm nuclei and non-sperm cells.

[0016] The objects are solved by the features of the independent claims. Preferred embodiments are defined in the dependent claims.

[0017] In one aspect the present disclosure refers to a method for treatment of a biological sample, comprising sperm cells and / or sperm nuclei and non-sperm cells, the method comprising: a) subjecting a biological sample comprising sperm cells and / or sperm nuclei and nonsperm cells to a lysis by applying one or more lysing agents or a lysis solution to the biological sample and / or incubating the biological sample with the one or more lysing agents or in the lysis solution, wherein the one or more lysing agents or the lysis solution are / is suitable to essentially lyse the non-sperm cells and to form a lysate comprising non-sperm DNA, sperm cells and / or sperm nuclei, wherein the one or more lysing agents or the lysis solution do / does not comprise any agent in an amount that causes a complete lysis of sperm cells and / or sperm nuclei and, preferably comprises at least one agent selected from a surfactant, a chaotropic agent, a non-sperm cell digesting enzyme, or any combination thereof; b) separating, removing, or separating and removing the lysate from a sperm cell and / or sperm nuclei enriched fraction; c) contacting the sperm cell and / or sperm nuclei enriched fraction with one or more binding agents or a binding solution and a solid support and / or incubating the sperm cell and / or sperm nuclei enriched fraction with a solid support in the presence of one or more binding agents or a binding solution and binding the sperm cell and / or sperm nuclei enriched fraction to the solid support; d) separating, removing, or separating and removing the binding agents or binding solution from the solid support; e) optionally, subjecting the sperm cell and / or sperm nuclei enriched fraction bound to the solid support to at least one washing step by applying one or more washing agents or a washing solution to the solid support and / or incubating the solid support with the one or more washing agents or in the washing solution, and separating, removing, or separating and removing the washing agents or washing solution from the solid support;f) subjecting the sperm cell and / or sperm nuclei enriched fraction bound to the solid support to at least one elution step by contacting the solid support with one or more elution agents or an elution solution and / or incubating the solid support in the presence of the one or more elution agents or the elution solution, wherein the one or more elution agents or the elution solution are / is suitable to elute the non-sperm DNA but not the sperm cells and / or sperm nuclei bound to the solid support, and removing, or separating and removing the eluate comprising the non-sperm DNA from the solid support; g) optionally, subjecting the sperm cells and / or sperm nuclei bound to the solid support to at least one elution step by contacting the solid support with one or more elution agents or an elution solution and / or incubating the solid support in the presence of the one or more elution agents or the elution solution, wherein the one or more elution agents or the elution solution are / is suitable to elute the sperm cells and / or sperm nuclei bound to the solid support; h) subjecting the sperm cells and / or sperm nuclei to a lysis by applying a lysis solution or one or more lysing agents and / or incubating the sperm cells and / or sperm nuclei in a sperm cell and / or sperm nuclei lysis solution or with one or more lysing agents, wherein the sperm cell and / or sperm nuclei lysis solution or the one or more lysing agents comprise(s) at least one lysing agent selected from a reducing agent, a sperm cell and / or sperm nuclei lysing enzyme, a chaotropic agent and / or an alkaline agent in a concentration suitable to lyse a sperm cell and / or sperm nuclei; an amino compound and / or an ammonium salt;, or a combination thereof, and optionally, separating, removing, or separating and removing the lysate comprising sperm DNA from the solid support.

[0018] In an embodiment the method of the disclosure further comprises purifying, isolating, detecting, analysing and / or quantifying the non-sperm cell and / or sperm cell DNA.In a preferred embodiment analysing comprises genotyping the DNA, preferably a short tandem repeat (STR) analysis, restriction fragment length polymorphism (RFLP) of genomic DNA, random amplified polymorphic detection (RAPD) of genomic DNA, amplified fragment length polymorphism (AFLP) detection, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR), DNA sequencing, allele specific oligonucleotide (ASO) probes, and hybridization to DNA microarrays or beads; or any combination thereof.

[0019] In a second aspect the present disclosure refers to a method for treatment of a biological sample, comprising sperm cells and / or sperm nuclei and non-sperm cells, the method comprising: a) subjecting a biological sample comprising sperm cells and / or sperm nuclei and nonsperm cells to a lysis by applying one or more lysing agents or a lysis solution to the biological sample and / or incubating the biological sample with the one or more lysing agents or in the lysis solution, wherein the one or more lysing agents or the lysis solution are / is suitable to essentially lyse the non-sperm cells and to form a lysate comprising non-sperm DNA, sperm cells and / or sperm nuclei, wherein the one or more lysing agents or the lysis solution do / does not comprise any agent in an amount that causes a complete lysis of sperm cells and / or sperm nuclei and, preferably comprises at least one agent selected from a surfactant, a chaotropic agent, a non-sperm cell digesting enzyme, or any combination thereof; b) separating, removing, or separating and removing the lysate from a sperm cell and / or sperm nuclei enriched fraction; c) contacting the sperm cell and / or sperm nuclei enriched fraction with one or more binding agents or a binding solution and particles and / or incubating the sperm cell and / or sperm nuclei enriched fraction with particles in the presence of one or more binding agents or a binding solution and binding the sperm cell and / or sperm nuclei enriched fraction to the particles;d) separating, removing, or separating and removing the binding agents or binding solution from the particles; e) optionally, subjecting the sperm cell and / or sperm nuclei enriched fraction bound to the particles to at least one washing step by applying one or more washing agents or a washing solution to the particles and / or incubating the particles with the one or more washing agents or in the washing solution, and separating, removing, or separating and removing the washing agents or washing solution from the particles; f) subjecting the sperm cell and / or sperm nuclei enriched fraction bound to the particles to at least one elution step by contacting the particles with one or more elution agents or an elution solution and / or incubating the particles in the presence of the one or more elution agents or the elution solution, wherein the one or more elution agents or the elution solution are / is suitable to elute the non-sperm DNA but not the sperm cells and / or sperm nuclei bound to the particles, and removing, or separating and removing the eluate comprising the non-sperm DNA from the particles; g) repeating steps c), d), e) and f) at least once, twice or several times; h) optionally, subjecting the sperm cells and / or sperm nuclei bound to the particles to at least one elution step by contacting the particles with one or more elution agents or an elution solution and / or incubating the particles in the presence of the one or more elution agents or the elution solution, wherein the one or more elution agents or the elution solution are / is suitable to elute the sperm cells and / or sperm nuclei bound to the particles ; i) subjecting the sperm cells and / or sperm nuclei to a lysis by applying a lysis solution or one or more lysing agents and / or incubating the sperm cells and / or sperm nuclei in a sperm cell and / or sperm nuclei lysis solution or with one or more lysing agents, wherein the sperm cell and / or sperm nuclei lysis solution or the one or more lysing agents comprise(s) at least one lysing agent selected from a reducing agent, a spermcell and / or sperm nuclei lysing enzyme, a chaotropic agent and / or an alkaline agent in a concentration suitable to lyse a sperm cell and / or sperm nuclei; an amino compound and / or an ammonium salt;, or a combination thereof, and optionally, separating, removing, or separating and removing the lysate comprising sperm DNA from the particles.

[0020] In yet another aspect, the present disclosure provides kits for treatment of biological samples, such as forensic samples, comprising non-sperm cells and sperm cells, the kit comprising one or more of: a) a non-sperm cell lysis solution or one or more non-sperm cell lysing agents capable of lysing non-sperm cells and essentially not suitable for completely lysing sperm cells and / or sperm nuclei; b) a solid support; c) binding agents or a binding solution capable of binding the sperm cell and / or sperm nuclei enriched fraction to the solid support, preferably the one or more binding agents or the binding solution have / has one or more, preferably at least two of the following characteristics i. to v.: i. it comprises a chaotropic agent, optionally under alkaline conditions; ii. it comprises a chaotropic agent selected from a group consisting of a sodium salt, a thiocyanate salt or a guanidinium salt or urea, preferably sodium iodide, sodium perchlorate, guanidinium hydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate and / or a mixture of two or more salts thereof; iii. it has an acidic pH value; iv. it has a first pH selected from a pH range from 3 to 7, 3.5 to 6.5, 4 to 6, preferably a pH value of 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7; and / or v. the binding solution is obtained by mixing two or more agents.; d) elution agents or an elution solution capable of eluting the non-sperm DNA but not the sperm cells and / or sperm nuclei bound to the solid support, the elution solution or the one or more elution agents has / have at least one or two or more of the following characteristics i. to vii:i. it comprises water, preferably is water, ii. it has a second pH value selected from a pH range of pH>8 and <13; pH>8 and <12,6; pH>8 and <12; >8 and <11; pH>8 and <10 and >8 and <9, iii. the second pH is higher than the first pH that was used for binding the nonsperm DNA to the solid support, iv. the second pH is lower than a third pH at which the sperm cells and / or sperm nuclei are eluted from the solid support, preferably at least 0.1 units lower than the third pH, at least 0.2 unit lower than the third pH, more preferably at least 0.25 units lower or at least 0.3 units lower than the third pH v. it comprises an aqueous solution comprising the buffering substance and optionally one or more further components such as a salt, in particular an alkali salt such as sodium chloride or potassium chloride, vi. it comprises an alkali hydroxide such as sodium hydroxide or potassium hydroxide, preferably sodium hydroxide, vii. it is an at least 1 mmol / 1 sodium hydroxide solution, preferably a 2 mmol / 1 sodium hydroxide solution; e) a sperm cell and / or sperm nuclei lysis solution or one or more sperm cell and / or sperm nuclei lysing agents capable of lysing sperm cells and / or sperm nuclei, preferably the sperm cell and / or sperm nuclei lysis solution or one or more lysing agents comprises at least one reduction agent selected from the group consisting of dithiothreitol (DTT), Dithioerythrit, P-mercaptoethanol (BME), glutathione (GSH), Tris(2-carboxyethyl)phosphine (TCEP) and combinations thereof; a sperm lysing enzyme selected from the group consisting of proteinase K, protease, zymolase, lyticase, chromopeptidase, trypsin, lysostaphin, lysozyme and / or combinations thereof; a chaotropic agent and / or an alkaline agent in a concentration suitable to lyse a sperm cell and / or sperm nuclei; an amino compound and / or an ammonium salt; or any combination thereof; f) optionally, elution agents or an elution solution capable of eluting the sperm cells and / or sperm nuclei bound to the solid support;g) optionally, one or more washing agents or a washing solution, preferably the one or more washing agents or the washing solution have / has at least one or two or more of the following characteristics i. to iv: i. it comprises an alcohol, preferably ethanol, ii. it comprises the binding solution and / or binding agents, iii. it comprises the binding agents in the same concentrations or amounts as the concentration or amount of the agents used as binding agents or in a binding solution, iv. it comprises the binding agents in a concentration or amount that is different to the concentration or amount of the agents used as binding agents or in a binding solution; h) optionally, one or more means for separating, removing, or separating and removing any one of the following: i. the lysate from a sperm cell and / or sperm nuclei enriched fraction, ii. the binding agents or binding solution from the solid support, iii. the lysate comprising sperm DNA from the solid support, and / or iv. the washing agents or washing solution from the solid support.BRIEF DESCRIPTION OF THE FIGURES

[0021] Fig. 1 shows sperm cell binding to silica beads.

[0022] Fig. 2 shows sperm cell binding to carboxy beads.

[0023] Fig. 3a shows sperm cell and / or nucleus binding and differential elution from anion-exchange beads.

[0024] Fig. 3b shows sperm cell and / or nucleus binding and elution from anion- exchange beads with NaCl.

[0025] Fig. 4 shows sperm cell and / or nucleus binding and differential elution from silica beads.

[0026] Fig. 5 shows sperm cell and / or nucleus binding and differential elution from silica beads.

[0027] Fig. 6 shows STR profiles of male and female DNA.

[0028] Fig. 7 shows sperm cell and / or nucleus binding to silica beads withRecBCD digestion.

[0029] Fig. 8 shows sperm cell and / or nucleus binding and elution from silica beads with DTT or TECEP.

[0030] Fig. 9 shows sperm cell lysis in the presence of L-arginine.DETAILED DESCRIPTION OF EMBODIMENTS

[0031] Reference will now be made in detail to the various preferred embodiments of the disclosure, one or more examples of which are illustrated in the figures. Further, features illustrated or described as part of one embodiment can be used on or in conjunction with other embodiments to yield yet a further embodiment. It is intended that the description includes such modifications and variations.

[0032] The term "biological sample" or “sample” as used in the present disclosure broadly refers to a sample which contains biological materials, preferably mammalian like human and animal biological material, such as blood, blood stains, saliva, saliva stains, skin debris, faeces, faeces stains, urine, vaginal specimens, perianal specimens, anorectal specimens, semen, sperm cells and non-sperm cells, such as epithelial cells. The term encompasses samples which are directly obtained from a human donor but also refers to samples in which biological conditions are artificially mimicked e.g. for in vitro testing, in particular for research purposes.

[0033] The term "biological sample" or “sample”, as used herein, further refers to a biological sample that is attached, adsorbed, absorbed or in any other contact with swabs, such as “samples swabs” or “forensic sample swabs”, preferably swabs comprised of various materials such as natural fibre (like cotton) or synthetic matrices, or any other material on which the sample may be collected or is found.

[0034] The term “forensic sample”, as used herein, refers to a biological sample collected for forensic analysis and examination. Collection of forensic samples is commonly performed by collecting a sample on a swab, using collection cards, like FTA® collection cards. Collecting cuttings of the area of interest, such as a biological fluid on a clothing or on a cut section of a clothing or from any other material on which the sample of interest is located or to which it is connected Forensic samples may also be found e.g. in soil, on any kind of fabric.

[0035] The term “non-sperm cells”, as used herein, refers to the all cell types that are not sperm cells, including but not limited to erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, chondrocytes, pancreatic islet cells, thyroid cells, parathyroid cells, parotid cells, tumour cells, neurons, glial cells, astrocytes, red blood cells, white blood cells, macrophages, epithelial cells, somatic cells, pituitary cells, adrenal cells, hair cells, bladder cells, kidney cells, retinal cells, rod cells, cone cells, heart cells, pacemaker cells, spleen cells, antigen presenting cells, memory cells, T cells, B cells, plasma cells, muscle cells, ovarian cells, uterine cells, prostate cells, vaginal epithelial cells, testicular cells, germ cells, egg cells, Leydig cells, Peritubular cells, Sertoli cells, lutein cells, cervical cells, endometrial cells, mammary cells, follicle cells, mucous cells, ciliated cells, nonkeratinized epithelial cells, keratinized epithelial cells, lung cells, goblet cells, columnar epithelial cells, squamous epithelial cells, osteocytes, osteoblasts, osteoclasts, and epithelial cells.

[0036] The term “sperm cell”, as used herein, refers to the reproductive cell of a male animal, preferably of a male mammal like a human. The term “sperm cell nuclei” or “sperm nuclei”, as used herein, refers to the cell nucleus located in the sperm cell head of two or more sperm cells.

[0037] As used herein, the term “non-sperm cell DNA” or “non-sperm DNA” refers to any DNA that is present in or resulting from “non-sperm cells”. During this application the terms “non-sperm cell DNA” and “non-sperm DNA” are used interchangeably. Non-sperm DNA may result from a female or male victim as well as from the male perpetrator in case of sexual assault samples.

[0038] As used herein the term “sperm cell and / or sperm nuclei enriched fraction” refers to a fraction of the sample after lysis, wherein in said fraction the ratio of spermcell and / or sperm nuclei to non-sperm DNA is equal or higher after the separation and / or removal of the lysate comprising non-sperm DNA than before the lysate removal.

[0039] The term “lysate” as used herein refers to a mixture containing lysed cell debris and DNA.

[0040] The term “complete lysis” refers to a lysis in which 100%, 99% or more, 98% or more, 97% or more, 96% or more, 95% or more, 94% or more, 93% or more, 92% or more, 91% or more, 90% or more, 88% or more of the cells, preferably the sperm cells and / or nuclei to be lysed have lost their cell integrity and / or the nucleic acids, in particular the sperm nucleic acids are exposed. The lysis of sperm cells and sperm nuclei may be independent from each other. I.e., sperm cells may have been lysed predominantly while the sperm nuclei are still intact.

[0041] Provided herein are improved methods and kits for treatment of a biological sample comprising sperm cells and / or sperm nuclei and non-sperm cells that allows the isolation of sperm cell DNA that is suitable for further analysis, such as genotyping.

[0042] In particular, the methods of the present disclosure are provided for treatment of a forensic sample, preferably forensic samples comprising sperm cells and non- sperm cells, more preferably a sexual assault sample, comprising sperm cells and / or sperm nuclei from a or multiple perpetrators that are in many cases mixed with non- sperm cells from the victim, the amount of victim non-sperm cells usually even tremendously exceeding the amount of the sperm cell and / or the sperm nuclei. The methods of the present disclosure allow for the isolation of sperm cell DNA that is suitable for further analysis, such as genotyping, to allow the identification of a perpetrator. In cases where more than one perpetrator is involved, sperm cells may originate from various men. The present method can be used to isolate male DNA present in a sample comprising a mixture of sperm cells.

[0043] Minimizing loss of DNA during extraction from a sample as well as ensuring complete lysis of DNA containing cells are two factors that must be considered when choosing a DNA extraction method. This is particularly relevant when DNA is extracted from forensic samples, such as sexual assault samples, where sperm cellDNA of the perpetrator is usually present together with non-sperm cell DNA of the victim. The purity and quality of the DNA will be paramount for detection, analysis, and profiling in order to clearly identify the individual e.g., based on their unique STR profile.

[0044] In forensic sample preparation the first step usually comprises the lysis of the non-sperm cells, like the epithelial cells, followed by the separation and removal of the lysate containing DNA obtained from said non-sperm cells from the sperm cells and / or sperm nuclei. After removal of the lysed non-sperm cells, such as the epithelial cells, their cell debris and other non-sperm cellular material the remaining sperm cell fraction, which contains the nucleus, is cleaned of non-sperm cell DNA by a series of centrifugation and wash steps. Finally, the sperm cells are lysed by addition of SDS, proteinase K, and a reducing agent like dithiothreitol (DTT) which disrupts the disulphide bonds protecting the sperm nucleus. In one or more subsequent steps the thereby released sperm DNA is purified and isolated.

[0045] A key feature of these methods is the attempt to separate and remove non- sperm DNA from the sperm cells and / or sperm nuclei before a sperm cell lysis is performed.

[0046] In a novel approach, in the method of the disclosure, after lysis of the biological sample, a sperm cell and / or sperm nuclei enriched fraction comprising sperm cells and / or sperm nuclei and residual non-sperm DNA is bound to a solid support, under conditions where both sperm cells and / or sperm nuclei as well as non-sperm cell DNA are bound to the support. Subsequently, the non-sperm cell DNA is selectively removed from the solid support while only the sperm cells and / or sperm nuclei remain bound to the support.

[0047] This approach enables an efficient elimination of non-sperm DNA from the enriched sperm and / or sperm nuclei fraction and thus leads to a higher yield and purity of the sperm cells and / or sperm nuclei.

[0048] The method of the present disclosure allows for efficient sperm cell and sperm nuclei detachment from forensic swabs, minimal sperm cell loss during treatment and a highly efficient sperm lysis after sperm cell and sperm nuclei purification.

[0049] The method of the present disclosure further allows an efficient isolation of sperm DNA from other types of DNA in a sample. Cross-contamination of the sperm DNA with non-sperm DNA is substantially eliminated. This maximises the yield and purity of the sperm DNA isolated from said samples and thus allows a reliable DNA detection, analysis and individual identification using e.g., STR profiling.

[0050] This is particularly important in the analysis of sexual assault samples, where separation of the perpetrator DNA from the DNA of the victim is crucial for subsequent genetic typing or profiling procedures.

[0051] In a first aspect, the present disclosure provides a method for treatment of a biological sample comprising sperm cells and / or sperm nuclei and non-sperm cells, the method comprising: a) subjecting a biological sample comprising sperm cells and / or sperm nuclei and non- sperm cells to a lysis by applying one or more lysing agents or a lysis solution to the biological sample and / or incubating the biological sample with the one or more lysing agents or in the lysis solution, wherein the one or more lysing agents or the lysis solution are / is suitable to essentially lyse the non-sperm cells and to form a lysate comprising non-sperm DNA, sperm cells and / or sperm nuclei, wherein the one or more lysing agents or the lysis solution do / does not comprise any agent in an amount that causes a complete lysis of sperm cells and / or sperm nuclei and, preferably comprises at least one agent selected from a surfactant, a chaotropic agent, a non-sperm cell digesting enzyme, or any combination thereof; b) separating, removing, or separating and removing the lysate from a sperm cell and / or sperm nuclei enriched fraction; c) contacting the sperm cell and / or sperm nuclei enriched fraction with one or more binding agents or a binding solution and a solid support and / or incubating the sperm cell and / or sperm nuclei enriched fraction with a solid support in the presence of one or more binding agents or a binding solution and binding the sperm cell and / or sperm nuclei enriched fraction to the solid support;d) separating, removing, or separating and removing the binding agents or binding solution from the solid support; e) optionally, subjecting the sperm cell and / or sperm nuclei enriched fraction bound to the solid support to at least one washing step by applying one or more washing agents or a washing solution to the solid support and / or incubating the solid support with the one or more washing agents or in the washing solution, and separating, removing, or separating and removing the washing agents or washing solution from the solid support; f) subjecting the sperm cell and / or sperm nuclei enriched fraction bound to the solid support to at least one elution step by contacting the solid support with one or more elution agents or an elution solution and / or incubating the solid support in the presence of the one or more elution agents or the elution solution, wherein the one or more elution agents or the elution solution are / is suitable to elute the non-sperm DNA but not the sperm cells and / or sperm nuclei bound to the solid support, and removing, or separating and removing the eluate comprising the non-sperm DNA from the solid support; g) optionally, subjecting the sperm cells and / or sperm nuclei bound to the solid support to at least one elution step by contacting the solid support with one or more elution agents or an elution solution and / or incubating the solid support in the presence of the one or more elution agents or the elution solution, wherein the one or more elution agents or the elution solution are / is suitable to elute the sperm cells and / or sperm nuclei bound to the solid support; and h) subjecting the sperm cells and / or sperm nuclei to a lysis by applying a lysis solution or one or more lysing agents and / or incubating the sperm cells and / or sperm nuclei in a sperm cell and / or sperm nuclei lysis solution or with one or more lysing agents, wherein the sperm cell and / or sperm nuclei lysis solution or the one or more lysing agents comprise(s) at least one lysing agent selected from a reducing agent, a sperm cell and / or sperm nuclei lysing enzyme, a chaotropic agent and / or an alkaline agent ina concentration suitable to lyse a sperm cell and / or sperm nuclei, an amino compound and / or an ammonium salt; or a combination thereof, and optionally, separating, removing, or separating and removing the lysate comprising sperm DNA from the solid support.

[0052] In a second aspect, the present disclosure refers to a method for treatment of a biological sample, comprising sperm cells and / or sperm nuclei and non-sperm cells, the method comprising: a) subjecting a biological sample comprising sperm cells and / or sperm nuclei and nonsperm cells to a lysis by applying one or more lysing agents or a lysis solution to the biological sample and / or incubating the biological sample with the one or more lysing agents or in the lysis solution, wherein the one or more lysing agents or the lysis solution are / is suitable to essentially lyse the non-sperm cells and to form a lysate comprising non-sperm DNA, sperm cells and / or sperm nuclei, wherein the one or more lysing agents or the lysis solution do / does not comprise any agent in an amount that causes a complete lysis of sperm cells and / or sperm nuclei and, preferably comprises at least one agent selected from a surfactant, a chaotropic agent, a non-sperm cell digesting enzyme, or any combination thereof; b) separating, removing, or separating and removing the lysate from a sperm cell and / or sperm nuclei enriched fraction; c) contacting the sperm cell and / or sperm nuclei enriched fraction with one or more binding agents or a binding solution and particles and / or incubating the sperm cell and / or sperm nuclei enriched fraction with particles in the presence of one or more binding agents or a binding solution and binding the sperm cell and / or sperm nuclei enriched fraction to the particles; d) separating, removing, or separating and removing the binding agents or binding solution from the particles;e) optionally, subjecting the sperm cell and / or sperm nuclei enriched fraction bound to the particles to at least one washing step by applying one or more washing agents or a washing solution to the particles and / or incubating the particles with the one or more washing agents or in the washing solution, and separating, removing, or separating and removing the washing agents or washing solution from the particles; f) subjecting the sperm cell and / or sperm nuclei enriched fraction bound to the particles to at least one elution step by contacting the particles with one or more elution agents or an elution solution and / or incubating the particles in the presence of the one or more elution agents or the elution solution, wherein the one or more elution agents or the elution solution are / is suitable to elute the non-sperm DNA but not the sperm cells and / or sperm nuclei bound to the particles, and removing, or separating and removing the eluate comprising the non-sperm DNA from the particles; g) repeating the series of steps c), d), optionally e) and f) at least once, twice or several times; h) optionally, subjecting the sperm cells and / or sperm nuclei bound to the particles to at least one elution step by contacting the particles with one or more elution agents or an elution solution and / or incubating the particles in the presence of the one or more elution agents or the elution solution; and i) subjecting the sperm cells and / or sperm nuclei to a lysis by applying a lysis solution or one or more lysing agents and / or incubating the sperm cells and / or sperm nuclei in a sperm cell and / or sperm nuclei lysis solution or with one or more lysing agents, wherein the sperm cell and / or sperm nuclei lysis solution or the one or more lysing agents comprise(s) at least one lysing agent selected from a reducing agent, a sperm cell and / or sperm nuclei lysing enzyme, a chaotropic agent and / or an alkaline agent in a concentration suitable to lyse a sperm cell and / or sperm nuclei, an amino compound and / or ammonium salt; or a combination thereof, andoptionally, separating, removing, or separating and removing the lysate comprising sperm DNA from the particles.

[0053] It was surprisingly found that repeating steps c) to f), with step e) being optional, resulted in a strengthening of the bond between the particles and the sperm cells and / or sperm nuclei. This prevents an undesired sperm cell or sperm nuclei elution from particles during washing steps. Furthermore, the repeated elution of the non-sperm DNA from the particles further reduces the amount of non-DNA that remains bound to the particles.

[0054] In some embodiments, the biological sample is a forensic sample, such as a forensic swab sample, preferably a sexual assault sample.

[0055] In an embodiment, the forensic sample is a sample collected on a sample holder, such as a swab, a cutting or a collection card, like an FTA® collection card. Preferably, the sample is collected on a swab.

[0056] In an embodiment, the forensic sample is a sexual assault sample comprising a mixture of cells from the victim and the perpetrator. In a further embodiment, the cells from a victim are non-sperm cells, such as epithelial cells, and the cells from the perpetrator comprise sperm cells and / or sperm nuclei yet may also comprise non- sperm cells like epithelial cells e.g. from the skin or hair.

[0057] In an embodiment of the disclosure, the biological sample is treated with a lysis solution or one or more lysing agents comprising a suitable amount of an agent under conditions that essentially promote lysis of the non-sperm cells, but do not promote complete lysis of the sperm cells and sperm nuclei.

[0058] In an embodiment of the disclosure the biological sample comprising sperm cells and / or sperm nuclei and non-sperm cells is subjected to a lysis by applying one or more lysing agents or a lysis solution to the biological sample and / or incubating the biological sample with the one or more lysing agents or in the lysis solution, wherein the one or more lysing agents or the lysis solution are / is suitable to essentially lyse the non-sperm cells and to form a lysate comprising non-sperm DNA released from said non-sperm cells. The one or more lysing agents or the lysis solution do / does notcomprise any agent in an amount that causes complete lysis of sperm cells and / or sperm nuclei and preferably comprises at least one agent selected from a surfactant, a chaotropic agent, a non-sperm cell digesting enzyme, or any combination thereof. Yet, the lysis may induce a partial lysis of the sperm cells and / or the sperm nuclei or of their membrane such that no marked amount of sperm DNA is released thereby.

[0059] In an embodiment of the disclosure, in method step a) the one or more lysing agents or the lysis solution is applied to or contacted with the biological sample to form a lysate comprising non-sperm DNA.

[0060] In an embodiment the lysing agents are not contacted with the biological sample in the form of a pre-prepared lysis solution, but the biological sample is contacted directly with the one or more lysing agents. This is in particular applicable if the biological sample is a liquid sample or dissolved in a liquid before being brought into contact with the one or more lysing agents. Preferably, the lysing agents are easily dissolvable in the liquid sample material or the dissolved sample. Alternatively, the lysing agents may be pre-lysed in a solvent and be contacted with the biological sample in the form of a lysing solution. The combination of the biological sample with the one or more lysing agents and / or the lysing solution results in the lysing mixture or the lysate once the lysis has proceeded.

[0061] In a further embodiment of the disclosure, in method step a) the one or more lysing agents or the lysis solution are / is applied to or contacted with the biological sample, and the biological sample is then incubated with the one or more lysing agents or in the lysis solution to form a lysate comprising non-sperm DNA.

[0062] Following the lysis of the sample with the lysis solution or the one or more lysing agents, the lysate comprising released non-sperm DNA is separated, removed or separated and removed from the sperm cell and / or sperm nuclei enriched fraction.

[0063] In certain embodiments of the disclosure, separation of the sperm cell and / or sperm nuclei enriched fraction from the lysate occurs through centrifugation. The lysed sample of step a) is subjected to centrifugation in order to separate the lysed sample into a lysate comprising non-sperm DNA and the sperm cell and / or sperm nucleienriched fraction, preferably by forming a pellet containing a sperm cell and / or sperm nuclei enriched fraction and a lysate supernatant comprising the non-sperm DNA.

[0064] In one embodiment the resulting supernatant comprising the non-sperm cell DNA is subsequently removed from the sperm cell and / or sperm nuclei enriched fraction or cell pellet by any suitable means, including but not limited to pipetting.

[0065] In other embodiments the sperm cell and / or sperm nuclei enriched fraction is separated by means of filtration.

[0066] The term “lysis solution” as used herein for lysing the biological sample, refers to a lysis solution comprising at least one agent capable of lysing cells, preferably non- sperm cells, and does not comprise any agent in an amount that causes complete lysis of sperm cells and / or sperm nuclei.

[0067] The term “one or more lysing agents” as used herein for lysing the biological sample, refers to lysing agents capable of lysing cells, preferably non-sperm cells, and does not comprise any agent in an amount that causes complete lysis of sperm cells and / or sperm nuclei.

[0068] In an embodiment of the present disclosure, the lysis solution or the one or more lysing agents comprise(s) at least an agent selected from a surfactant, a chaotropic agent, a non-sperm cell digesting enzyme, or any combination thereof.

[0069] Some of the indicated suitable lysing agents may also be able to completely lyse sperm cells and / or sperm nucleic depending on their concentration, e.g. chaotropic agents. However, when using them for lysing the biological sample they are supposed to be used in a concentration that does not result in a complete lysis of the sperm cells and / or sperm nuclei.

[0070] In an embodiment of the present disclosure, the lysis solution or the one or more lysing agents comprise(s) at least an agent selected from surfactants. Surfactants, often also designated as detergents, comprise ionic surfactants such as anionic and cationic surfactants, zwitterionic surfactants and non-ionic surfactants.

[0071] In another embodiment of the present disclosure, the lysis solution or the one or more lysing agents comprise(s) at least an agent selected from surfactants, wherein the surfactant(s) has / have one or more of the subsequent features: i. the surfactant is selected from an anionic surfactant; ii. the surfactant is selected from a non-ionic surfactant; iii. the surfactant is selected from an anionic and a non-ionic surfactant; iv. the surfactant is selected from a cationic surfactant; v. the surfactant is selected from a zwitter-ionic surfactant; vi. the surfactant is selected from two or more of the surfactants indicated in i. to v.

[0072] In an embodiment of the disclosure, the lysis solution or the one or more lysing agents comprise(s) an agent selected from anionic surfactants comprising e.g. sodium dodecyl sulfate (SDS), sarkosyl, deoxycholate, sodium laureth sulfate (SLS), sodium octyl sulfate (SOS), sodium methyl cocoyl taurate and disodium coco sulfosuccinates, or a combination thereof.

[0073] In an embodiment of the disclosure, the lysis solution or the one or more lysing agents comprise(s) an agent selected from cationic surfactants comprising for example cetyltrimethylammonium bromide (CTAB), cetyl trimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), 5-bromo-5-nitro-l,3 dioxane, dimethyldioctadecylammonium chloride, cetrimonium bromide, dioctadecyldimethylammonium bromide (DODAB), Dodecyltrimethylammonium bromide (DOTAB), or a combination thereof.

[0074] In an embodiment of the disclosure, the lysis solution or the one or more lysing agents comprise(s) an agent selected from non-ionic surfactants comprising for example Triton X-100, Triton X-114, N-octylglucoside, Tween 20 (polysorbate 20, polyoxyethylene (20) sorbitan monolaurate), Tween 40, Tween 80, Span 80, Span 85, Ecosurf EH-6, Brij-35 (polyalkylenglycolether), Brij 58 (polyalkylenglycolether), NP- 40 (nonyl phenoxypolyethoxyl ethanol), Nonidet P-40 (octylphenoxypolyethoxyethanol), Ecosurf EH-9, Ecosurf SA-9, Tergitol 15-S-7, Tergitol 15-S-9, Tergitol 15-S-15, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol 15-S-5, Brij35, Brij CIO (=Brij 56), Brij58, BrijS20 (=Brij78), Brij98, Poloxamer 188 (=Synperonic F68), Genapol X-080, Genapol X-100, Genapol X-150, Genapol C-100, Genapol C-200, Genapol X-050, Genapol X-060, Pluronic F-127, MEGA-8, MEGA- 9, MEGA- 12, MEGA- 10, APO- 10, APO- 12, Big CHAP, Big CHAP Deoxy, Pluronic P-123, Pluronic L64 and Pluronic 17R4, or a combination thereof.

[0075] In an embodiment of the disclosure, the lysis solution or more lysing agents comprise(s) an agent selected from zwitterionic surfactants comprising for example Zwittergent 3-08, Zwittergent 3-12, Zwittergent 3-16, Zwittergent 3-10, phospholipids phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelins, 3-[(3-Cholamidopropyl)-dimethylammonio]-propansulfonat (CHAPS), and 3-([3- Cholamidopropyl]dimethylammonio)-2-hydroxy-l-propansulfonat (CHAPSO), or a combination thereof.

[0076] Suitable concentrations and amounts of the surfactant may vary depending on the nature of the samples or lysis parameters, with concentrations of the surfactant in the range of from 0,lmM to 5M, or in the range of from 0,005% to 4%, generally being favourable, based on the total volume of the lysis solution or the lysing mixture with the biological sample comprising the one or more lysing agents, respectively. The concentrations and amounts of the surfactant are in the range of from 0,05% to 4%, 0,05 to 3%, 0,05 to 2%, 0,05 to 1,0%, 0,05 to 0,5%, 0,05 to 0,1% , preferably in the range of from 0,1 to 2,5%, 0,1 to 2,0%, 0,1 to 1,5%, 0,1 to 1,0%, 0,1 to 0,5%, more preferably in the range of from 0,30 to 2%, 0,3 to 1,75%, 0,3 to 1,5%, 0,3 to 1,0%, or any suitable concentration within this range, based on the total volume of the lysis solution or the lysing mixture with the biological sample comprising the one or more lysing agents, respectively. In case more than one surfactant is contained in the lysing mixture their total amount is summed up to the indicated range.

[0077] In an embodiment of the present disclosure, the lysis solution or the one or more lysing agents comprise(s) at least an agent selected from non-sperm cell digesting enzymes, in particular for example proteinase K (e.g. from QIAGEN), protease, zymolase, lyticase, chromopeptidase, lysostaphin, lysozyme, or any combination thereof.

[0078] In an embodiment of the present disclosure, the lysis solution or one or more lysing agents comprise(s) at least one chaotropic compound. In a preferred embodiment, the chaotropic compound is selected from a group consisting of one or more sodium salts, one or more thiocyanate salts or one or more guanidinium salts or urea, preferably sodium iodide, sodium perchlorate, guanidinium hydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate and / or a mixture of two or more salts thereof. The chaotropic compound is preferably a guanidinium salt, such as a guanidinium hydrochloride, guanidinium thiocyanate and / or guanidinium isothiocyanate.

[0079] Suitable concentrations and amounts of the chaotropic compounds may vary depending on the nature of the samples or lysis parameters, with a total concentration of the chaotropic compounds in the range of > 0,0001 mM to < 10 M generally being favourable, based on the total volume of the lysis solution or the lysing mixture with the biological sample, respectively. Preferably, the total concentration of the chaotropic compounds is in the range of > 0,005 mM to < 8 M, preferably in the range of > 0.1 M to < 8 M, more preferably in the range of > 0.4 M to = 7 M, particularly preferably in the range of > 0.4 M to 6 M, such as 0,5M, IM, 1,5M, 2M, 2,5M, 3M, 3,5M, 4M, 4,5M, 5M, 5,5M and 6M , based on the total volume of the lysis solution or the lysing mixture with the biological sample, respectively. In case more than one chaotropic compound is contained in the lysing mixture their total amount is summed up to the indicated range.

[0080] In a preferred embodiment of the present disclosure, the lysis solution or the one or more lysing agents in steps a) comprises at least one agent selected from a surfactant selected from the group consisting of non-ionic surfactants or ionic surfactants, such as anionic -, cationic- or zwitterionic surfactants; a chaotropic agent selected from the group consisting of one or more sodium salts, thiocyanate salts or guanidinium salts or urea; a non-sperm cell digesting enzyme selected from the group consisting of proteinase K, protease, zymolase, lyticase, chromopeptidase, lysostaphin, lysozyme, or any combination thereof.

[0081] The lysing agents may also successively be added to or contacted with the biological mixture either by subsequently adding one or more of the lysing agents or by adding one or more further lysing solutions to result in the final lysing mixture.

[0082] According to a further preferred embodiment, the lysis solution, the one or more lysing agents or the lysing mixture with the biological sample has a pH in the range of > 4 to = 12, in particular in the range of > 4 to = 11, preferably in the range of > 4 to = 10, especially preferably in the range of > 4 to < 9.

[0083] In an embodiment of the disclosure, the biological sample is incubated during lysis for a period of 10 minutes to 2 hours and more, preferably 10 minutes, 20 minutes, 30 minutes 40 minutes, 50 minutes 60, minutes 75 minutes 90 minutes or 120 minutes, or any suitable incubation time within this range.

[0084] In a preferred embodiment of the disclosure essentially all, but at least 90%, such as 92%, 94%, 96%, 98%, or 99,99999% of the non-sperm cells present in the sample are lysed after lysing the biological sample.

[0085] In an embodiment of the disclosure after lysis in method step a) the lysate is separated, removed, or separated and removed from a sperm cell and / or sperm nuclei enriched fraction.

[0086] In one embodiment the lysate and sperm cells and / or sperm nuclei are separated by means of centrifugation into a lysate comprising non-sperm DNA and a sperm cell and / or sperm nuclei enriched fraction, preferably by forming a pellet containing a sperm cell and / or sperm nuclei enriched fraction and a lysate supernatant comprising non-sperm DNA.

[0087] In one embodiment the resulting supernatant comprising the non-sperm cell DNA is subsequently removed from the sperm cell and / or sperm nuclei enriched fraction or cell pellet by any suitable means, including but not limited to pipetting.

[0088] In one embodiment, after the majority of the supernatant is removed, a buffer solution is carefully, preferably by automated pipetting, added to the pellet and the remaining supernatant that has not been removed by the former separation step. The buffer solution is allowed to mix with the supernatant in order to wash the sperm pelletwithout disturbing the pellet. Any buffer or solution can be used that does not interfere with the subsequent method steps. This method is also known as “Sperm pellet Wash” and may be repeated several times.

[0089] In a preferred embodiment the buffer solution is selected from the non-sperm cell lysis solution as described above and / or the binding solution, as described below.

[0090] In one embodiment the resulting supernatant comprising non-sperm cell DNA may subsequently be removed from the sperm cell and / or sperm nuclei enriched fraction or cell pellet by any suitable means, including but not limited to pipetting. This step can be repeated once, twice or several times.

[0091] In other embodiments the lysate and sperm cells and / or sperm nuclei cells are separated by means of filtration.

[0092] It is preferred to not only separate the lysed and non-lysed cell fractions after the lysis step from each other, but moreover to remove non-sperm DNA from the sperm cells and / or sperm nuclei and thereby reducing or removing the majority of non- sperm DNA contamination connected with the sperm cells or sperm nuclei. The sperm cell and / or sperm nuclei enriched fraction however may contain small amounts of residual non-sperm cell DNA.

[0093] In order to further purify the sperm cells and / or sperm nuclei and to remove the residual non-sperm cell DNA from the sperm cell enriched fraction the inventors have chosen a different approach to what is known from the state-of-the-art methods. Mostly, non-sperm cell DNA is removed through repeated washing steps and / or treatment of the fraction with DNA digesting enzyme. As mentioned before, this will inevitably lead to loss of sperm cells and / or nuclei or deterioration of the sperm cells or nuclei. Furthermore, this removal of non-sperm cell DNA is often not sufficient, especially in samples in which there are few sperm / sperm cell nuclei and a high amount of non-sperm DNA present. As a result of insufficient removal of non-sperm cell DNA, the non-sperm cell DNA will still be present in the enriched sperm fraction even after the process in an amount that impedes the further analysis. When residual non-sperm cell DNA is present during sperm DNA analysis in such amounts, this will make a sperm DNA donor identification very challenging or even impossible.

[0094] Surprisingly it was found that binding the sperm cell and / or sperm nuclei enriched fraction to a solid support, under conditions where both sperm cells and / or sperm nuclei as well as non-sperm cell DNA bind to said support and subsequently selectively removing the non-sperm cell DNA from the solid support, while the sperm cells and / or sperm nuclei remain bound to the solid support, leads to a higher yield and purity of the sperm cells and / or sperm nuclei.

[0095] While not being preferred, it is also possible to bind the whole or most of the non-sperm DNA contained in the supernatant to the solid support in case the non- sperm DNA is separated from the sperm cell and / or sperm nucleic enriched pellet although not removed. Yet, it is preferred to remove the supernatant containing most of the non-sperm DNA before binding the sperm cell and / or sperm nuclei to the solid support to reduce the amount of non-sperm DNA contamination in advance before it is bound to the solid support.

[0096] In an embodiment of the present disclosure, the sperm cell and / or sperm nuclei enriched fraction is contacted with one or more binding agents or a binding solution and a solid support.

[0097] In an embodiment of the present disclosure, the sperm cell and / or sperm nuclei enriched fraction is incubated with a solid support in the presence of one or more binding agents or a binding solution.

[0098] Any solid support suitable for reversibly binding DNA as well as sperm cells and / or sperm nuclei may be used.

[0099] The term “solid support” is not intended to imply any limitation regarding its form or design. Thus, the term solid support encompasses appropriate materials that are porous or non-porous, permeable or impermeable, including but not limited to membranes, filters, sheets, particles, magnetic particles, beads, gels, powders, fibers and the like.

[0100] Preferred formats of the solid support include but are not limited to particles such as beads, membranes, filters, plates, columns, vessels and dipsticks.

[0101] Preferably the solid support is in the form of beads or particles, more preferred magnetic beads or magnetic particles.

[0102] In an embodiment of the present disclosure, the solid support, preferably in the form of particles, is formed of a solid support material selected from a material including, but not limited to, silica, silica gel, borosilicates, silicates, inorganic glasses, organic polymers such as poly(meth)acrylates, polyurethanes, polystyrene, agarose, polysaccharides such as cellulose, metal oxides such as aluminum oxide, magnesium oxide, titanium oxide and zirconium oxide, agarose, sephadex, sepharose, polyacrylamide, divinylbenzene polymers, styrene divinylbenzene polymers, dextran, resin and derivatives thereof. The solid support may be present in the form of a gel, particles, beads, membranes, plates, a surface coating and capillary tubes; for example silica solid supports, such as glass particles, silica particles, silica beads, silica plates, silica membranes and silica capillary tubes. The solid support may be magnetizable or magnetic (e.g. paramagnetic, superparamagnetic, ferromagnetic or ferrimagnetic), preferably when it is in the form of particles. The solid support material, including but not limited to polystyrene, agarose, polyacrylamide, dextran, and / or silica materials, may have a magnetic material incorporated therein or associated therewith or the solid support may be formed of two or more layers of solid support materials with one or more layers, preferably an inner layer, more preferred the core, containing a magnetic material. The solid support material of one or more layers may also be mixed homogeneously or inhomogeneously with the magnetic material.

[0103] In an embodiment of the present disclosure, the solid support materials are selected from materials capable of reversibly binding to nucleic acids and to sperm cells and / or sperm cell nuclei.

[0104] In an embodiment of the present disclosure, solid supports that can be used in conjunction with the present disclosure include, but are not limited to, solid supports comprising a 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.

[0105] According to one embodiment of the present disclosure, the use of a silica based solid phase in the form of particles, in particular magnetic particles, is preferred.

[0106] According to one embodiment, particles are used that may have the form of beads. Preferably, said particles or beads have a size of about 0.02 to 30 pm, more preferred 0.05 to 15 pm and most preferred of 0.1 to 10 pm. To ease the processing of the binding solid phase, preferably magnetic particles or magnetic beads, more preferred silica magnetic particles or silica magnetic beads are used.

[0107] Suitable silica beads are commercially available e.g from QIAGEN (QIAGEN EZ1&2 DNA Investigator Kit, Cat. No.952034).

[0108] Suitable magnetic silica particles are for example described in WO 01 / 71732, WO 2004 / 003231 and WO 2003 / 004150. Other magnetic silica particles are also known from the prior art and are e.g. described in WO 98 / 31840, WO 98 / 31461, EP 1 260 595, WO 96 / 4181 1 and EP 0 343 934 and also include for example magnetic silica glass particles.

[0109] The use of magnetic particles is convenient, because in general magnetic particles including bound nucleic acids and / or cells and / or nuclei can be processed easily by the aid of a magnetic field, e.g. by using a permanent magnet. In the context of this application, magnetic particles to which nucleic acids and sperm cells and / or sperm nuclei are bound are particularly useful. This embodiment is compatible with established robotic systems capable of processing magnetic particles. Here, different robotic systems exist in the prior art that can be used in conjunction with the present disclosure to process the magnetic particles to which nucleic acids and sperm cells and / or sperm nuclei were bound.

[0110] In an embodiment of the present disclosure, the solid support has a functionalized surface.

[0111] According to another embodiment of the present disclosure, the solid support is formed of one or materials as described above that have a surface functionalized with carboxylic acid groups (COOH groups). Suitable carboxy beads are commercially available e.g. from QIAGEN (QIAGEN Cat. No. 333923, 333903 or 333927).

[0112] According to another embodiment of the present disclosure, the solid support is formed of one or more materials as described above that have a surfacefunctionalized with one or more types of anion exchange moieties, preferably in the form of particles which comprise one or more types of anion exchange moieties at their surface.

[0113] Preferably, the surface is functionalized with one or more types of anion exchange moieties, each moiety comprising one or more anion exchange groups. The term "moiety" does not include any restrictions with respect to size. The same or different types of anion exchange groups may be present within one moiety if the moiety comprises more than one type of anion exchange group. Various moieties may comprise the same or different numbers and types of anion exchange groups. Examples of suitable anion exchange groups within one or more moieties include but are not limited to monoamines, diamines, polyamines, and nitrogen- containing aromatic or aliphatic heterocyclic groups as well as cyclic amines, aromatic amines and heterocyclic amines. Preferably, an anion exchange moiety comprises at least one anion exchange group which is selected from a primary, secondary and / or tertiary amino group.

[0114] In preferred embodiments, the anion exchange moiety comprises or consists of one anion exchange group selected from a primary, secondary or tertiary amine.

[0115] Preferably, the surface comprises one or more anion exchange moieties each comprising one polyethyleneimine anion exchange group.

[0116] Preferably, at least the surface functionalized with the anion exchange moieties contains one of the above-described solid support materials or a mixture thereof. Also, any other solid support material suitable for anion exchange chromatography may be used to provide the surface of the solid support that comprises and / or is functionalized with one or more types of anion exchange moieties.

[0117] In a preferred embodiment, the solid support comprising the functionalized surface is provided by a solid support that can be provided as suspension and can be separated from a liquid phase. When contacted with a liquid phase such as for example a cell and nucleic acids containing liquid, the surface of the solid support comprising the functional groups or types of anion exchange moieties is in contact with the liquid phase in order to allow cell and nucleic acid binding thereto.

[0118] Preferably, the solid support is provided in the form of particles having a functionalized surface, preferably in the form of particles which comprise types of anion exchange moieties or COOH groups at their surface. The particles may have an average size that is selected from a range of 100 nm to 50 pm, 200 nm to 40 pm, 300 nm to 35 pm, 400 nm to 30 pm, 450 nm to 25 pm, 500 nm to 20 pm, 550 nm to 15 pm, 600 nm to 12.5 pm, 650 nm to 10 pm, 700 nm to 7.5 pm, 750 nm to 5 pm, 800 nm tom3.5 ppm, 800 nm to 3 pm, 800 to 2,5 pm, 800nm to 2 pm and 800 nm to 1 ,5 pm. Particles of the respective sizes and in particular of a smaller size such as 10pm or less, 7.5pm or less, preferably 5pm or less, 2.5pm or less or 1.5pm or less are easy to handle and can be well resuspended in the sample.

[0119] The particles may also comprise more than one of the above-described solid support materials, e.g. comprising two or more layers comprising or consisting of different materials to provide the particle body.

[0120] Preferably, magnetic particles are used to provide a solid support with a functionalized surface, preferably a surface functionalized with types of anion exchange moeities or a surface comprising COOH groups that binds cells as well as nucleic acids.

[0121] Using functionalized magnetic particles as solid support has the advantage, that they can be processed and moved by the aid of a magnetic field. The magnetic particles can for example have superparamagnetic, paramagnetic, ferrimagnetic or ferromagnetic characteristics. The magnetic particles may comprise a magnetic material that is incorporated in the particles and / or is associated with the particles. To avoid leaching of the magnetic material e.g. when being in contact with a liquid like a sample material, the magnetic material is preferably completely encapsulated e. g. by the solid support material providing the surface of the solid support such as e.g. silica, polysilicic acid, glass or a polymeric material such as polyacrylate. The magnetic material may provide the core(s) of the particles, may be comprised in the core e.g. mixed with another material like a solid support material, and / or may be applied onto the core of the particle. In case the solid support is formed of two or more layers of solid support materials, the magnetic material may also be mixed with a solid supportmaterial used in at least one of the layers. Said mixture may be inhomogeneously or homogeneously.

[0122] According to one embodiment, the particle comprises a polymer core e.g. made of polystyrol, which is surrounded by at least one polymeric layer, preferably at least two polymeric layers, preferably a polyethylenimine layer and / or a poly acrylate layer.

[0123] In one embodiment, the particle surface is made of or comprises a polyacrylate, and the surface is then functionalized with one or more types of anion exchange moieties.

[0124] Suitable carboxyl particles comprising COOH groups are commercially available e.g. from QIAGEN (QIAGEN QIAseq Beads, 333923, 333903, 333927)

[0125] In an embodiment of the present disclosure, the surface of the solid support comprising one or more types of anion exchange moieties has at least one of the following characteristics: i. a type of anion exchange moieties comprises amine groups, preferably comprises or consists of a primary, secondary or tertiary amine or mixtures thereof; ii. the surface comprises polyethylenimine as anion exchange moiety; and at least one of the following characteristics: a. the solid support is provided in the form of particles, preferably magnetic particles; b. the solid support is provided in the form of particles which are provided in a suspension; c. the solid support is provided in the form of a membrane.

[0126] Binding the sperm cell and / or sperm nuclei as well as the non-sperm cell DNA to the solid support occurs in the presence of a suitable binding agent or binding solution.

[0127] While the lysing agent(s) or the lysing solution, respectively, are / is not supposed to lyse the sperm cells completely, it may occur that the sperm cells to someextent are pre-lysed in such a way that sperm nuclei may be released, while the sperm nucleic themselves are not supposed to be completely lysed. In such a case both, the sperm cells as well as the sperm nuclei, shall be bound to the solid support together with any residual amount of non-sperm DNA that might be present in the lysate. In order to reduce the amount of non-sperm DNA in the binding step, it is preferred though, to remove the lysate from the sperm cell and / or sperm nuclei enriched fraction before the binding step is initiated.

[0128] In an embodiment of the present disclosure, the binding solution or the one or more binding agents comprise(s) at least one chaotropic compound. In a preferred embodiment, the chaotropic compound is selected from a group consisting of a sodium salt, a thiocyanate salt or a guanidinium salt or urea, preferably sodium iodide, sodium perchlorate, guanidinium hydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate or a mixture of two or more salts thereof. The chaotropic compound is preferably a guanidinium salt, such as a guanidinium hydrochloride, guanidinium thiocyanate and / or guanidinium isothiocyanate.

[0129] Suitable concentrations and amounts of the chaotropic compounds may vary and are typically in the range of in the range of > 1 M to < 10 M generally being favourable, based on the total volume of the binding solution or the binding mixture, respectively. Preferably, the concentrations of the chaotropic compound is in the range of > 1 M to < 8 M, preferably in the range of > 2 M to < 8 M, more preferably in the range of > 2 M to7 M, particularly preferably in the range of > 2 M to6 M, such as IM, 1,5M, 2M, 2,5M, 3M, 3,5M, 4M, 4,5M, 5M, 5,5M and 6M , based on the total volume of the binding solution.

[0130] In one embodiment the binding solution and the one or more binding agents is / are suitable to bind sperm cells and / or sperm nuclei as well as non-sperm DNA to a silica solid support, preferably silica beads, more preferably magnetic silica beads. In a preferred embodiment the solid support in method step c) is a solid support selected from materials comprising silica, borosilicate, silicates, inorganic glasses, preferably in the form of particles, and binding of the sperm cell and / or sperm nuclei enriched fraction occurs in the presence of a binding solution and one or more binding agents comprising a chaotropic agent selected from a group consisting of a sodium salt, athiocyanate salt or a guanidinium salt or urea, preferably sodium iodide, sodium perchlorate, guanidinium hydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate and / or a mixture of two or more salts thereof and / or the binding solution having an alkaline pH value.

[0131] In another embodiment of the present disclosure the binding solution and the one or more binding agents is / are suitable to bind sperm cells and / or sperm nuclei as well as non-sperm DNA to a solid support comprising one or more types of anion exchange moieties, preferably anion exchange beads, more preferably magnetic anion exchange beads.

[0132] The solid support carrying the one or more type of anion exchange moieties as described herein can be provided in form of a suspension, the “solid support suspension”, preferably the “particle suspension” or more preferred the “bead suspension”. In a preferred embodiment, the solid support suspension comprises the anion exchange functionalized solid support, preferably the anion exchange functionalized beads, in an aqueous solution. In a simple embodiment, the anion exchange functionalized solid support, preferably the anion exchange functionalized in the form of beads is provided in water.

[0133] According to one embodiment, the solid support suspension comprising the anion exchange functionalized solid support, preferably in the form of anion exchange functionalized particles or beads, has a pH value of 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8.5 or less, 8 or less, 7.5 or less, 7 or less and preferably has a pH value below 6.5, more preferred below 6. Preferred pH values can be selected from a pH range from 3 to 10, 3.5 to 9, 3.75 to 8, 4 to 7, 3 to 6.5, 3.5 to 6, preferably the pH value is 3, 3.25, 3.5, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 6, 6.25, 6.5, 6.75, 7.

[0134] In some embodiments the binding solution has a pH value of 8 or less, 7 or less and preferably has a pH value below 6.5. Preferred pH values can be selected from a pH range from 3 to 8.5, 3.5 to 8, 3.5 to 7.75, 3.5 to 7.5, 3.5 to 7.25, 3.75 to 7, 4 to 6, preferably has a pH value of 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7.

[0135] In one embodiment the anion exchange functionalized solid support suspension, preferably the anion exchange functionalized particle or bead suspension, is mixed with the binding solution to form a binding mixture. The binding mixture has a pH value of 6 or less and preferably has a pH value below 5.75. Preferred pH values can be selected from a pH range from 3 to 6, 3.5 to 5.75, 4 to 5.5, preferably the pH value is 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6.

[0136] In one embodiment the sperm cell and / or sperm nuclei enriched fraction is contacted and / or incubated with the binding mixture.

[0137] According to one embodiment, binding solutions may further comprise one or more additives selected from the group consisting of buffers, in particular biological buffers as described herein (e.g. TRIS, MES or MOPS), detergents, preferably nonionic detergents (e.g. in a concentration of 2.5% or less, more preferred 1 % or less, 0.5% or less, 0.25% or less or 0,175% or less) and preservatives such as azides (e.g. in a concentration of 0.5% or less, 0.25% or less, 0.2%or less, 0.1 % or less, 0.05% or less).

[0138] In an embodiment, the binding solution in step c) further comprises one or more additives, preferably a chelating agent, a buffering agent, a surfactant, and / or a preservative.

[0139] In one embodiment, the solid support in method step c) is a solid support selected from materials having a surface functionalized with one or more types of anion exchange moieties, preferably in the form of particles which comprise one or more types of anion exchange moieties at their surface, and binding of the sperm cell and / or sperm nuclei enriched fraction occurs at a first pH selected from a pH range from 3 to 7, 3.5 to 6.5, 4 to 6, preferably a first pH value of 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7.

[0140] In an embodiment of the present disclosure, the one or more binding agents or the binding solution in step c) has one or more, preferably at least two of the following characteristics i. to v.: i. it comprises a chaotropic agent;ii. it comprises a chaotropic agent selected from a group consisting of sodium salts, a thiocyanate salts or guanidinium salts or urea, preferably a sodium iodide, sodium perchlorate, guanidinium hydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate and / or a mixture of two or more salts thereof; iii. it has an alkaline pH value; iv. it has a pH selected from a pH range from 3 to 7, 3.5 to 6.5, 4 to 6, preferably a pH value of 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7; v. the binding solution is obtained by mixing two or more agents with a solvent, preferably an aqueous solvent, more preferred water.

[0141] In some embodiments the solid support with bound non-sperm DNA and bound sperm cells and / or sperm nuclei is subjected to one or more washing steps.

[0142] In an embodiment a suitable washing solution or washing buffer comprises ethanol, chaotropic compounds, surfactants, or any combination thereof.

[0143] In a preferred embodiment the washing agents or the washing solution has at least one or two or more of the following characteristics i. to iv: i. it comprises an alcohol, preferably ethanol; ii. it comprises the binding solution and or binding agents; iii. it comprises the binding agents in the same concentration or amounts; iv. it comprises the binding agents in a concentration or amount that is different to the concentration or amount of the agents used as binding agents or in a binding solution.

[0144] In an embodiment of the disclosure the washing step is repeated once, twice or multiple times. This will ensure that impurities, such as cell debris that is present on the solid support are removed. Furthermore, by using the binding solution or agents as washing buffers, will strengthen the bond between the sperm cells and / or sperm nuclei and the non-sperm DNA bound to the solid support.

[0145] In an embodiment, after the washing in method step e) the washing agents and / or washing solution is separated, removed, or separated and removed from the solid support.

[0146] [In an embodiment of the present disclosure, the bound non-sperm DNA is eluted from the solid support, wherein the one or more elution agents or the elution solution are / is suitable to elute the non-sperm DNA but not the sperm cells and / or sperm nuclei bound to the solid support.

[0147] Any suitable elution method, elution agents and / or elution solution can be used that allows the elution of the non-sperm DNA from the support but not the sperm cells and / or sperm nuclei bound to the solid support. Suitable elution methods are known and can be determined by the skilled person.

[0148] In an embodiment of the disclosure, the elution solution or the one or more elution agents in step f) has at least one or two or more of the following characteristics i. to vii: i. it comprises water, preferably is water; ii. it has a second pH value selected from a pH range consisting of pH>8 and <13; pH>8 and <12,6; pH>8 and <12; >8 and <11; pH>8 and <10 and >8 and <9; iii. the second pH is higher than a first pH that was used for binding the non- sperm DNA to the solid support; iv. the second pH is lower than a third pH at which the sperm cells and / or sperm nuclei are eluted, wherein in this case the second pH value is not higher than 13, preferably the second pH is at least 0.1 units lower than the third pH, at least 0.2 unit lower than the third pH, more preferably at least 0.25 units lower or at least 0.3 units lower than the third pH; v. it comprises an aqueous solution comprising the buffering substance and optionally one or more further components such as a salt, in particular an alkali salt such as sodium chloride or potassium chloride; vi. it comprises an alkali hydroxide such as sodium hydroxide or potassium hydroxide, preferably sodium hydroxide;vii. it is added at least a 1 mmol / 1 sodium hydroxide solution, preferably a 2 mmol / 1 sodium hydroxide solution.

[0149] The choice of suitable elution agents or a suitable elution solution will mainly depend on the solid support that was used to bind the non-sperm DNA.

[0150] In an embodiment of the present disclosure, where the solid support is a solid support selected from silica materials the elution agents or elution solution comprises water.

[0151] In a preferred embodiment of the present disclosure, where the solid support is a solid support selected from silica materials the elution agent is water.

[0152] In a further preferred embodiment solid support selected from materials comprising silica, borosilicate, silicates, inorganic glasses, preferably in the form of particles, and the elution of the non-sperm DNA occurs in the presence of one or more elution agents or an elution solution comprising water, preferably is water.

[0153] In an embodiment of the present disclosure, where the solid support is a solid support selected from materials having a surface functionalized with anion exchange moieties, preferably in the form of particles which comprise anion exchange moieties at their surface, elution of the non-sperm DNA is pH dependent.

[0154] Elution of non-sperm DNA from anion exchange supports preferably occurs at a second pH which is higher than the first pH that was used for binding the non- sperm DNA to the solid support and lower than a third pH at which the sperm cells and / or sperm nuclei are eluted from the anion exchange support. The choice of the second pH value that is suitable for eluting the non-sperm DNA but not sperm cells or sperm nuclei from the anion exchange groups inter alia depends on the nature of the anion exchange groups present on the solid support, the density of the anion exchange groups on the surface of the solid support and the ionic strength of the elution solution. Suitable pH values for the second pH can be determined by the skilled person.

[0155] Preferably, non-sperm DNA elution from anion exchange supports occurs at a pH range selected from the group consisting of pH>8 and <13; pH>8 and <12,6; pH>8 and <12; >8 and <11; pH>8 and <10 and >8 and <9 and the second pH preferablyis at least 0.1 units lower than the third pH, at least 0.2 unit lower than the third pH, more preferably at least 0.25 units lower or at least 0.3 units lower than a third pH, at which the sperm cells and / or sperm nuclei are eluted.

[0156] In a preferred embodiment the solid support is a solid support selected from materials having a surface functionalized with one or more anion exchange moieties, preferably in the form of particles which comprise one or more types of anion exchange moieties at their surface, and the elution of the non-sperm DNA occurs at a second pH which is higher than the first pH that was used for binding the non-sperm DNA to the solid support, and the second pH is in a range selected from the group consisting of pH 2s 8 and 13; pH 5= 8 and 12,6;12;11;and the second pH preferably is at least 0.1 units lower than the third pH, at least 0.2 unit lower than the third pH, more preferably at least 0.25 units lower or at least 0.3 units lower than the third pH, at which the sperm cells and / or sperm nuclei are eluted.

[0157] The pH value that is used for elution may also depend on the intended further application of the eluate.

[0158] In an embodiment, the non-sperm DNA elution solution or the one or more elution agents preferably comprises a buffering substance which is capable of adjusting the second pH. In particular, biological buffers can be used. A suitable example is tri s(hydroxymethyl)aminom ethane (Tris) in a concentration of about 1 mM to 1 M, preferably 10 mM to 500 mM, more preferred 50 mM to about 250 mM, most preferred 75 mM to 150 mM, adjusted to the desired pH using e.g. HC1.

[0159] According to one embodiment, an elution solution comprising Tris as free base is used. The pH value of said elution solution containing the free base may be e.g. in a range of 10 to 10.5.

[0160] The non-sperm DNA elution solution preferably is an aqueous solution comprising the buffering substance and optionally further components such as a salt,in particular an alkali salt such as sodium chloride or potassium chloride, for example in a concentration of about 0.05 to about 0.5 M, preferably about 0.1 to about 0.2 M, more preferably about 0.16 M. The used salt concentration has an influence on the elution efficiency, in particular the elution efficiency of longer nucleic acids. The higher the salt concentration, the more efficient is the elution of longer nucleic acids.

[0161] Other possible buffers include but are not limited to HEPES, Tris-Borate and MOPS. Hence, preferably elution takes place at a low salt condition. Using a low salt concentration has the advantage that the eluate can be directly used in a downstream assay such as e.g. a PCR reaction.

[0162] According to one embodiment, a non-sperm DNA elution solution is used which comprises an alkali hydroxide such as sodium hydroxide or potassium hydroxide.

[0163] In one embodiment, elution is achieved by using a 1 mmol / 1 sodium hydroxide solution, preferably a 2 mmol / 1 sodium hydroxide solution.

[0164] Elution can also be assisted by heating and / or shaking.

[0165] In one embodiment of the present disclosure, elution step f) is repeated once, twice or multiple times.

[0166] In one embodiment of the present disclosure, the series of steps c), d), e) and f) is repeated at least once, twice or several times.

[0167] Once the non-sperm DNA is removed from the solid support, the sperm cells and / or sperm nuclei are subjected to a lysis by applying a sperm cell and / or sperm nuclei lysis solution or one or more lysing agents capable of lysing sperm cells and / or sperm nuclei. Lysis comprises applying or treating the sperm cell and / or sperm nuclei with a sperm cell lysis solution or with one or more sperm cell and / or sperm nuclei lysing agents in order to lyse sperm cells and / or sperm nuclei and to form a lysate comprising sperm DNA.

[0168] The sperm cell and / or sperm nuclei lysis solution or the one or more lysing agents comprise(s) at least one sperm cell and / or sperm nuclei lysing agent selectedfrom a reducing agent, a sperm cell and / or sperm nuclei lysing enzyme, a chaotropic agent, an alkaline agent in a concentration suitable to lyse a sperm cell and / or sperm nuclei, or a combination thereof.

[0169] In an embodiment of the disclosure, the sperm cell lysis solution or the one or more sperm cell lysing agents is / are applied to the sperm cells or sperm nuclei to form a lysate comprising sperm DNA.

[0170] In an embodiment of the disclosure, the sperm cell and / or sperm nuclei lysis solution or the one or more sperm cell and / or sperm nuclei lysing agents is / are applied to the sperm cells and / or sperm nuclei and the sperm cells and / or sperm nuclei are incubated in the sperm cell and / or sperm nuclei lysis solution or the one or more lysing agents to lyse sperm cells and / or sperm nuclei and to form a lysate comprising sperm DNA.

[0171] In an embodiment of the disclosure, the sperm cells and / or sperm nuclei are bound to the solid support during lysis.

[0172] In an embodiment of the disclosure, the sperm cells and / or sperm nuclei have been eluted from the solid support before lysis.

[0173] In the methods of the present disclosure, the sperm cell and / or sperm nuclei lysis solution or the one or more sperm cell and / or sperm nuclei lysing agents comprise(s) a chemical reducing agent. In some embodiments, the chemical reducing agent is selected from the group consisting of dithiothreitol (DTT), dithioerythrit, [3- mercaptoethanol (BME), glutathione (GSH), tris(2-carboxyethyl)phosphine (TCEP) and combinations thereof. Any suitable amount of reducing agent may be added to the sperms as long as the concentration of the reducing agent is sufficient to break the disulfide bonds present in the sperm cells and sperm nuclei. In certain embodiments, the concentration of the chemical reducing agent is between 0.1 mM and 0.1M (calculated within the total lysate).

[0174] In an embodiment of the present disclosure, the sperm cell and / or sperm nuclei lysis solution or the one or more sperm cell and / or sperm nuclei lysing agents comprise(s) at least an agent selected from sperm digesting enzymes, such as trypsin.

[0175] In a preferred embodiment the sperm cell and / or sperm nuclei lysis solution or the one or more sperm cell and / or sperm nuclei lysing agents further comprise(s) a chaotropic compound. The chaotropic compound is preferably a guanidinium salt, preferably selected from the group comprising guanidinium hydrochloride, guanidinium thiocyanate and / or guanidinium isothiocyanate or mixtures thereof. Preferably, the total concentration of the chaotropic compound(s) of the lysis solution or the one or more lysing agents is in the range that is sufficiently high to lyse at least 80% of the sperm cells and / or sperm nuclei, preferably in a concentration of > 0,005 mM to < 8 M, more preferably in a range of > IM to < 8 M based on the total volume of the lysis solution or the total lysate.

[0176] In an embodiment of the present disclosure, the sperm cell and / or sperm nuclei lysis solution or the one or more sperm cell and / or sperm nuclei lysing agents comprise(s) at least one chemical reducing agent and at least one sperm cell digesting enzyme.

[0177] In one embodiment of the present disclosure the sperm cell and / or sperm nuclei lysis solution or composition or the one or more sperm cell and / or sperm nuclei lysing agents comprise(s) an amine compound and / or an ammonium salt and one or more cell digesting enzymes.

[0178] As used herein, the term “amine” or “amino compound” refers to an organic compound characterized by the presence of a nitrogen atom bonded to one or more alkyl or aryl groups. The term "amino", is used herein as a prefix when the amine group is a substituent on a molecule with a higher-priority functional group, such as e.g. carboxylic acids, ketones, alcohols.

[0179] Amines may be classified as primary (-NH2), secondary (-NHR), or tertiary (-NR2), depending on the number of carbon-containing substituents (R) attached to the nitrogen atom. Unless otherwise specified, the term “amine” or “amino compound” includes aliphatic amines, aromatic amines, and heterocyclic amines, as well as their salts, protonated forms (ammonium ions), and derivatives that may participate in hydrogen bonding, coordination, or acid-base reactions within the context of the invention.

[0180] In one embodiment of the present disclosure the amino compound and / or ammonium salt has a pKa value of pKa 5= 8, preferably in a range of pKa 5= 8 and pKa13, more preferably a pKa value of 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.2, 10.4, 10.6, 10.8, 11.0, 11.2, 11.4, 11.6, 11.8, 12.0, 12.2, 12.4, 12.6, 12.8 or 13.0.

[0181] In an embodiment of the present disclosure the amine or amino compound is selected from a group comprising one or more amino alcohols selected from the group consisting of a primary amino alcohol, a secondary amino alcohol, a tertiary amino alcohol; one or more amino acids selected from the group consisting of alanine (ala - A), arginine (arg - R), asparagine (asn - N), aspartic acid (asp - D), cysteine (cys - C), glutamine (gin - Q), glutamic acid (glu - E), glycine (gly - G), histidine (his - H), isoleucine (ile - I), leucine (leu - L), lysine (lys - K), methionine (met - M), phenylalanine (phe - F), proline (pro - P), serine (ser - S), threonine (thr - T), tryptophan (trp - W), tyrosine (tyr - Y), valine (val - V); one or more amino sulfonic acids; and / or one or more ammonium salts; or any combination of the aforementioned amines or amino compounds.

[0182] In a further embodiment the sperm cell and / or sperm nuclei lysis solution comprises a composition and the composition has one or more of the following characteristics: a. the composition is a solution, preferably an aqueous solution; b. the composition further comprises : i. at least one solvent, preferably water; and / or ii. at least one pH regulating buffer. c. the composition comprises one or more amine compounds and / or an ammonium salt in a total concentration of 5mMol-50mMol, preferably one or more amino acids in a concentration range of 5mMol-50mMol, 5mMol- 40mMol, 10mMol-30mMol, preferably in a concentration of 5mMol, lOMmol, 15mMol, 20mMol, 25mMol, 30mMol, 35mMol, 40mMol, 45mMol or 50mMol; if more than one amine compound and / or ammonium salt are used in the composition, their amounts add up to the indicated concentration.d. the composition having a pH in a range of pH>8 and <13; pH>8 and <12; pH >8 and <11; pH>8 and <10, preferably a pH within the range of 1.5 pH unit above or below the pKa of the amine compound (pH = pKa ± 1.5); more preferably a pH within the range of 1 pH units above or below the pKa of the amine compound (pH = pKa ± 1). e. the composition contains, consists essentially of, or consists of one or more of the following components: i. an amino acid; ii. an amino acid having a pKa value in a range of pKa > 8 and pKa < 13; iii. L-arginine, glycine or alanine, or any combination thereof; iv. an amino acid and a solvent; v. L-arginine, glycine or alanine, or any combination thereof, and an aqueous solvent; vi. an amino acid and one or more pH regulators; vii. L-arginine, glycine or alanine, and one or more pH regulators; viii. a pH regulator selected from Tris, HEPES, MOPS or a combination thereof; ix. an amino acid and one or more cell digesting enzymes^ x. L-arginine, glycine or alanine, and one or more cell digesting enzymes; xi. one or more cell digesting enzymes selected from proteinase K, protease, zymolase, lyticase, chromopeptidase, trypsin, lysostaphin, lysozyme and / or any combinations thereof; xii. an ammonium salt; xiii. ammonium chloride or ammonium acetate, or a combination thereof; xiv. an ammonium salt, a solvent, optionally one or more pH regulators, and one or more cell digesting enzymes; xv. ammonium chloride or ammonium acetate, water, otionally Tris, HEPES or MOPS or any combination thereof, and one or more cell digesting enzymes selected from proteinase K, protease, zymolase, lyticase, chromopeptidase, trypsin, lysostaphin or lysozyme or any combination thereof; xvi. ethanolamine, a solvent, optionally one or more pH regulators, and one or more cell digesting enzymes;xvii. ethanolamine, water, optionally Tris, HEPES or MOPS or any combination thereof, and one or more cell digesting enzymes selected from proteinase K, protease, zymolase, lyticase, chromopeptidase, trypsin, lysostaphin or lysozyme or any combination thereof; xviii. taurine, a solvent, optionally one or more pH regulators, and one or more cell digesting enzyme, ; xix. taurine, water, optionally Tris, HEPES or MOPS or any combination thereof, and one or more cell digesting enzymes selected from proteinase K, protease, zymolase, lyticase, chromopeptidase, trypsin, lysostaphin or lysozyme or any combination thereof; xx. an aqueous solvent; xxi. water; xxii. PEG f. the composition does not contain a reducing agent; g. the composition does not contain a chaotropic agent; h. the composition does not contain any detergents, preferably no amine containing detergents.

[0183] In an embodiment of the present disclosure, the sperm cells and / or sperm nuclei are subjected to an alkaline lysis.

[0184] In an embodiment of the disclosure, lysis step h) is repeated once, twice or several times to ensure complete lysis of the sperm cells and sperm nuclei.

[0185] In an embodiment of the disclosure, lysis of the biological sample or the sperm cells and / or sperm nuclei is performed at room temperature or at a temperature sufficient to ensure efficient lysis of the cells and nuclei. In a preferred embodiment lysis is performed at a temperature in a range of 30°C to 80°C, preferably between 37° to 75°C, more preferably at 37°C, 56°C, 70°C, 75°C and / or 80°C.

[0186] In an embodiment of the disclosure, before lysis, the sperm cells and / or sperm nuclei bound to the solid support are subjected to at least one elution step by contacting the solid support with one or more elution agents or an elution solution and / or incubating the solid support in the presence of the one or more elution agents or theelution solution, wherein the one or more elution agents or the elution solution are / is suitable to elute the sperm cells and / or sperm nuclei bound to the solid support.

[0187] In an embodiment of the present disclosure, where the solid support is a solid support selected from materials having a surface functionalized with COOH groups preferably the solid support is in the form of particles having a surface functionalized with COOH groups.

[0188] In an embodiment of the present disclosure, where the solid support is a solid support selected from materials having a surface functionalized with anion exchange moieties, preferably in the form of particles which comprise anion exchange moieties on their surface, elution of the sperm cells and / or sperm nuclei is pH dependent.

[0189] In a preferred embodiment, elution of sperm cells and / or sperm nuclei from anion exchange supports occurs at a third pH which is higher than the second pH that was used for eluting the non-sperm DNA from the solid support or the particles comprising one or more anion-exchange moieties. The choice of the third pH value that is suitable for eluting the sperm cells and / or sperm nuclei from the anion exchange groups inter alia depends on the nature of the anion exchange groups present on the solid support or the particle, the density of the anion exchange groups on the surface of the solid support or the particles and the ionic strength of the elution solution.

[0190] In an embodiment the third pH for eluting sperm cells and / or sperm nuclei from anion exchange supports has a pH value selected from a pH range consisting of pH>8 and <14; pH>8 and <12,6; pH>8 and <12; >8 and <11; pH>8 and <10 and >8 and <9 and preferably is at least 0.1 units higher than the second pH, at least 0.2 unit higher than the second pH, more preferably at least 0.25 units higher or at least 0.3 units higher than the second pH at which non-sperm DNA is eluted.

[0191] In an embodiment of the present disclosure, the solid support is a solid support selected from materials comprising silica, borosilicate, silicates, inorganic glasses, preferably in the form of particles, and elution of the sperm cells and / or sperm nuclei occurs in the presence of an elution solution and one or more elution agents comprising H2O. Vigorously mixing and / or washing the sperm cells and / or sperm nuclei bound tothe solid support with water, preferably pure water, may also lead to an elution or partial elution of the sperm cells and / or sperm nuclei.

[0192] In an embodiment of the present disclosure, after method step a) and before method step b) the non-sperm DNA present in the sperm cell and / or sperm nuclei enriched fraction is digested or decomposed, preferably using one or more nucleases.

[0193] In an embodiment, after method step b) and before method step c) the nonsperm DNA present in the sperm cell and / or sperm nuclei enriched fraction is digested or decomposed, preferably using one or more nucleases.

[0194] In an embodiment, after method step c) and before method step d) and / or e) the non-sperm DNA present in the sperm cell and / or sperm nuclei enriched fraction is digested or decomposed, preferably using one or more nucleases.

[0195] In an embodiment, after method step d) and before optional method step e) and / or method step f) the non-sperm DNA present in the sperm cell and / or sperm nuclei enriched fraction is digested or decomposed, preferably using one or more nucleases.

[0196] In an embodiment, the non-sperm DNA is digested or decomposed during method steps c), and / or e), and / or f), and / or g), preferably using one or more nucleases.

[0197] In an embodiment, the non-sperm DNA is digested or decomposed after method step g) and before method step h),

[0198] In an embodiment, the method of the present disclosure further comprises one, two or more of the following steps i. the non-sperm DNA present in the sperm cell and / or sperm nuclei enriched fraction is digested or decomposed after method step a) and before method step b); ii. the non-sperm DNA present in the sperm cell and / or sperm nuclei enriched fraction is digested or decomposed after method step b) and before method step c); iii. the non-sperm DNA bound to the solid support is digested or decomposed after method step c) and before method step d) and / or e);iv. the non-sperm DNA is digested or decomposed after method step d) and before method step e) and / or f); v. the non-sperm DNA is digested or decomposed in method step c) and / or e), and / or f), and / or g); vi. the non-sperm DNA is digested or decomposed after method step g) and before method step h); vii. the non-sperm DNA is digested or decomposed using one or more nucleases.

[0199] In a preferred embodiment the non-sperm DNA is digested or decomposed using one or more nucleases selected from nucleases comprising for example Turbonuclease, all members of the DNase I family, all members of the DNase II family, Mikrococcus-Nuclease, Nuclease Pl, Nuclease SI, Phosphodiesterase I, Phosphodiesterase II, Restriction Endonucleases, Salt Activated Nunlease (SAN), , RNase I, Turbo Nuclease (=Benzonase ®), BAL 31 Nuclease, Cryonase Cold-Active Nuclease, , Mung Bean Nuclease, Thermolabile Exonuclease I, Mst Exonuclease I, Exonuclease T, , RecJf, T7 Exonuclease, RecBCD (Exonuclease V), truncated Exonuclease VIII, Lambda Exonuclease, T5 Exonuclease, DNase LXT, DNaseMe, Saltonase, Masterase, Azotobacter nuclease , SM nuclease, Neurospora nuclease, Bacillus nucleases, Strptomycex Nucleases, Cl Nuclease, Yeast Nuclease, Leishmania Nuclease, Staphylococcal Nuclease, Bal 31 nuclease, Anabaena Nuclease, Lysobacter Nuclease, Vibrio Nuclease, Schizosaccharomyces Nuclease, Nuclease Rsn, SR nuclease, Rye Germ Nuclease-I, Rye Germ Nuclease-II, Peunia Pollen Nuclease, Barley Nuclease, Wheat Nuclease, Potato Nuclease, Tobacco Pollen Nuclease, Barley Microspore Nuclease, Nuclease A, Drosophila Nuclease, Drosophila embryo nuclease, Shrimp Nuclease, Rat Liver Nuclease, Endonuclease G, Calf Thymus Nuclease, Phospholipase D Family, Caspase-activated DNase (CAD), DNase gamma (DNase y), Msz Exonuclease I, Exonuclease I, Exonuclease II, Exonuclease III, Exonuclease IV, Exonuclease VI, Exonuclease VII, Exonuclease VIII, Exonuclease IX, Exonuclease X, Cyclophilin, NUC-18 and Lactoferrin, more preferably a non-sperm cell digesting enzyme selected from the group consisting of proteinase K, protease, zymolase, lyticase, chromopeptidase, lysostaphin, lysozyme, nuclease, such as DNase, Benzonase(R), RNase, endonucleases, and exonucleases; or any combination thereof.

[0200] In one embodiment the one or more nucleases are selected from nucleases that have a large size, preferably of 50kDa or above, such as e.g., of 60kDa, 80kDa, lOOkDa, 150kDa, 200kDa, 250kDa, 300kDa, 350kDa or preferably of 400kDa or above, such as nucleases having a size of 70kDa, 90kDa, HOkDa, 120kDa, 130kDa, 140kDa,160kDa, 170kDa, 180kDa, 190kDa, 210kDa, 220kDa, 230kDa, 240kDa,260kDa, 270kDa, 280kDa, 290kDa, 310kDa, 320kDa, 330kDa, 340kDa, 360kDa,370kDa, 380kDa, 390kDa, 410kDa, 420kDa, 430kDa, 440kDa, 450kDa, 460kDa,470kDa, 480kDa, 49kDa, or 500kDa, or having any of the sizes in between.

[0201] In one embodiment the one or more nucleases are selected from nucleases that have a large size, of preferably over 50kDa, such as EcoRl restriction endonuclease, Phosphodiesterase I, Phosphodiesterase II, Exonuclease I, Exonuclease V (RecBCD), Exonuclease VII, and Lactoferrin.

[0202] In another embodiment the one or more nucleases are selected from nucleases that have multiple independent functions, such as a helicase activity, and / or an endonuclease activity and / or an exonuclease activity, preferably nucleases that have a helicase activity and an endonuclease activity, a helicase activity and an exonuclease activity, an endonuclease and an exonuclease activity or a helicase activity, an endonuclease activity and an exonuclease activity.

[0203] In yet another embodiment the one or more nucleases are selected from nucleases that are large in size and have multiple independent functions, such as a helicase activity, and / or an endonuclease activity and / or an exonuclease activity.

[0204] In a preferred embodiment the one or more nucleases are selected from nucleases that have a size of 50kDa or above, such as of 60kDa, 80kDa, lOOkDa, 150kDa, 200kDa, 250kDa, 300kDa, 350 kDa, or preferably of 400kDa or above, like having 70kDa, 90kDa, HOkDa, 120kDa, 130kDa, 140kDa,160kDa, 170kDa, 180kDa, 190kDa, 210kDa, 220kDa, 230kDa, 240kDa, 260kDa, 270kDa, 280kDa, 290kDa, 310kDa, 320kDa, 330kDa, 340kDa, 360kDa, 370kDa, 380kDa, 390kDa, 410kDa, 420kDa, 430kDa, 440kDa, 450kDa, 460kDa, 470kDa, 480kDa, 49kDa, or 500kDa, or having any of the sizes in between, and in addition have multiple independent functions, such as a helicase activity, an endonuclease activity and / or an exonuclease activity.

[0205] Without being bound by any particular theory, it may be advantageous that nucleases that are desired to digest non-sperm DNA but not to digest sperm DNA within sperm cells or sperm nuclei, are selected from large molecules that due to their size may be prevented from getting close to the sperm DNA, as this forms a coherent, tightly packed formation as the sperm nucleus. Furthermore, in order for the nuclease to be able to exert an endonuclease activity, it is assumed that at first a segregation of the DNA double strand into single strands needs to take place. This may e.g. be achieved by a helicase, preferably by a nuclease that has a helicase activity. However, this might be difficult with sperm DNA, as sperm DNA is condensed within the sperm nucleus. It is therefore believed that due to the fact that sperm DNA is protected as a tightly packed sperm nucleus, it is difficult for a small nuclease or a nuclease that has no exonuclease activity to find and access the free starting points on the sperm DNA.

[0206] In an embodiment of the invention the nuclease has at least one of the subsequent features: i) it has a size of 50kDa or above; ii) it has a size of 60kDa, 80kDa, lOOkDa, 150kDa, 200kDa, 250kDa, 300kDa, 350kDa or preferably of 400kDa or above, or any size in between; iii) it is selected from the group comprising EcoRl restriction endonuclease, Phosphodiesterase I, Phosphodiesterase II, Exonuclease I, Exonuclease V (RecBCD), Exonuclease VII, and Lactoferrin; iv) it has multiple independent functions; v) it has a helicase activity; vi) it has an endonuclease activity; vii) it has an exonuclease activity; viii) it has a helicase activity and an endonuclease activity; ix) it has a helicase activity and an exonuclease activity; x) it has an endonuclease and an exonuclease activity; xi) it has a helicase activity, an endonuclease activity and an exonuclease activity; xii) the nuclease is chosen such that it does not get access to the packed DNA.

[0207] Digesting and / or decomposing and removing non-sperm DNA from the sperm cells or sperm nuclei reduces or removes contamination of the sperm cells or sperm nuclei with non-sperm DNA prior to lysing the sperm fraction.

[0208] The present disclosure also provides kits for treatment of forensic samples comprising non-sperm cells and sperm cells and sperm nuclei comprising one or more of a) a non-sperm cell lysis solution or one or more non-sperm cell lysing agents capable of lysing non-sperm cells and essentially not suitable for completely lysing sperm cells and / or sperm nuclei; b) a solid support; c) binding agents or a binding solution capable of binding the sperm cell and / or sperm nuclei enriched fraction to the solid support, preferably the one or more binding agents or the binding solution have / has one or more, preferably at least two of the following characteristics i. to v.: i. it comprises a chaotropic agent, optionally under alkaline conditions; ii. it comprises a chaotropic agent selected from a group consisting of sodium salts, a thiocyanate salts or guanidinium salts or urea, preferably a sodium iodide, sodium perchlorate, guanidinium hydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate and / or a mixture of two or more salts thereof; iii. it has an acidic pH value; iv. it has a first pH selected from a pH range from 3 to 7, 3.5 to 6.5, 4 to 6, preferably a pH value of 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7; v. the binding solution is obtained by mixing two or more agents; d) elution agents or an elution solution capable of eluting the non-sperm DNA but not the sperm cells and / or sperm nuclei bound to the solid support, the elution solution or the one or more elution agents has / have at least one or two or more of the following characteristics i. to v: i. it comprises water, preferably is water; ii. it has a second pH value selected from a pH range consisting of pH>8 and <13; pH>8 and <12,6; pH>8 and <12; >8 and <11; pH>8 and <10 and >8 and <9;iii. the second pH is higher than the first pH that was used for binding the nonsperm DNA to the solid support; iv. the second pH is lower than a third pH at which the sperm cells and / or sperm nuclei are eluted from the solid support, preferably at least 0.1 units lower than the third pH, at least 0.2 unit lower than the third pH, more preferably at least 0.25 units lower or at least 0.3 units lower than the third pH; iv. it comprises an aqueous solution comprising the buffering substance and optionally one or more further components such as a salt, in particular an alkali salt such as sodium chloride or potassium chloride; v. it comprises an alkali hydroxide such as sodium hydroxide or potassium hydroxide, preferably sodium hydroxide, such as a 1 mmol / 1 sodium hydroxide solution, preferably a 2 mmol / 1 sodium hydroxide solution; e) a sperm cell and / or sperm nuclei lysis solution or one or more sperm cell and / or sperm nuclei lysing agents capable of lysing sperm cells and / or sperm nuclei, preferably the sperm cell and / or sperm nuclei lysis solution or one or more lysing agents comprises at least one reduction agent selected from the group consisting of dithiothreitol (DTT), Dithioerythrit, P-mercaptoethanol (BME), glutathione (GSH), Tris(2-carboxyethyl)phosphine (TCEP) and combinations thereof; a sperm lysing enzyme selected from the group consisting of proteinase K, protease, zymolase, lyticase, chromopeptidase, trypsin, lysostaphin, lysozyme; a chaotropic agent and / or an alkaline agent in a concentration suitable to lyse a sperm cells and / or sperm nuclei; one or more amino compounds selected from the group comprising amino alcohols selected from the group consisting of a primary amino alcohol, a secondary amino alcohol and a tertiary amino alcohol; one or more amino acids selected from the group consisting of alanine (ala - A), arginine (arg - R), asparagine (asn - N), aspartic acid (asp - D), cysteine (cys - C), glutamine (gin - Q), glutamic acid (glu - E), glycine (gly - G), histidine (his - H), isoleucine (ile - I), leucine (leu - L), lysine (lys - K), methionine (met - M), phenylalanine (phe - F), proline (pro - P), serine (ser - S), threonine (thr - T), tryptophan (trp - W), tyrosine (tyr - Y) and valine (val - V),; one or more amino sulfonic acids and / or one or more ammonium salts; or any combination of the aforementioned lysing agents;f) optionally, elution agents or an elution solution capable of eluting the sperm cells and / or sperm nuclei bound to the solid support; g) optionally, one or more washing agents or a washing solution, preferably the one or more washing agents or the washing solution have / has at least one or two or more of the following characteristics i. to iv: i. it comprises an alcohol, preferably ethanol; ii. it comprises the binding solution and or binding agents; iii. it comprises the binding agents in the same concentration or amounts as the concentration or amount of the agents used as binding agents or in a binding solution; iv. it comprises the binding agents in a concentration or amount that is different to the concentration or amount of the agents used as binding agents or in a binding solution; h) optionally, one or more means for separating, removing, or separating and removing any one of the following: i. the lysate from a sperm cell and / or sperm nuclei enriched fraction, and / or ii. the binding agents or binding solution from the solid support, and / or iii. the lysate comprising sperm DNA from the solid support, and / or iv. the washing agents or washing solution from the solid support.

[0209] The lysis, binding, elution and washing solutions or the lysing, binding, elution and / or washing agents used in the kits of the disclosure are described in detail above and apply to this aspect of the disclosure as well.

[0210] The lysis, binding, elution and / or washing solutions present in the kit may be provided as ready to use mixtures or as separate components or compositions to be mixed (before use) or to be added directly to the material to be lysed, bound, eluted or washed by the user according to instructions present in the kit. The lysis, binding, elution or washing solutions and / or their individual agents or compositions are provided in suitable containers.

[0211] In an embodiment, the kit further comprises suitable containers, collection tubes, sample tubes etc, spin columns or baskets and other material useful for sampleprocessing according to the method of the disclosure. The kit also includes instructions for use.

[0212] In some embodiments, the present disclosure includes an apparatus for processing a forensic sample including sperm cells and sperm nuclei and non-sperm cells. The method can be easily adapted to automation and low or high throughput processing with a robotic system.

[0213] In a preferred embodiment one or more steps of the methods of the present disclosure are automated.

[0214] In an embodiment the method of the disclosure further comprises purifying, isolating, detecting, analysing and / or quantifying the non-sperm cell and / or sperm DNA.

[0215] In some embodiments, sperm DNA is obtained from the purified sperm cells and / or sperm nuclei and further analysed. For example, sperm DNA may be quantified or genotyped.

[0216] In a further embodiment, non-sperm DNA obtained from the sample is further purified and / or analysed. For example, non-sperm DNA may be quantified or genotyped.

[0217] Methods for quantifying DNA are known in the art and include, for example, spectrophotometry, fluorescence, real-time PCR (also known as quantitative PCR (qPCR)), and digital PCR technologies. Genotyping DNA may be performed using any suitable method known in the art. Such methods include, for example, short tandem repeat (STR) analysis, restriction fragment length polymorphism (RFLP) of genomic DNA, random amplified polymorphic detection (RAPD) of genomic DNA, amplified fragment length polymorphism (AFLP) detection, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR), DNA sequencing, allele specific oligonucleotide (ASO) probes, and hybridization to DNA microarrays or beads.

[0218] EXAMPLES

[0219] Each example is provided by way of explanation of the disclosure and is not meant as a limitation of the disclosure.

[0220] In the examples the Investigator Quantiplex Pro Kit (QIAGEN Cat. No. 387216) was used for quantification of autosomal and male DNA in the individual samples.

[0221] The quantification of the qPCR markers on autosomal chromosomes and on the Y chromosome is shown in Figures 1 to 9.

[0222] Example 1

[0223] This example demonstrates that sperm cells and / or sperm nuclei can be bound to silica beads and DNA from sperm cells can be extracted and purified after binding.

[0224] Ejaculate was diluted with RNase free water to a ratio of 1 / 10 and a 20 pl aliquot was treated with a buffer containing 25 pl Proteinase K (20 mg / ml) and sodium dodecyl sulphate (SDS, 0.4%) and incubated for Ih at 56 °C and 900 rpm.

[0225] After pelletisation of the sperm cells and / or sperm nuclei most of the supernatant containing the non-sperm DNA from non-sperm cells of the ejaculate was removed, with a residue of up to 30 pl of the supernatant remaining in the tube. The pellet is resuspended in a buffer containing sodium dodecyl sulphate (SDS, 0.4%) and incubated and mixed at 56 °C.

[0226] The Suspension was then mixed with a silica beads suspension. In this example silica beads from the QIAGEN EZ1&2 DNA Investigator Kit were used (QIAGEN Cat. No.952034)) and treated with 770 pl of one or more chaotropic buffers (e.g. QIAGEN, Buffer G2 (260 pl) Cat. No. / ID: 1014636G2 and QIAGEN, Buffer MTL Cat. No. / ID: 1014636).

[0227] Sperm cells and / or sperm nuclei are bound to the silica beads with 720 pl of a chaotropic buffer comprising 5,5 M GTC (QIAGEN, Buffer MTL Cat. No. / ID: 1014636).

[0228] The silica beads with bound sperm cells and / or sperm nuclei were washed twice with washing buffers comprising 5.6M GuHCl and 24 Vol-% Ethanol and a detergent (e.g. Tween) and subsequently with washing buffers comprising 80 Vol-% Ethanol (e.g. QIAGEN, washing buffers from in the EZ1&2 DNA Investigator Kit; QIAGEN Cat. No.952034).

[0229] In a first step, the so called “Elution sperm lysis” (see Figure 1, middle column), the sperm cells and / or sperm nuclei were partly eluted by vigorously mixing the beads with H2O. During this step, only a fraction of sperms is eluted from the beads.

[0230] This fraction of sperm cells was then lysed using 200pl of a lysis solution comprising DTT (final concentration 200 mM), one or more chaotropic buffers (e.g., QIAGEN, Buffer G2, Cat. No. / ID: 1014636) and 10 pl Proteinase K (20 mg / ml).

[0231] Sperm DNA present in the lysate was purified using the QIAGEN EZ1&2 DNA Investigator Kit Trace protocol (QIAGEN Cat. No.952034).

[0232] The elution with water did not result in a complete removal of the sperm cells from the silica beads and sperm cells were still bound to the silica beads.

[0233] In a second step, the so called “Bead sperm lysis” (see Figure 1, right column) the sperm cells that were not eluted and still bound to the silica beads were lysed without prior elution. The sperm cells were lysed and DNA from sperm cells and / or sperm nuclei still bound to the beads was extracted and purified using the same lysis buffers and protocols as in the first step.

[0234] Results are shown in Figure 1.

[0235] Figure 1 clearly shows that sperm cells bind to silica beads. DNA from sperm cells can be extracted and purified after binding of the sperm cells to the beads. The extraction of the DNA is possible either after eluting the sperm cells from the beads or while the sperm cells are still bound to the silica beads.

[0236] The middle column (“Elution sperm lysis”) shows the concentration of the targeted male DNA purified from the sperm fraction after elution from the silica beads.

[0237] The right column (“Bead sperm lysis”) shows the concentration of the targeted male DNA purified in the second step while sperm cells were still bound to the silica beads.

[0238] The left column shows the amount of male DNA concentration extracted from sperm cells that did not bind to silica beads and thus remained in the supernatant after sperm cell binding.

[0239] The very low concentration of 0.02 ng / pl of male DNA extracted from sperm cells remaining in the supernatant after binding proves that sperm cells are effectively bound to silica beads.

[0240] Example 2

[0241] This example demonstrates that sperm cells and / or sperm nuclei can be bound to carboxy beads.

[0242] Ejaculate was diluted with RNase free water to a ratio of 1 / 10 and a 20 pl aliquot was treated with a buffer containing 25 pl Proteinase K (20 mg / ml) and sodium dodecyl sulphate (SDS, 0.4%) and incubated for Ih at 56 °C and 900 rpm.

[0243] After pelletisation of the sperm cells and / or sperm nuclei most of the supernatant containing the non-sperm DNA from non-sperm cells of the ejaculate was removed with a residue of up to 30 pl of the supernatant remaining in the tube.

[0244] The pellet is resuspended in 220 pl of a buffer containing sodium dodecyl sulphate (SDS, 0.4%) and mixed with 250 pl of carboxy beads and incubated for 10 minutes on an rotating shaker. In this example a QIAseq Bead suspension was used (QIAGEN Cat. No. 333923, 333903 or 333927).

[0245] Afterwards the supernatant was removed, and the beads were washed 3x with a buffer comprising lOmM TrisHCl, ImM EDTA at pH 8.0.

[0246] Following the washing steps the sperm cells on the beads were lysed using 200pl of a lysis solution comprising DTT (final concentration 200 mM), one or more chaotropic buffers (e.g., QIAGEN, Buffer G2, Cat. No. / ID: 1014636) and 10 pl Proteinase K (20 mg / ml).

[0247] Sperm DNA present in the lysate was purified using the EZ1&2 DNA Investigator Kit (QIAGEN Cat. No.952034) Trace protocol.

[0248] As a control, the pellet was lysed directly after centrifugation and removal of supernatant prior to the binding of sperm cells and / or sperm nuclei to beads. The sperm cells were lysed and DNA from sperm cells and / or sperm nuclei was extracted and purified using the same lysis buffers and protocols as mentioned above (QIAGEN, EZ1&2 DNA Investigator Kit (QIAGEN Cat. No.952034) Trace protocol).

[0249] The DNA concentration was determined using the Investigator Quantiplex Pro Kit (QIAGEN Cat. No. 387216).

[0250] Results are shown in Figure 2.

[0251] Figure 2 clearly shows that sperm cells bind to carboxy beads. DNA from sperm cells can be extracted and purified after binding of the sperm cells to the beads. The extraction of the DNA is possible while the sperm cells are still bound to the carboxy beads.

[0252] In Figure 2 the concentration of purified DNA after lysis of the bound sperm cells is shown in the right column as “Sperm lysis on beads”. The male DNA concentration measured for this fraction is 1.79 ng / pl.

[0253] The left column in Figure 2 shows the concentration of purified DNA from the sperm pellet, without binding to the beads, the “Pellet control”. The male DNA concentration measured is 2.69 ng / pl and represents the sperm cells which are present in the ejaculate used in this experiment.

[0254] Example 3

[0255] This example demonstrates that sperm cells and / or sperm nuclei together with non-sperm DNA can be bound to anion exchange beads and non-sperm cell DNA and sperm cells can be eluted at different pH values and with different binding and elution agents.

[0256] The results are shown in Figures 3a and 3b.

[0257] Ejaculate was diluted 1 / 10 with RNase free water and 5 pl mixed with nonsperm DNA.

[0258] The non-sperm DNA was extracted from vaginal swabs. The swabs were incubated with a buffer containing 25 pl Proteinase K (20 mg / ml) and sodium dodecyl sulphate (SDS, 0.4%) at 56 °C and 900 rpm for 1 h.

[0259] The mix of ejaculate and non-sperm DNA was pre-treated for Ih at 56 °C and 900 rpm with a buffer containing 25 pl Proteinase K (20 mg / ml) and sodium dodecyl sulphate (SDS, 0.4%). After pelletization of the sperm cells and / or sperm nuclei the supernatant was removed with up to 30 pl of the supernatant remaining in the tube. The pellet is resuspended in 400 pl of a 100 mM Tris buffer, pH8.0 and mixed with 60 pl of a buffer comprising citrate acid (330 mM) and a detergent at pH 4.5 (e.g. QIAGEN, QSB4 buffer in EZ1&2 ccfDNA Kit Cat. no. 954854).

[0260] Binding of sperm cells and non-sperm DNA to the anion exchange beads:

[0261] The suspension was mixed with 60 pl of anion exchange beads suspension (e.g. QIAGEN, Magnetic Bead Suspension EZ from EZ1&2 ccfDNA Kit Cat. no. 954854) and incubated for 15 min at RT.

[0262] Afterwards the beads were separated from the supernatant and the supernatant further processed resulting in the so called “supernatant after bead binding” results (see Figure 3a and 3b).

[0263] The supernatant was lysed using 400 pl of a lysis solution comprising DTT (final concentration 40 mM) and one or more chaotropic buffers (e.g. QIAGEN, Buffer MTL, Cat. No. / ID: 1014636). Sperm DNA present in the lysate was purified using the EZ1&2 DNA Investigator Kit (QIAGEN Cat. No.952034) Large Volume protocol.

[0264] As a control, another sample was processed similarly, but without incubating the mix of ejaculate and non-sperm DNA with beads. Instead, the mix of ejaculate and non-sperm DNA was directly subjected to lysis and the lysate was purified in the same way as described above.

[0265] In Figure 3a, the second from the left two columns (“supernatant after bead binding”) shows the concentration of autosomal DNA obtained to be 0.10 ng / pl. No male DNA concentration could be measured.

[0266] In Figure 3b, the third double columns (“supernatant after bead binding”) show the concentration of autosomal DNA obtained to be 0,1 ng / pl.

[0267] This result shows that most of non-sperm DNA and all sperm cells are bound to anion exchanger beads and a very low residual amount of non-sperm DNA is present in the supernatant after binding.

[0268] Elution of non-sperm cell DNA and sperm cells bound to the anion exchange beads:

[0269] As shown in Figure 3a a sample of sperm cells and / or sperm nuclei and non- sperm DNA bound to the anion exchanger beads was treated with NaOH as the elution buffer. Different concentrations of NaOH are applied to the beads in order to elute the two DNA species at different pH values.

[0270] The non-sperm DNA is eluted with a 2 mM NaOH elution solution. The sperm cells were eluted when the beads were treated with a 3mM NaOH elution solution.

[0271] The DNA of 500 pl of 2 mM and 3 mM eluates (named 2 mM NaOH and 3 mM in Figure 3) were lysed and purified purified using the EZ1&2 DNA Investigator Kit (QIAGEN Cat. No.952034) Large Volume protocol.

[0272] In Figure 3b, the elution results are shown with sodium chloride (NaCl) used in an elution solution.The respective DNA concentration was determined using the Investigator Quantiplex Pro Kit (QIAGEN Cat. No. 387216).

[0273] Results in Figures 3a and 3b highlight that sperm cells and non-sperm cell DNA can be bound to anion-exchange beads and non-sperm cell DNA and sperm cells can be eluted at different pH values and using different elution agents.

[0274] In Figure 3a, the first two columns (“no beads control”) show the concentration of the measured DNA: 1.29 ng / pl of autosomal DNA and 0.18 ng / pl male DNA. This shows the total amount of non-sperm DNA and sperm cell DNA that can be extracted from the used samples. The second from the left two columns (“supernatant after bead binding”) show the concentration of autosomal DNA obtained to be 0.10 ng / pl. No male DNA concentration could be measured.

[0275] This result shows that most of non-sperm DNA and all sperm cells are bound to anion exchanger beads and a very low residual amount of non-sperm DNA is present in the supernatant after binding.

[0276] In Figure 3 a the second from the right two columns (“2mM NaOH”) show the results after the elution with a 2mM NaOH elution buffer. The concentration of autosomal DNA purified after this elution is 1,01 ng / pl whereas only 0,03 ng / pl male DNA was measured.

[0277] The two right columns (“3mM NaOH”) show the results after the elution with a 3mM NaOH elution buffer. The concentration of autosomal DNA purified after this elution is 0,22 ng / pl whereas 0,11 ng / pl male DNA was measured. When comparing these concentrations to the controls it is obvious that most of the autosomal DNA is eluted at a lower NaOH concentration, whereas the sperm DNA is eluted at the higher NaOH concentration.

[0278] By selecting the pH accordingly, non-sperm DNA can be eluted from the anion exchange beads, while the sperm cells remain bound to the beads. As shown, non-sperm DNA can be eluted at a concentration of 2 mM NaOH while sperm cells are eluted at a higher concentration of NaOH (3 mM) and thus at a higher pH.

[0277] Fig. 3b shows in the right two columns (“supernatant after bead binding”) the concentration of autosomal DNA obtained to be 0.10 ng / pl and the one of the male DNA to be 0.08 ng / pl. This again indicates that most of the nucleic acid has been bound to the solid support.

[0278] Figure 3b shows that elution with a NaCl solution is also possible to obtain a sufficient yield of male DNA. While the left and middle two columns in Figure 3bshow that the obtained yield of the male DNA decreases with increasing NaCl concentration, it also shows that the amount of the autosomal DNA is in a similar order of magnitude than the male DNA.

[0279] Example 4

[0280] This example showcases an embodiment of the method of the invention where sperm cells and non-sperm DNA are jointly bound to silica beads. Once non-sperm DNA is removed from the beads, sperm cell DNA can be extracted and purified. The extraction of the DNA is possible either after eluting the sperm cells from the beads and / or while the sperm cells are still bound to the silica beads.

[0281] Binding of the sperm cells and the non-sperm DNA:

[0282] Ejaculate (20 pl) and vaginal swabs containing epithelial cells were separately treated Ih at 900 rpm and 56 °C with 500 pl of a buffer containing 25 pl Proteinase K (20 mg / ml) and sodium dodecyl sulphate (SDS, 0.4%). This treatment resulted in the lysis of the epithelial cells but not the sperm cells or sperm nuclei

[0283] 15 pl of the lysate of epithelial cells and 35 pl of the suspension of sperm cells and / or sperm nuclei were mixed and 140 pl of a binding buffer (e.g. e.g. QIAGEN Buffer G2 (260 ml) Cat. No. / ID: 1014636) and 10 pl Proteinase K (QIAGEN) (20 mg / ml) were added to the mixture. The non-sperm DNA and sperm cells and / or sperm nuclei are bound to the silica beads with 720 pl of a chaotropic buffer comprising 5,5 M GTC (QIAGEN, Buffer MTL Cat. No. / ID: 1014636).

[0284] The silica beads with bound sperm cells and / or sperm nuclei were washed twice with washing buffers comprising 5.6M GuHCl and 24 Vol-% Ethanol and a detergent (e.g. Tween) and subsequently with washing buffers comprising 80 Vol-% Ethanol (e.g. QIAGEN, washing buffers from in the EZ1&2 DNA Investigator Kit; QIAGEN Cat. No.952034).

[0285] Non-sperm DNA elution and lysis:

[0286] In a first step, the non-sperm DNA is eluted from the beads by rinsing the beads with 800 pl H2O.

[0287] As a control for sperm elution, 50 pl of this eluate was treated with sperm lysis buffer containing reducing agent DTT and 10 pl Proteinase K (20 mg / ml) in order to lyse sperm cells that were eluted during this step.DNA was purifies using the EZ1&2 DNA Investigator Kit Trace.

[0288] In Figure 4 the “Non-Sperm DNA elution sperm lysis” is shown in the left two columns. DNA concentrations measured were multiplied with the factor of 16, because 50 pl of the 800 pl rinse was used. The autosomal DNA concentration was at 3.87 ng / pl and the male DNA concentration 0.02 ng / pl.

[0289] The results show that female DNA can be reduced when non-sperm DNA is eluted and the sperm cells and / or sperm nuclei remain bound to the silica beads.

[0290] Sperm cell elution and lysis:

[0291] After elution of non-sperm DNA, the beads with the bound sperm cells were subjected to a sperm cell elution step by vigorously mixing the beads with H2O.

[0292] This fraction of sperm cells was then lysed using 200pl of a lysis solution comprising DTT (final concentration 200 mM), one or more chaotropic buffers (e.g., QIAGEN, Buffer G2, Cat. No. / ID: 1014636) and 10 pl Proteinase K (20 mg / ml).

[0293] Sperm DNA present in the lysate was purified using the QIAGEN EZ1&2 DNA Investigator Kit Trace protocol (QIAGEN Cat. No.952034).

[0294] The concentration of DNA after purification is shown in Figure 4, right two columns as “Elution sperm lysis”. The autosomal DNA concentration is measured as 0.48 ng / pl and the mal DNA concentration 0.23 ng / pl.

[0295] The results show that a sperm fraction with minimal non-sperm DNA contamination was eluted.

[0296] The elution with water did not result in a complete removal of the sperm cells from the silica beads and sperm cells were still bound to the silica beads.

[0297] Sperm cells that were not eluted and remained bound to the silica beads were then lysed without prior elution. DNA from sperm cells and / or sperm nuclei still boundto the beads was extracted by using 200 pl of a lysis solution comprising DTT (final concentration 200 mM), one or more chaotropic buffers (e.g. QIAGEN, Buffer G2, Cat. No. / ID: 1014636) and 10 pl Proteinase K (20 mg / ml).

[0298] Sperm DNA present in the lysate was purified using the QIAGEN EZ1&2 DNA Investigator Kit Trace protocol (QIAGEN Cat. No.952034). DNA concentrations were determined using the Quantiplex Pro Kit (QIAGEN Cat. No. 387216).

[0299] The concentration of DNA after purification is shown in Figure 4, middle two columns as “Beads sperm lysis”. The autosomal DNA concentration is measured as 0.43ng / pl and the mal DNA concentration 0.27 ng / pl.

[0300] The results show that a sperm fraction with minimal non-sperm DNA contamination is still bound to the silica beads.

[0301] The results of this Example are shown in Figure 4.

[0302] The right two columns (“Elution sperm lysis”) show the concentration of the targeted male DNA purified from the sperm fraction after elution from the silica beads.

[0303] The middle two columns (“Bead sperm lysis”) show the concentration of the targeted male DNA purified while sperm cells were still bound to the silica beads.

[0304] The left two columns (Non-Sperm DNA elution sperm lysis”) show the amount of non-sperm DNA and male DNA obtained from the lysate after the non- sperm DNA elution step.

[0305] The very low concentration of 0.02 ng / pl of male DNA extracted from sperm cells remaining in the supernatant after binding proves that sperm cells are effectively bound to silica beads. Furthermore, a low amount of autosomal contamination was observed in the samples after differential elution of the autosomal DNA.

[0306] The sperm fraction could be lysed and purified after binding to the silica beads.

[0307] Example 5

[0308] This example highlights a complete workflow capable of purifying DNA from sperm cells.

[0309] For this experiment vaginal swabs were spiked with 20 pl of ejaculate diluted 1 / 100 or 1 / 500 with RNase free water. These swabs were treated with a buffer containing 25 pl Proteinase K (20 mg / ml) and sodium dodecyl sulphate (SDS, 0.4%) and incubated for Ih at 56 °C and 900 rpm. After pelletisation of the sperm cells and / or sperm nuclei most of the supernatant containing the non-sperm DNA was removed, with a residue of up to 30 pl of the supernatant remaining in the tube.

[0310] The pellet was washed three times with a buffer containing sodium dodecyl sulphate (SDS, 0.4%). After the washing of the pellet, a residual volume of 30 pl containing the sperm cells and / or sperm nuclei and non-sperm DNA remains in the tube.

[0311] For the c+ pellet control, the pellet of sperm cells and residual non sperm DNA in the remaining 30 pl of the supernatant was treated with 200 pl of a lysis solution comprising DTT (final concentration 200 mM), one or more chaotropic buffers (e.g. QIAGEN, Buffer G2, Cat. No. / ID: 1014636) and 10 pl Proteinase K (20 mg / ml). This results in the so called “c+ pellet control“(see Figure 5).

[0312] Sperm DNA present in the lysate was purified using the EZ1&2 DNA Investigator Kit (QIAGEN Cat. No.952034) Trace protocol.

[0313] In Figure 5 the DNA concentration of purified DNA from this fraction is shown as “c+ pellet control”.

[0314] The autosomal DNA concentration for the 1 / 100 diluted ejaculate sample is 1.07 ng / pl and 1.05 ng / pl for the 1 / 500 diluted ejaculate sample.

[0315] The male DNA concentration is 0.29 ng / pl for the sample with ejaculate diluted 1 / 100 and 0.06 for the 1 / 500 diluted ejaculate sample. This result shows remaining non-sperm DNA and DNA from sperm cells which can be purified in the workflow.

[0316] For the so called “Bead capture” (Figure 5) sample sperm cells and / or sperm nuclei pellets were resuspended in remaining buffer containing sodium dodecyl sulfate (SDS, 0.4%) with non-sperm DNA and 620 pl of Buffer MTL (QIAGEN, Cat. No. / ID: 1014636)

[0317] Sperm cells and / or sperm nuclei are bound to the silica beads. Silica beads with DNA and sperm cells and / or sperm nuclei were washed with washing buffers comprising 5.6M GuHCl and 24 Vol-% Ethanol and a detergent (e.g. Tween) subsequently two times with washing buffers comprising 80 Vol-% Ethanol (e.g. QIAGEN, washing buffers from in the EZ1&2 DNA Investigator Kit; QIAGEN Cat. No.952034).

[0318] Non-sperm DNA is eluted with 800 pl H2O. Afterwards the beads were resuspended in buffer MTL (QIAGEN, Cat. No. / ID: 1014636) to rebind the sperm cells and / or sperm nuclei and non- sperm DNA. The washing steps are repeated with washing buffers comprising 5.6M GuHCl and 24 Vol-% Ethanol and a detergent (e.g. Tween) and subsequently with washing buffers comprising 80 Vol-% Ethanol (e.g. QIAGEN, washing buffers from in the EZ1&2 DNA Investigator Kit; QIAGEN Cat. No.952034). After washing the residual non-sperm DNA was eluted with 800 pl H2O.

[0319] The sperm cells and / or sperm nuclei bound to the beads were then lysed using the lysis buffer of the QIAGEN Investigator Casework GO! Kit (QIAGEN Cat. No. 386546) and after lysis DNA from sperm cells was eluted.

[0320] In Figure 5 DNA concentration of purified DNA from this samples are shown as “Bead capture”.

[0321] The autosomal DNA concentration for the 1 / 100 diluted ejaculate sample is 0.59 ng / pl and 0.17 ng / pl for the 1 / 500 diluted ejaculate sample. The male DNA concentration is 0.46 ng / pl for the sample with ejaculate diluted 1 / 100 and 0.08 for the 1 / 500 diluted ejaculate sample.

[0322] This shows that the remaining non-sperm DNA concentration is lower compared to the corresponding “c+ pellet controls”. Which highlights the differential elution of non-sperm DNA and lysis and elution of sperm cell associated DNA.

[0323] Samples from “Bead capture” purification and “c+ Pellet control” were used for DNA quantification (QIAGEN Investigator Quantiplex Pro Kit) and STR profiling (QIAGEN Investigator 24plex QS Kit Cat. No. 382415)

[0324] The samples were subjected to a capillary electrophoresis (CE) on a ABI PRISM Genetic Analyzer 3500 XL and the STR- Alleles of the blue channel was used for identification (see Table 1).

[0325] DNA isolated from” Bead capture” and “c+ Pellet control” fractions was amplified and the STR profiles were generated. For the creation of the STR profiles, 500 pg DNA was used.

[0326] The STR profiles of male and female DNA are shown in Figure 6 and Tables 1 and 2.

[0327] Figure 6 shows exemplary STR profiles after purification with silica bead capture workflow with differential elution of DNA from sperm and non-sperm cells. The Profiles were generated using the Investigator 24plex QS Kit (QIAGEN Cat. No. 382415).

[0328] Where possible, 500 pg. of DNA was used for STR profiling, DNA concentrations and samples from figure 5 were used. C+ indicates the pellet control of the different ejaculate dilutions while bead capture indicates the samples treated with the full differential elution workflow. The shown electropherograms are single replicates exemplary for results of a one experiment (n=3).

[0329] Table 1 : STR-Allele (extract blue channel) of male and female donor profiles

[0330] Table 2: STR- Allele (extract blue channel) of male and female donor profiles

[0331] These results show that by conducting at least two elution procedures on the non-sperm cell DNA before lysing the sperm cells it is significantly easier to obtain a specific genetic profile of a perpetrator without being hindered by the underlying genetic profile of the victim in sexual assault analyses.

[0332] This approach leads to a higher yield and purity of the DNA extracted from sperm cells and / or sperm nuclei. Furthermore, this allows an efficient elimination on non-sperm DNA from the enriched sperm and / or sperm nuclei fraction.

[0333] Example 6

[0334] This example demonstrates that digesting the non-sperm DNA (but not the sperm DNA) with an exonuclease, such as RecBCD, ensures a more favourable ratio of sperm DNA to non-sperm DNA than without a nuclease digestion.

[0335] A 30 pl aliquot of a non-sperm cells containing fraction was mixed with lOpl sperm cells and a first lysis was performed with Proteinase K in the absence of a reducing agent. The sample was lysed in a total volume of 500 pl in a buffer containing 25 pl Proteinase K (20 mg / ml) and sodium dodecyl sulphate (SDS, 0.4%) and incubated for Ih at 56 °C and 900 rpm.

[0336] DNA from the non-sperm fraction and sperm cells and / or sperm nuclei was mixed with a silica beads suspension, bound to the magnetic beads and purified.

[0337] In this example silica beads from the QIAGEN EZ1&2 DNA Investigator Kit were used (QIAGEN Cat. No.952034)) and treated with 770 pl of one or more chaotropic buffers (e.g. QIAGEN, Buffer G2 (260 pl) Cat. No. / ID: 1014636G2 and QIAGEN, Buffer MTL Cat. No. / ID: 1014636).

[0338] Sperm cells and / or sperm nuclei were bound to the silica beads with 720 pl of a chaotropic buffer comprising 5.5M GTC (QIAGEN, Buffer MTL Cat. No. / ID: 1014636).

[0339] The silica beads with bound sperm cells and / or sperm nuclei were washed twice with washing buffers comprising 5.6M GuHCl and 24 Vol-% ethanol and a detergent (e.g. Tween) and subsequently with washing buffers comprising 80 Vol-% ethanol (e.g. QIAGEN, washing buffers from in the EZ1&2 DNA Investigator Kit; QIAGEN Cat. No.952034).

[0340] After purification, the beads with bound sperm cells and / or sperm nuclei were resuspended in 80 pl 10 mM Tris buffer. To this suspension 275 pl of a buffer was added comprising 50 mM Tris (pH 8.5), 10 mM MgCh and 8.7 pl of an ATP solution (lOOmM). 2.5 pg RecBCD enzyme was added to this suspension resulting in a total volume of 420 pl.

[0341] Sperm cells and / or nuclei were then incubated at 37°C for 1 hour and lysed using 200pl of a lysis solution comprising DTT (final concentration 200 mM), one or more chaotropic buffers (e.g., QIAGEN, Buffer G2, Cat. No. / ID: 1014636) and lOpl proteinase K (20 mg / ml) to lyse sperm cells and open up sperm nuclei.

[0342] Sperm DNA present in the lysate was purified using the QIAGEN EZ1&2 DNA Investigator Kit Trace protocol (QIAGEN Cat. No.952034).

[0343] Remaining procedure followed the protocol of Example 1.

[0344] Results are shown in Figure 7. Figure 7 clearly shows that the RecBCD digested samples showed similar quantification values for autosomal and male DNA, indicating that the DNA from the non-sperm fraction has been removed.

[0345] The male DNA concentration was only slightly reduced compared to samples not digested with RecBCD, and the pellet control, indicating that DNA present in sperm cells has not been degraded by the DNase.

[0346] Without RecBCD digestion, non-sperm DNA was still present in the final eluate in a comparatively high amount compared to the amount of male DNA, leading to a high autosomal / male ratio.

[0347] Example 7

[0348] This example shows the effective use of sperm lysis solutions comprising different reduction agents to lyse sperm cells and / or sperm nuclei.

[0349] Ejaculate samples were treated in accordance with the protocol used in Example 1, except for the lysis and elution of the sperm cells and / or sperm nuclei from the silica beads.

[0350] After lysis and removal of the non-sperm DNA from the sample by following the steps described in Example 1, the sperm cells and / or sperm nuclei bound to the beads were lysed and DNA from sperm cells was eluted.

[0351] Sperm nuclei were lysed and eluted using 100 pL of QIAGEN Investigator Casework GO! Lysis Buffer (Cat. No. 386546, PCR-compatible) with either 10 mM DTT or 10 mM TCEP, with or without 3.5 pL proteinase K.

[0352] As shown in Figure 8, DNA from sperm cells could be extracted using a lysis solution comprising a reducing agent and an enzyme.

[0353] In this experiment it is shown that samples treated with a combination of TECEP / proteinase K and DTT / proteinase K showed equivalent efficiency, whereas samples treated with DTT or TECEP without proteinase K showed a very low yield. It can also be seen that the obtained amounts of autosomal and male DNA were in a similar order of magnitude.

[0354] Example 8

[0355] This example demonstrates the effective lysis of sperm cells with a lysis composition comprising L-arginine as the lysing agent.

[0356] In this Example, sperm cells and / or sperm nuclei were lysed with L-arginine in a concentration of lOmM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM (see Figure 9).

[0357] For lysis of the sperm cells and / or sperm nuclei, a lysis composition was prepared per reaction, comprising 47pl of 6mM HEPES, 0.165mM PEG600, L- arginine at a concentration ranging from 5mM - 50mM (as indicated in Figure 9) and 3.5 pl proteinase K (0.07mg), resulting in 50 pl lysis mixture.

[0358] In preparation for the lysis of the cells, the ejaculate (containing sperm cells and / or sperm nuclei) was diluted 1 : 10 and 1 :200, where 20pL of the respective ejaculate dilutions were used per reaction. For the ejaculate dilutions, 80pL and 7.5pL of ejaculate were respectively diluted with 720pL and 1493pL of water. As the negative control, a lysis buffer without L-arginine, but with 6mM HEPES, 0.165mM PEG600 and 3.5 pl proteinase K (0.07mg) was used (labelled as „w / o“ in Figure 9).

[0359] For cell lysis, the 50pl lysis buffer mixture was added to each individual sample (diluted ejaculates). Lysis took place at 60°C for 25 minutes at 900rpm in a heating shaker. This was followed by inactivation of proteinase K at 80°C for 5 minutes.

[0360] For quantification of DNA the QIAGEN Quantiplex Pro Kit (QIAGEN Cat. No. 387216) was used.

[0361] Results are shown in Table 3 and Figure 9.

[0362] Table 3: Sperm cell lysis in the presence of arginine

[0363] Table 3 and Figure 9 clearly show that when L-arginine was present in the lysis buffer, sperm lysis was achieved in both, the 1 :200-diluted ejaculate and the 1 : 10- diluted ejaculate. Furthermore, with the l :200-diluted ejaculate, a plateau phase was already reached at a concentration of 20mM L-arginine, whereas with a l : 10-diluted mixture this plateau phase was only reached at a concentration of 30mM L-arginine.

[0364] The example also shows that without the presence of L-arginine in the lysis composition a very low DNA yield was obtained. The DNA found here possibly originated only from non-sperm cells (e.g. epithelial cells), as ejaculate comprises the sperm fraction (SF, sperm cells / sperm nuclei) and the non-sperm fraction.

Claims

CLAIMS1. A method for treatment of a biological sample comprising sperm cells and / or sperm nuclei and non-sperm cells, the method comprising: a) subjecting a biological sample comprising sperm cells and / or sperm nuclei and nonsperm cells to a lysis by applying one or more lysing agents or a lysis solution to the biological sample and / or incubating the biological sample with the one or more lysing agents or in the lysis solution, wherein the one or more lysing agents or the lysis solution are / is suitable to essentially lyse the non-sperm cells and to form a lysate comprising non-sperm DNA, sperm cells and / or sperm nuclei, wherein the one or more lysing agents or the lysis solution do / does not comprise any agent in an amount that causes a complete lysis of sperm cells and / or sperm nuclei and, preferably comprises at least one agent selected from a surfactant, a chaotropic agent, a non- sperm cell digesting enzyme, or any combination thereof; b) separating, removing, or separating and removing the lysate from a sperm cell and / or sperm nuclei enriched fraction; c) contacting the sperm cell and / or sperm nuclei enriched fraction with one or more binding agents or a binding solution and a solid support and / or incubating the sperm cell and / or sperm nuclei enriched fraction with a solid support in the presence of one or more binding agents or a binding solution and binding the sperm cell and / or sperm nuclei enriched fraction to the solid support; d) separating, removing, or separating and removing the binding agents or binding solution from the solid support; e) optionally, subjecting the sperm cell and / or sperm nuclei enriched fraction bound to the solid support to at least one washing step by applying one or more washing agents or a washing solution to the solid support and / or incubating the solid support with the one or more washing agents or in the washing solution, and separating, removing, or separating and removing the washing agents or washing solution from the solid support;f) subjecting the sperm cell and / or sperm nuclei enriched fraction bound to the solid support to at least one elution step by contacting the solid support with one or more elution agents or an elution solution and / or incubating the solid support in the presence of the one or more elution agents or the elution solution, wherein the one or more elution agents or the elution solution are / is suitable to elute the non-sperm DNAbut not the sperm cells and / or sperm nuclei bound to the solid support, and removing, or separating and removing the eluate comprising the non-sperm DNA from the solid support; g) optionally, subjecting the sperm cells and / or sperm nuclei bound to the solid support to at least one elution step by contacting the solid support with one or more elution agents or an elution solution and / or incubating the solid support in the presence of the one or more elution agents or the elution solution, wherein the one or more elution agents or the elution solution are / is suitable to elute the sperm cells and / or sperm nuclei bound to the solid support; h) subjecting the sperm cells and / or sperm nuclei to a lysis by applying a lysis solution or one or more lysing agents and / or incubating the sperm cells and / or sperm nuclei in a sperm cell and / or sperm nuclei lysis solution or with one or more lysing agents, wherein the sperm cell and / or sperm nuclei lysis solution or the one or more lysing agents comprise(s) at least one lysing agent selected from a reducing agent, a sperm cell and / or sperm nuclei lysing enzyme, a chaotropic agent and / or an alkaline agent in a concentration suitable to lyse a sperm cell and / or sperm nuclei, an amino compound and / or an ammonium salt; or a combination thereof, and optionally, separating, removing, or separating and removing the lysate comprising sperm DNA from the solid support.

2. The method of claim 1, wherein the biological sample is a forensic sample, such as a forensic swab sample, preferably a sexual assault sample.

3. The method of claims 1 to 2, further comprising purifying, isolating, detecting, analysing and / or quantifying the non-sperm DNA and / or the sperm-DNA.

4. The method of claim 3, wherein analysing comprises genotyping the DNA, preferably a short tandem repeat (STR) analysis, restriction fragment length polymorphism (RFLP) of genomic DNA, random amplified polymorphic detection (RAPD) of genomic DNA, amplified fragment length polymorphism (AFLP) detection, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR), DNA sequencing, allele specific oligonucleotide (ASO) probes, and hybridization to DNA microarrays or beads; or any combination thereof.

5. The method of claims 1 to 4, wherein the lysis solution or the one or more lysing agents in step a) comprises at least one agent selected from a surfactant selected from the group consisting of non-ionic surfactants or ionic surfactants, such as anionic -, cationic- or zwitterionic surfactants; a chaotropic agent selected from the group consisting of one or more sodium salts, thiocyanate salts, guanidinium salts or urea; a non-sperm cell digesting enzyme selected from the group consisting of proteinase K, protease, zymolase, lyticase, chromopeptidase, lysostaphin, lysozyme; or any combination thereof.

6. The method of claims 1 to 5, wherein the solid support is selected from materials capable of reversibly binding to nucleic acids and to sperm cells and / or sperm cell nuclei, optionally having one or more of the subsequent features: i. the solid support is selected from a group of materials comprising silica, materials having a surface functionalized with one or more types of anion exchange moieties and materials having a surface functionalized with carboxylic acid groups; ii. the solid support is in the form of particles, magnetic particles, beads or magnetic beads.

7. The method of claims 1 to 6, wherein the solid support is formed of a solid support material selected from materials including, but not limited to, silica, silica gel, borosilicates, silicates, inorganic glasses, organic polymers such as poly(meth)acrylates, polyurethanes, polystyrene, agarose, polysaccharides such as cellulose, metal oxides such as aluminium oxide, magnesium oxide, titanium oxide and zirconium oxide, agarose, polystyrene, sephadex, sepharose, polyacrylamide, divinylbenzene polymers, styrene divinylbenzene polymers, dextrans, resins, and derivatives thereof,; and preferably present in the form of particles, beads, membranes, plates, a surface coating and capillary tubes, more preferably magnetizable or magnetic (e.g. paramagnetic, superparamagnetic, ferromagnetic or ferrimagnetic) particles, including but not limited to polystyrene, agarose, polyacrylamide, dextran, and / or silica materials having a magnetic material incorporated therein and / or associated therewith.

8. The method of claims 1 to 7, wherein the solid support is selected from one or more materials having a surface functionalized with one or more types of anion exchange moieties, preferably in the form of particles which comprise one or more types of anion exchange moieties at their surface.

9. The method of claims 1 to 8, wherein the surface of the solid support comprising one or more types of anion exchange moieties has at least one of the following characteristics: i. a type of anion exchange moieties comprises amine groups, preferably comprises or consists of a primary, secondary or tertiary amine or mixtures thereof; ii. the surface comprises polyethyleneimine as anion exchange moiety; and at least one of the following characteristics: a. the solid support is provided in the form of particles, preferably magnetic particles; b. the solid support is provided in the form of particles which are provided in a suspension; c. the solid support is provided in the form of a membrane.

10. The method of claims 1 to 9, wherein the one or more binding agents or the binding solution in step c) has one or more, preferably at least one of the following characteristics i. to v.: i. it comprises a chaotropic agent, optionally under alkaline conditions; ii. it comprises a chaotropic agent selected from a group consisting of a sodium salt, a thiocyanate salt or a guanidinium salt or urea, preferably sodium iodide, sodium perchlorate, guanidinium hydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate or a mixture of two or more salts thereof; iii. it has an acidic pH value; iv. it has a pH selected such that the resulting binding mixture has a pH range from 3 to 7, 3.5 to 6.5, 4 to 6, preferably a pH value of 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7; v. the binding solution is obtained by mixing two or more agents.

11. The method of claims 1 to 10, wherein i. the solid support in method step c) is a solid support selected from materials comprising silica, borosilicate, silicates, inorganic glasses, preferably in the form of particles, and binding of the sperm cell and / or sperm nuclei enriched fraction occurs in the presence of a binding solution and one or more binding agents comprising a chaotropic agentselected from a group consisting of a sodium salt, a thiocyanate salt, a guanidinium salt or urea, preferably sodium iodide, sodium perchlorate, guanidinium hydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate or a mixture of two or more salts thereof and the binding solution having an alkaline pH value, or ii. the solid support in method step c) is a solid support selected from materials having a surface functionalized with one or more types of anion exchange moieties, preferable in the form of particles which comprise one or more types of anion exchange moieties at their surface, and binding of the sperm cell and / or sperm nuclei enriched fraction occurs at a first pH selected from a pH range from 3 to 7, 3.5 to 6.5, 4 to 6, preferably a first pH value of 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7.

12. The method of claims 1 to 11, wherein the one or more washing agents or the washing solution in step e) has at least one or two or more of the following characteristics i. to iv: i. it comprises an alcohol, preferably ethanol; ii. it comprises the binding solution and / or binding agents; iii. it comprises the binding agents in the same concentrations or amounts as the concentration or amount of the agents used as binding agents or in a binding solution; iv. it comprises the binding agents in a concentration or amount that is different to the concentration or amount of the agents used as binding agents or in a binding solution.

13. The method of claims 1 to 12, wherein the elution solution or the one or more elution agents in step f) has at least one or two or more of the following characteristics i. to vii: i. it comprises water, preferably is water; ii. it has a second pH value selected from a pH range of pH>8 and <13; pH>8 and <12,6; pH>8 and <12; >8 and <11; pH>8 and <10 and >8 and <9; iii. the second pH is higher than a first pH that was used for binding the non-sperm DNA to the solid support; iv. the second pH is lower than a third pH at which the sperm cells and / or sperm nuclei are eluted, wherein in this case the second pH value is not higher than 13, preferably the second pH is at least 0.1 units lower than the third pH, at least 0.2 unit lower than the third pH, more preferably at least 0.25 units lower or at least 0.3 units lower than the third pH;v. it comprises an aqueous solution comprising the buffering substance and optionally one or more further components such as a salt, in particular an alkali salt such as sodium chloride or potassium chloride; vi. it comprises an alkali hydroxide such as sodium hydroxide or potassium hydroxide, preferably sodium hydroxide; vii. it is added as an at least 1 mmol / 1 sodium hydroxide solution, preferably a 2 mmol / 1 sodium hydroxide solution.

14. The method of claims 1 to 13, wherein i. the solid support in method step f) is a solid support selected from materials comprising silica, borosilicate, silicates, inorganic glasses, preferably in the form of particles, and the elution of the non-sperm DNA occurs in the presence of one or more elution agents or an elution solution comprising water, preferably is water, or ii. the solid support is a solid support selected from materials having a surface functionalized with one or more types of anion exchange moieties, preferably in the form of particles which comprise one or more types of anion exchange moieties at their surface, and the elution of the non-sperm DNA occurs at a second pH which is higher than the first pH that was used for binding the non-sperm DNA to the solid support, and the second pH is in a range selected from the group consisting of pH>8 and <13; pH>8 and <12,6; pH>8 and <12; >8 and <11; pH>8 and <10 and >8 and <9, and the second pH preferably is at least 0.1 units lower than the third pH, at least 0.2 unit lower than the third pH, more preferably at least 0.25 units lower or at least 0.3 units lower than the third pH, at which the sperm cells and / or sperm nuclei are eluted.

15. The method of claims 1 to 14, wherein the sperm cell and / or sperm nuclei lysis solution or one or more lysing agents comprises one or more reduction agents selected from the group consisting of dithiothreitol (DTT), Dithioerythrit, P-mercaptoethanol (BME), glutathione (GSH) and Tris(2-carboxyethyl)phosphine (TCEP) ; one or more sperm lysing enzymes selected from the group consisting of proteinase K, protease, zymolase, lyticase, chromopeptidase, trypsin and lysostaphin; one or more chaotropic agents; one or more alkaline agents in a sufficiently high concentration to lyse sperm cells and / or sperm nuclei; one or moreamino compounds selected from the group comprising amino alcohols selected from the group consisting of a primary amino alcohol, a secondary amino alcohol and a tertiary amino alcohol ; one or more amino acids selected from the group consisting of alanine (ala - A), arginine (arg - R), asparagine (asn - N), aspartic acid (asp - D), cysteine (cys - C), glutamine (gin - Q), glutamic acid (glu - E), glycine (gly - G), histidine (his - H), isoleucine (ile - I), leucine (leu - L), lysine (lys - K), methionine (met - M), phenylalanine (phe - F), proline (pro - P), serine (ser - S), threonine (thr - T), tryptophan (trp - W), tyrosine (tyr - Y) and valine (val - V); one or more amino sulfonic acids and / or one or more ammonium salts; or any combination of the aforementioned lysing agents.

16. The method of claims 1 to 15, wherein after method step f) and before method step h), the sperm cells and / or sperm nuclei bound to the solid support are subjected to at least one elution step by contacting the solid support with one or more elution agents or an elution solution and / or incubating the solid support in the presence of the one or more elution agents or the elution solution, wherein the one or more elution agents or the elution solution are / is suitable to elute the sperm cells and / or sperm nuclei bound to the solid support.

17. The method of claims 1 to 16, wherein method step g) having at least one or two of the following characteristics i. to ii: i. the solid support is a solid support selected from materials having a surface functionalized with anion exchange moieties, preferably in the form of particles which comprise one or more types of anion exchange moieties at their surface, and elution of the sperm cells and / or sperm nuclei occurs at a third pH which is higher than the second pH that was used for eluting the non-sperm DNA from the solid support comprising one or more types of anion-exchange moieties, preferably the third pH is at least 0.1 units higher than the second pH, at least 0.2 unit higher than the second pH, more preferably at least 0.25 units higher or at least 0.3 units higher than the second pH; ii. the solid support is a solid support selected from materials comprising silica, borosilicate, silicates, inorganic glasses, preferably in the form of particles, and elution of the sperm cells and / or sperm nuclei occurs in the presence of an elution solution comprising water and optionally one or more elution agents, preferably pure water. The method of any one of claims 1 to 17, comprising one, two or more of the following stepsi. the non-sperm DNA present in the sperm cell and / or sperm nuclei enriched fraction is digested or decomposed after method step a) and before method step b); ii. the non-sperm DNA present in the sperm cell and / or sperm nuclei enriched fraction is digested or decomposed after method step b) and before method step c); iii. the non-sperm DNA bound to the solid support is digested or decomposed after method step c) and before method steps d) and / or e); iv. the non-sperm DNA is digested or decomposed after method step d) and before method steps e) and / or f); v. the non-sperm DNA is digested or decomposed in method step c) and / or e), and / or f), and / or g); vi. the non-sperm DNA is digested or decomposed after method step g) and before method step h); vii. the non-sperm DNA is digested or decomposed using one or more nucleases.

19. The method of any one of claims 1 to 18, wherein the solid support is in the form of particles, and the series of steps c), d), optionally e) and f) is repeated at least once, twice or several times.

20. The method of claims 1 to 19, wherein one or more steps are automated.

21. A kit comprising one or more of: a) a non-sperm cell lysis solution or one or more non-sperm cell lysing agents capable of lysing non-sperm cells and essentially not suitable for completely lysing sperm cells and / or sperm nuclei; b) a solid support; c) binding agents or a binding solution capable of binding the sperm cell and / or sperm nuclei enriched fraction to the solid support, preferably the one or more binding agents or the binding solution have / has one or more, preferably at least two of the following characteristics i. to v.: i. it comprises a chaotropic agent, optionally under alkaline conditions; ii. it comprises a chaotropic agent selected from a group consisting of a sodium salt, a thiocyanate salt, a guanidinium salt or urea, preferably sodium iodide, sodium perchlorate, guanidinium hydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate and / or a mixture of two or more salts thereof; iii. it has an acidic pH value;iv. it has a first pH value selected from a pH range from 3 to 7, 3.5 to 6.5, 4 to 6; v. the binding solution is obtained by mixing two or more agents; d) elution agents or an elution solution capable of eluting the non-sperm DNA but not the sperm cells and / or sperm nuclei bound to the solid support, the elution solution or the one or more elution agents has / have at least one or two or more of the following characteristics i. to vii: i. it comprises water, preferably is water; ii. it has a second pH value selected from a pH range of pH>8 and <13; pH>8 and <12,6; pH>8 and <12; >8 and <11; pH>8 and <10 and >8 and <9; iii. the second pH is higher than a first pH that was used for binding the non-sperm DNA to the solid support; iv. the second pH is lower than a third pH at which the sperm cells and / or sperm nuclei are eluted from the solid support, preferably at least 0.1 units lower than the third pH, at least 0.2 unit lower than the third pH, more preferably at least 0.25 units lower or at least 0.3 units lower than the third pH; v. it comprises an aqueous solution comprising the buffering substance and optionally one or more further components such as a salt, in particular an alkali salt such as sodium chloride or potassium chloride; vi. it comprises an alkali hydroxide such as sodium hydroxide or potassium hydroxide, preferably sodium hydroxide; vii. it is an at least 1 mmol / 1 sodium hydroxide solution, preferably a 2 mmol / 1 sodium hydroxide solution; e) a sperm cell and / or sperm nuclei lysis solution or one or more sperm cell and / or sperm nuclei lysing agents capable of lysing sperm cells and / or sperm nuclei, preferably the sperm cell and / or sperm nuclei lysis solution or one or more lysing agents comprises one or more reduction agents selected from the group consisting of dithiothreitol (DTT), Dithioerythrit, P- mercaptoethanol (BME), glutathione (GSH) and Tris(2-carboxyethyl)phosphine (TCEP) ; one or more sperm lysing enzymes selected from the group consisting of proteinase K, protease, zymolase, lyticase, chromopeptidase, trypsin, lysostaphin, lysozyme and, depending on the application, nucleases, such as DNase, Benzonase(R), RNase and / or restriction endonucleases ; one or more chaotropic agents one or more alkaline agents in a sufficiently high concentration to lyse sperm cells and / or sperm nuclei; one or more amino compounds selected from the groupcomprising amino alcohols selected from the group consisting of a primary amino alcohol, a secondary amino alcohol and a tertiary amino alcohol; one or more amino acids selected from the group consisting of alanine (ala - A), arginine (arg - R), asparagine (asn - N), aspartic acid (asp - D), cysteine (cys - C), glutamine (gin - Q), glutamic acid (glu - E), glycine (gly - G), histidine (his - H), isoleucine (ile - I), leucine (leu - L), lysine (lys - K), methionine (met - M), phenylalanine (phe - F), proline (pro - P), serine (ser - S), threonine (thr - T), tryptophan (trp - W), tyrosine (tyr - Y) and valine (val - V),; one or more amino sulfonic acids and / or one or more ammonium salts; or any combination of the aforementioned lysing agents; f) optionally, elution agents or an elution solution capable of eluting the sperm cells and / or sperm nuclei bound to the solid support; g) optionally, one or more washing agents or a washing solution, preferably the one or more washing agents or the washing solution have / has at least one or two or more of the following characteristics i. to iv: i. it comprises an alcohol, preferably ethanol; ii. it comprises the binding solution and / or binding agents; iii. it comprises the binding agents in the same concentrations or amounts as the concentration or amount of the agents used as binding agents or in a binding solution; iv. it comprises the binding agents in a concentration or amount that is different to the concentration or amount of the agents used as binding agents or in a binding solution; h) optionally, one or more means for separating, removing, or separating and removing any one of the following: i. the lysate from a sperm cell and / or sperm nuclei enriched fraction, and / or ii. the binding agents or binding solution from the solid support, and / or iii. the lysate comprising sperm DNAfrom the solid support, and / or iv. the washing agents or washing solution from the solid support.

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