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. Moreover, the present disclosure relates to the use of specific enzymes for the decomposition of non-sperm DNA in a sample comprising a mixture of non-sperm DNA and sperm DNA, the sperm DNA being contained in sperm cells and / or sperm nuclei, and a corresponding method, therefore.

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. Moreover, the present disclosure relates to the use of specific enzymes for the decomposition of non-sperm DNA in a sample comprising a mixture of non-sperm DNA and sperm DNA, the sperm DNA being contained in sperm cells and / or sperm nuclei, and a corresponding method therefor.BACKGROUND

[0002] The most common type of DNA profiling today for forensic uses is "STR" (short tandem repeat) analysis. The patterns of short tandem repeats (STR) differ between individuals and therefore DNA is used to identify individuals.

[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 (usually mostly 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 incomparison 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 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 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 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 inefficient 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. 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 invention 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 particularly 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 invention to provide a DNA profiling method that allows for reliable identification of a sperm donor and a non-sperm cell donor, particularly an epithelial 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 invention is to provide a kit for the processing of 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 of the present disclosure, a method for treatment of a biological sample, comprising sperm cells and / or sperm nuclei and non-sperm cells, the method comprising:a) subjecting the biological sample comprising the sperm cells and / or sperm nuclei and the non-sperm cells to a first 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 first 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) optionally separating and / or removing the non-lysed cells and / or sperm nuclei from the first lysate comprising non-sperm DNA and / or decomposing or separating or removing or separating and removing the non-sperm DNA from the first lysate comprising non-sperm DNA, sperm cells and / or sperm nuclei, preferably by subjecting the non-sperm DNA in the first lysate to a nuclease digestion; c) subjecting the non-lysed cells and / or sperm nuclei to at least a second lysis by applying one or more lysing agents or a lysis solution to the non-lysed cells and / or sperm nuclei and / or incubating the non-lysed cells and / or sperm nuclei 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 non-sperm cells and to form a second or further lysate comprising non-sperm DNA, wherein the one or more lysing agents or the lysis solution do / does not comprise 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; d) decomposing or separating or removing or separating and removing the non- sperm DNA from the second or further lysate comprising non-sperm DNA, preferably by subjecting the non-sperm DNA in the second or further lysate to a nuclease digestion; and e) subjecting the non-lysed cells to at least a third or further lysis by applying a sperm cell and / or sperm nuclei lysis solution or one or more lysing agents to lyse spermcells and / or sperm nuclei completely and to form a third or further lysate comprising sperm DNA, wherein 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 selected from a reduction agent, a sperm cell and / or sperm nuclei lysing enzyme, a chaotropic agent in a concentration suitable to lyse a sperm cell and / or sperm nuclei, and / or an alkaline agent in a concentration suitable to lyse sperm cells and or sperm nuclei or a combination thereof.

[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 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 first 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 second and / or further non-sperm cell lysis solution or one or more non- sperm cell lysing agents capable of lysing non-sperm cells and not suitable for completely lysing sperm cells and / or sperm nuclei, wherein the second and / or further non-sperm cell lysis solution or the one or more non-sperm cell lysing agents is / are identical to the first non-sperm cell lysis solution or the one or more non-sperm cell lysing agents used in a), or wherein the second and / or further non-sperm cell lysis solution or the one or more non-sperm cell lysing agents is / are different from the first non-sperm cell lysis solution or the one or more non-sperm cell lysing agents used in a);c) optionally, one or more washing compositions; d) optionally one or more DNA digesting enzymes; e) a sperm cell and / or sperm nuclei lysing 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 in a concentration suitable to lyse sperm cells and or sperm nuclei and / or an alkaline agent in a concentration suitable to lyse a sperm cell and / or sperm nuclei; or any combination thereof.BRIEF DESCRIPTION OF THE FIGURES

[0020] Fig. 1 (A&B) shows the amount of DNA isolated from a female cell sample after the sample was subjected to a first lysis (“supernatant”), after DNase digestion and after second lysis (“2nd lysis”) of the sample. Parts A and B show the same data but the DNA yield is either plotted in a logarithmic scale (A) or linear scale with broken Y axis (B).

[0021] Fig. 2 shows STR DNA profiles of an isolated epithelial cell fraction subjected to a first lysis (“supernatant”), DNase digestion and second lysis (“2nd lysis”).

[0022] Fig. 3a shows the amount of DNA recovered from the non-sperm fraction of a mixed epithelial and sperm cell containing sample.

[0023] Fig. 3b shows the amount of autosomal DNA and male DNA recovered from the sperm-cell fraction of a mixed epithelial and sperm-cellcontaining sample after 1 lysis step and 1st and 2nd lysis of the nonsperm fraction.

[0024] Fig. 4 shows STR profiles of DNA recovered from the non-sperm fraction and the sperm fraction of a sample containing epithelial and spermcell that was subjected to a 1st and 2nd lysis of the non-sperm fraction.

[0025] Fig. 5 (A&B) shows the amount of the remaining DNA isolated from female cell samples after the sample was subjected to a first lysis followed by a DNase digestion (“after DNase digestion”) and after second lysis (“2nd lysis”), wherein different lysis buffers were used. Parts A and B show the same data, but the DNA yield is either plotted in a logarithmic scale (A) or linear scale with broken Y axis (B).

[0026] Fig. 6a shows the concentration of autosomal DNA and male DNA recovered from a sperm-cell fraction of a sperm-cell containing sample after non-sperm cell lysis and washing of the obtained sperm cell pellet, spiked-in with female gDNA, without digestion and after digestion either with RecBCD or after digestion with DNase I.

[0027] Fig. 6b shows the DNA degradation ratio of autosomal DNA and male DNA recovered from a sperm-cell fraction of a sperm-cell containing sample after non-sperm cell lysis and washing of the obtained sperm cell pellet, spiked-in with female gDNA, without digestion and after digestion either with RecBCD or after digestion with DNase I.shows STR profiles of DNA recovered from a sperm-cell fraction of a sperm-cell containing sample after non-sperm cell lysis and washing of the obtained sperm cell pellet, spiked with female gDNA, without digestion and after digestion either with RecBCD or after digestion with DNase I.DETAILED DESCRIPTION OF EMBODIMENTS

[0029] 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.

[0030] The term "biological sample" or “sample” as used in the present invention 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.

[0031] The term "biological sample" or “sample”, as used herein, preferably 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.

[0032] 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 for example by collecting a sample on a swab or other collection means 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 other kind of fabric, like sheets, blankets, covers, towels, handkerchiefs or similar, or on other non-fabric clothing, like shoes, leather wear, synthetic material, or on any other kind of solid support that was in contact with a crime scene attendee.

[0033] 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.

[0034] 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.

[0035] As used herein, the term “non-sperm cell DNA” refers to any DNA that is present in “non-sperm cells”.

[0036] The term “lysate” as used herein refers to a mixture containing lysed cell debris and DNA.

[0037] 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 such that the sperm nucleic acids, at least to some extent, are not yet exposed.

[0038] Provided herein is a method and kits for treatment of a biological sample comprising sperm cells and / or sperm nuclei and non-sperm cells that allows the selective isolation of sperm cell DNA that is essentially free of non-sperm cell DNA.

[0039] In particular, the methods of the present disclosure are provided for treatment of a forensic sample, preferably a sexual assault sample, comprising sperm cells and / or sperm nuclei from the perpetrator 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 nucleic. The methods of the present disclosure allow for the isolation of sperm cell DNA that that is suitable for further analysis, such as genotyping, i.e. to allow the identification of the perpetrators.

[0040] Minimizing loss of target DNA during extraction as well as ensuring complete lysis of target DNA containing cells are two factors that must be considered when choosing an extraction method. This is particularly relevant when DNA is extracted from forensic samples, such as sexual assault samples, where sperm cell DNA of the perpetrator(s) 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.

[0041] In forensic sample preparation the first step usually comprises the lysis of the non-sperm cells, like the epithelial cells, followed by the subsequent lysis of the remaining intact sperm cells.

[0042] In a novel approach, as specified in the method of the present disclosure, the non-sperm cells present in a biological sample comprising sperm cells and / or sperm nuclei and non-sperm cells are lysed in a first lysis step followed by at least a second lysis step under conditions where intact or essentially intact non-sperm cells are lysed, yet sperm cells and sperm nuclei are not or not completely lysed.

[0043] Surprisingly it has been found that the first lysis step does not result in a complete lysis of the non-sperm cells. Instead, enough non-sperm cells remain which, if not being removed in a second or further lysis procedure, are found to contaminate sperm cells and sperm nuclei. Not sufficiently removing said contaminating non-spermcells will also result in a contamination of sperm DNA with non-sperm DNA once the sperm cells and sperm nuclei are lysed.

[0044] Consequently, a second or further lysis will ensure that any intact or essentially intact non-sperm cells that are not lysed during the first treatment step will be lysed during a second or further step.

[0045] Once essentially all non-sperm cells present in the biological sample are lysed and non-sperm DNA is separated, removed and / or decomposed and / or digested the remaining sperm cells and / or cell nuclei are lysed to obtain sperm DNA.

[0046] Repeated treatment of the sample according to the method of the disclosure, and in particular treating the non-sperm fraction present in the sample, with a lysis solution or one or more lysing agents suitable to lyse non-sperm cells, optionally decomposing and / or removing the non-sperm DNA after each non-sperm cell lysis step and before the sperm cells or sperm nuclei are lysed, will maximise the yield and purity of the sperm DNA isolated from said samples and in particular reduce the contamination of sperm DNA with non-sperm DNA. 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.

[0047] 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.

[0048] 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 will maximise 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.

[0049] In one embodiment, 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 the biological sample comprising the sperm cells and / or sperm nuclei and the non-sperm cells to a first 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 first 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) optionally separating and / or removing the non-lysed cells and / or sperm nuclei from the first lysate comprising non-sperm DNA and / or decomposing or separating or removing or separating and removing the non-sperm DNA from the first lysate comprising non-sperm DNA, sperm cells and / or sperm nuclei, preferably by subjecting the non-sperm DNA in the first lysate to a nuclease digestion; c) subjecting the non-lysed cells and / or sperm nuclei to at least a second lysis by applying one or more lysing agents or a lysis solution to the non-lysed cells and / or sperm nuclei and / or incubating the non-lysed cells and / or sperm nuclei 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 non-sperm cells and to form a second or further lysate comprising non-sperm DNA, wherein the one or more lysing agents or the lysis solution do / does not comprise 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; d) decomposing or separating or removing or separating and removing the non- sperm DNA from the second or further lysate comprising non-sperm DNA, preferably by subjecting the non-sperm DNA in the second or further lysate to a nuclease digestion; and e) subjecting the non-lysed cells to at least a third or further lysis by applying a sperm cell and / or sperm nuclei lysis solution or one or more lysing agents to lyse spermcells and / or sperm nuclei completely and to form a third or further lysate comprising sperm DNA, wherein 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 selected from a reduction agent, a sperm cell and / or sperm nuclei lysing enzyme, a chaotropic agent in a concentration suitable to lyse a sperm cell and / or sperm nuclei, or a combination thereof.

[0050] In some embodiments, the biological sample is a forensic sample, such as a forensic swab sample, preferably a sexual assault sample.

[0051] 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.

[0052] In an embodiment, the forensic sample is a sexual assault sample comprising a mixture of cells from the victim and one perpetrator, in some cases even more than one 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.

[0053] 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. In a novel approach, the non-lysed cells are subjected to repeated lysis under conditions that essentially promote lysis of the non-sperm cells, but do not promote complete lysis of the sperm cells and / or sperm nuclei.

[0054] In an embodiment of the disclosure the biological sample comprising sperm cells and / or sperm nuclei and non-sperm cells is subjected to a first 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 first lysate comprising non-sperm DNA. Theone or more lysing agents or the lysis solution do / does not comprise 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 nonsperm 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.

[0055] 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 first lysate comprising non-sperm DNA.

[0056] 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.

[0057] 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 first lysate comprising non-sperm DNA.

[0058] In a further embodiment of the disclosure, following the first lysis of the sample with the lysis solution or the one or more lysing agents, the non-lysed cells are separated from the first lysate comprising non-sperm DNA and / or the first lysate comprising released non-sperm DNA is removed from the sperm cell or sperm nuclei fraction and / or non-sperm DNA comprised in the first lysate is decomposed or separated or removed or separated and removed from the first lysate comprising non- sperm DNA, sperm cells and / or sperm nuclei, preferably by subjecting the non-sperm DNA in the first lysate to a nuclease digestion. In certain embodiments of thedisclosure, separation of the non-lysed cells from the first 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 non-lysed cells, preferably by forming a pellet containing non-lysed cells and a lysate supernatant comprising the non-sperm DNA.

[0059] In one embodiment the resulting supernatant comprising the lysed non-sperm cell DNA is subsequently removed from the non-lysed cells or cell pellet by any suitable means, including but not limited to pipetting. In other embodiments the non- lysed cells are separated by means of filtration.

[0060] In yet another embodiment the non-lysed cells are separated by any suitable method or means, such as bead separation, preferably magnetic bead separation, or by binding specific non-lysed cells to a solid matrix, such as a membrane or beads, preferably magnetic beads, more preferably with the help of antibodies (e.g. selective cell capture).

[0061] Removal or separation of DNA, in particular the non-sperm DNA, may occur with any suitable means including precipitation, centrifugation, filtration, bead separation, preferably magnetic bead separation, binding to a non-particulate solid matrix, e.g., a membrane, or incubation with a nuclease.

[0062] In a preferred embodiment non-sperm DNA comprised in the first lysate is decomposed, preferably by subjecting the non-sperm DNA to a nuclease digestion.

[0063] Following the first lysis, the non-lysed cells are subjected to a second or further lysis step, by applying one or more lysing agents or a second or further lysis solution to the non-lysed cells and / or incubating the non-lysed cells with the one or more lysing agents or in the second or further lysis solution. The one or more lysing agents or the second or further lysis solution are / is suitable to essentially lyse non-sperm cells and to form a second or further lysate comprising non-sperm DNA. The second or further lysis solution does not comprise 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.

[0064] In an embodiment of the disclosure, in method step c) the second or further lysis solution is applied to the non-lysed cells to form a second or further lysate comprising non-sperm DNA.

[0065] In an embodiment the lysing agents are not contacted with the non-lysed cells in the form of a pre-prepared lysing solution, but the one or more lysing agents are contacted directly with the non-lysed cells. This in particular is applicable if the non- lysed cells have been re-suspended in a liquid before or during being brought into contact with the one or more lysing agents.

[0066] In one embodiment the non-lysed cells remaining after the first lysis step have not been separated from the (liquid) lysate comprising the non-sperm DNA but the second lysis solution or the lysing agents for the second lysis are contacted with the lysate from the first lysis step a). Said lysate includes the non-sperm DNA released from the non-sperm cells, any non-sperm cells that haven’t been lysed in the first lysing step and the non-lysed sperm cells and / or sperm nuclei.

[0067] In one embodiment the non-lysed cells remaining after the second or any further lysis step have not been separated from the (liquid) lysate comprising the non- sperm DNA but the lysis solution, the second or further lysis solution or the lysing agents for the second or further lysis are contacted with the lysate from the previous lysis step(s). Said lysate includes the non-sperm DNA released from the non-sperm cells, any non-sperm cells that haven’t been lysed in the previous lysing step(s) and the non-lysed sperm cells and / or sperm nuclei.

[0068] In an embodiment of the disclosure, in method step c) the lysis solution, the second or further lysis solution or the one or more lysing agents is / are applied to the separated non-lysed cells, the one or more lysing agents for the second or further lysis are contacted with the non-lysed cells, which are preferably re-suspended, or the one or more lysis agents for the second or further lysis in a pre-dissolved form or directly are contacted with the complete lysate including the lysate of the first or any previous lysis step(s), and the non-lysed cells are incubated with the one or more lysing agents or in the second or further lysis solution to essentially lyse non-sperm cells and to form a second or further lysate comprising non-sperm DNA.

[0069] In an embodiment, the non-sperm DNA present in the first lysate is decomposed or separated or removed or separated and removed from the first lysate comprising said non-sperm DNA, preferably by subjecting the non-sperm DNA in the first lysate to a nuclease digestion, more preferably to an Exonuclease V (RecBCD) digestion.

[0070] In an embodiment, the non-sperm DNA present in the first lysate is decomposed or separated or removed or separated and removed from the first lysate comprising said non-sperm DNA, preferably after step a) and before step c). Preferably the non-sperm DNA in the first lysate as such is subjected to a nuclease digestion, more preferably to an Exonuclease V (RecBCD). Optionally, after the nuclease digestion the non-lysed cells, including the non-lysed sperm cells and / or sperm nuclei, are further separated and removed from the digested lysate for further treatment. Alternatively, in an embodiment, after the nuclease digestion the complete resulting mixture, including digested non sperm DNA, is used for treatment in accordance with step c), i.e. without separation or removal of the non-lysed cells, including the non- lysed sperm cells and / or sperm nuclei, from the digested lysate and the digested non- sperm DNA.

[0071] In a further embodiment, the non-sperm DNA present in the first lysate is separated and removed from the first lysate comprising said non-sperm DNA in accordance with step b), and before step c), the non-sperm DNA still contaminating the non-lysed cells, including the non-lysed sperm cells and / or sperm nuclei is decomposed preferably by subjecting the non-sperm DNA in the separated and removed non-lysed cells to a nuclease digestion, more preferably to a Exonuclease V (RecBCD) digestion. Optionally, after the nuclease digestion the non-lysed cells, including the non-lysed sperm cells and / or sperm nuclei, are further separated and removed from the digested non-lysed cell mixture for further treatment, i.e. the digested non-sperm DNA is removed from the mixture of the remaining non-lysed cells.

[0072] Alternatively, in an embodiment, after the nuclease digestion the complete resulting mixture, i.e. including the digested non-sperm DNA as well as the mixture of non-lysed cells, is used for treatment in accordance with step c), i.e. without theseparation or removal of the non-lysed cells, including the non-lysed sperm cells and / or sperm nuclei, from the digested non-sperm DNA in the mixture of non-lysed cells.

[0073] In a further embodiment, the non-sperm DNA present in the first lysate is decomposed or separated or removed or separated and removed from the first lysate comprising said non-sperm DNA, preferably after step a) and / or b) and before step c) preferably by subjecting the non-sperm DNA in the first lysate to a nuclease digestion, more preferably to an Exonuclease V (RecBCD). Thus, the step of decomposing or decomposing and removing, optionally decomposing, separating and removing, the non-sperm DNA from the first lysate may be repeated once, twice or several times to ensure decomposing and removing of as much of the non-sperm DNA as possible.

[0074] In an embodiment of the disclosure, this further lysis step c) is repeated once, twice or several times to ensure complete lysis of the non-sperm DNA.

[0075] The term “lysis solution”, “first lysis solution" and / or “second lysis solution” as used herein, 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.

[0076] The term “one or more lysing agents” as used herein, 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.

[0077] In an embodiment of the present disclosure, the lysis solution, first lysis solution, second lysis solution or one or more lysing agents comprise(s) at least a chaotropic compound. In a preferred embodiment, the chaotropic compound is 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. The chaotropic compound is preferably a guanidinium salt, such as a guanidinium hydrochloride, guanidinium thiocyanate and / or guanidinium isothiocyanate.

[0078] Suitable concentrations and amounts of the chaotropic compounds may vary depending on the nature of the samples or lysis parameters, with concentrations of the chaotropic compound in the range of > 0,0001 mM to < 10 M generally being favourable, based on the total volume of the lysis solution or the lysate, respectively. Preferably, the concentrations of the chaotropic compound 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 0,5 M, 1 M, 1,5 M, 2 M, 2,5 M, 3 M, 3,5 M, 4 M, 4,5 M, 5 M, 5,5 M and 6 M , based on the total volume of the lysis solution or the lysate, respectively.

[0079] In another embodiment of the present disclosure, the lysis solution, first lysis solution, second 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.

[0080] In another embodiment of the present disclosure, the lysis solution, first lysis solution, second 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 / or 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); vii) the anionic surfactant is selected from the group consisting of sodium dodecyl sulfate (SDS), sarkosyl, deoxycholate, sodium laureth sulfate (SLS), sodiumoctyl sulfate (SOS), sodium methyl cocoyl taurate and disodium coco sulfosuccinates, or a combination thereof; viii) the non-ionic surfactant is selected from the group consisting of Triton X- 100, Triton X-l 14, N-octylglucoside, Tween 20 (polysorbate 20, polyoxyethylene (20) sorbitan monolaurate), Tween 40, Tween 80, Span 80, Span 85, Ecosurf EH-6, Brij- 35 (polyalkylengly col ether), NP-40 (nonyl phenoxypolyethoxyl ethanol), Nonidet P- 40 (octylphenoxypoly ethoxy ethanol), 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.

[0081] In an embodiment of the disclosure, the lysis solution, the first lysis solution, the second 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.

[0082] In an embodiment of the disclosure, the lysis solution, the first lysis solution, the second 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.

[0083] In an embodiment of the disclosure, the lysis solution, the first lysis solution, the second 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-l 14, N- octylglucoside, Tween 20 (polysorbate 20, polyoxyethylene (20) sorbitan monolaurate), Tween 40, Tween 80, Span 80, Span 85, Ecosurf EH-6, Brij -35(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(polyalkylenglycolether), 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.

[0084] In an embodiment of the disclosure, the lysis solution, the first lysis solution, the second lysis solution or the one 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.

[0085] 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 0,lmM to 5M, or in the range 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 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 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 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.

[0086] In an embodiment of the present disclosure, the lysis solution, the first lysis solution, the second lysis solution or the one or more lysing agents comprise(s) at leastan agent selected from non-sperm cell digesting enzymes, in particular for example proteinase K (QIAGEN), protease, zymolase, lyticase, chromopeptidase, lysostaphin, lysozyme, or any combination thereof.

[0087] 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.

[0088] According to a further preferred embodiment, the lysis solution, which may be the first lysis solution comprising one or more lysing agents, the second lysis solution comprising one or more lysing agents or any further lysing solution comprising one or more lysing agents, the one or more lysing agents or the mixture of the lysis solution or the lysing agents with the biological sample has a pH in the range of > 4 to < 12, in particular in the range of > 5 to < 11, preferably in the range of > 6 to < 10, especially preferably in the range of > 6 to < 9. Each used lysis solution, lysing agent of mixture of biological sample with lysing solution or lysing agent, may have the same pH value as specified or the single lysis solutions may have pH values different from each other while still preferably being in the specified range.

[0089] In an embodiment of the disclosure, the biological sample and / or the non-lysed cells are 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.

[0090] 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.

[0091] In one embodiment of the disclosure, the one or more lysing agents or the lysis solution applied in method step c) comprise(s) the same agents in the same concentration and / or amounts as the one or more lysing agents or the lysis solution applied in method step a).

[0092] In another embodiment the one or more lysing agents or the lysis solution applied in method step a) and the one or more lysing agents or the lysis solution appliedin method step c) are different in at least one agent and / or at least one agent concentration or amount.

[0093] The lysis solution may be pre-prepared by dissolving the lysing agents in a solvent or the lysing agents may be added directly to the sample in step a) or to the non-lysed cells in step c) or their respective re-suspension to result in a lysate.

[0094] In an embodiment a pre-prepared lysis solution or the one or more lysing agents are added directly to the lysate obtained in step a). In this case preferably no separation and / or removal of the first lysate comprising non-sperm DNA from the non-lysed cells obtained from the first lysing step takes place.

[0095] In an embodiment, the non-lysed cells in step c) are incubated in the lysis solution or the one or more lysing agents used in method step a). In a further embodiment the non-lysed cells in step c) are incubated in a lysis solution or with one or more lysing agents that is / are different in at least one agent and / or at least one agent concentration or amount from that used in method step a).

[0096] In one embodiment of the disclosure, the lysis solution applied in method step c) or the one or more lysing agents is / are free of nuclease inhibiting agents, preferably free of chelating agents, such as ethylenediaminetetraacetic acid (EDTA), and / or free of anionic detergents, such as sodium dodecyl sulfate (SDS), sarkosyl, deoxycholate, sodium laureth sulfate (SLS), sodium octyl sulfate (SOS), sodium methyl cocoyl taurate and di sodium coco sulfosuccinates, or any mixtures thereof.

[0097] After lysis step a) and / or c) the non-sperm DNA present in the first and / or second and / or further lysate is decomposed or separated or removed or separated and removed or decomposed and removed or decomposed, separated and removed from the first and / or second and / or further lysate comprising said non-sperm DNA.

[0098] Removal or separation of DNA, in particular the non-sperm DNA, generally and in particular in step b) and / or d) may occur with any suitable means including precipitation, centrifugation, filtration, bead separation, preferably magnetic bead separation, binding to a non-particulate solid matrix, e.g., a membrane, or incubation with a nuclease.

[0099] In an embodiment of the disclosure, separating, removing, or separating and removing the non-lysed cells occurs through centrifugation, preferably by forming a pellet of the non-lysed cells and / or sperm nuclei, through filtration, through binding to a solid matrix, preferably to a membrane or beads, preferably to magnetic beads.

[0100] In a preferred embodiment the non-sperm DNA present in the first and / or second and / or further lysate is digested or decomposed using one or more nucleases. Preferably the one or more nucleases are 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.

[0101] In one embodiment the one or more nucleases are selected from nucleases that have a large size, preferably of 50 kDa or above, such as e.g., of 60 kDa, 80 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa or preferably of 400 kDa orabove, such as nucleases having a size of 70 kDa, 90 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa, or 500 kDa, or having any of the sizes in between.

[0102] In one embodiment the one or more nucleases are selected from nucleases that have a large size, of preferably over 50 kDa, such as EcoRl restriction endonuclease, Phosphodiesterase I, Phosphodiesterase II, Exonuclease I, Exonuclease V (RecBCD), Exonuclease VII, and Lactoferrin.

[0103] In a preferred embodiment the one or more nucleases are selected from nucleases that do not or only moderately digest or decompose sperm DNA when still being contained in sperm cells or sperm nuclei, but predominantly digest and / or decompose non-sperm DNA, such as are typically present together in forensic samples.

[0104] The present disclosure also provides a nuclease that is particularly useful in the treatment of forensic samples comprising non-sperm DNA and sperm DNA together, where the aim is to digest and decompose non-sperm DNA without digesting or decomposing the sperm DNA present in the sperm cell, more specifically in the sperm nucleus of the sperm cell, of the same sample.

[0105] Currently, in workflows for isolating sperm DNA from samples which contain sperm cells as well as non-sperm cells while the non-sperm cells are usually in a tremendous excess, such as in forensic and in particular sexual assault samples, the nuclease which is used to digest non-sperm DNA (in particular DNase I) to a huge extent also digests sperm DNA although the sperm DNA may still be within the sperm nucleus, i.e. before the lysis of the sperm cell and / or sperm nucleus. This has the major disadvantage that when attempting to reduce the contamination of non-sperm DNA in a forensic sample in order to improve the sensitivity of the subsequent downstream analysis, the yield of sperm DNA may inadvertently be reduced as well, which in turn reduces the sensitivity. Thus, in the workflows known in the state of the art there is usually the conflict / competition between on one hand reducing the amount of contaminating non-sperm DNA and on the other hand reducing the loss of sperm DNA,i.e. receiving a maximum yield of sperm DNA with minimum non-sperm DNA contamination.

[0106] 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. The nuclease can preferably also recognize and be altered by the Chi DNA sequence.

[0107] Preferably nucleases that have a helicase activity, an endonuclease activity and an exonuclease activity and the nuclease in addition can also recognize and be altered by the Chi DNA sequence.

[0108] 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.

[0109] In a preferred embodiment the one or more nucleases are selected from nucleases that have a size of 50 kDa or above, such as of 60 kDa, 80 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300kDa, 350 kDa, or preferably of 400 kDa or above, like having 70 kDa, 90 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 160 kDa, 170 kDa,180 kDa, 190 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 260 kDa, 270 kDa, 280 kDa,290 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa,410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa, or 500 kDa, 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.

[0110] In a preferred embodiment the one or more nucleases are selected from nucleases that do not or only moderately digest or decompose sperm DNA when still being contained in sperm cells or sperm nuclei but predominantly digest and / or decompose non-sperm DNA, such as are typically present together in forensic samples.

[0111] 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.

[0112] In an embodiment of the invention the nuclease has at least one or more of the subsequent features: i) it has a size of 50 kDa or above; ii) it has a size of 60 kDa, 80 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa or preferably of 400 kDa 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; xiii) the nuclease is chosen such that it digests and / or decomposes non-sperm DNA but not sperm DNA; xiv) the nuclease can recognize and be altered by the Chi DNA sequence.

[0113] More specifically, Exonuclease V (RecBCD), which has a helicase and exonuclease activity, does not digest and / or decompose sperm DNA present in the sperm nucleus, but digests and / or decomposes any other type of non-sperm DNA present in a sample. Exonuclease V (RecBCD) in particular can be used in methods, workflows, and kits for the treatment of forensic or sexual assault samples comprising non-sperm cells and sperm cells, where selective isolation of sperm cell DNA that is essentially free from non-sperm cell DNA is paramount.

[0114] Separating the lysed and non-lysed cell fractions after the first and / or the second and / or further lysis step, and thereby 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.

[0115] Once the non-sperm DNA is removed, the non-lysed cell fraction is subjected to at least a third or further 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. A third or further lysis comprises applying or treating the non-lysed cells with the sperm cell and / or sperm nuclei lysis solution or the 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 third or further lysate comprising sperm DNA.

[0116] 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 selected from a reduction agent, a sperm cell and / or sperm nuclei lysing enzyme, a chaotropic agent and / or an alkaline agent in a concentration suitable to lyse sperm cells and / or sperm nuclei, or a combination thereof.

[0117] In an embodiment of the disclosure, in method step e) the sperm cell lysis solution or the one or more sperm cell lysing agents is / are applied to the non-lysed cells or sperm nuclei to form a third or further lysate comprising sperm DNA.

[0118] In an embodiment of the disclosure, in method step e) 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 non-lysed cells and / or sperm nuclei and the non-lysed 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 third or further lysate comprising sperm DNA.

[0119] 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 0.1 M (calculated within the total lysate).

[0120] 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.

[0121] 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 of > 0,005 mM to < 8 M, more preferably in a range of > 1 M to < 5 M based on the total volume of the lysis solution or the total lysate.

[0122] 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.

[0123] In an embodiment of the present disclosure, the sperm cells and / or sperm nuclei are subjected to an alkaline lysis. Preferably, the alkaline conditions are established using hydroxide salts like NaOH, KOH or similar. The hydroxide concentration in the lysis mixture should be in the range of from 10 mM to 1 M, preferably from 25 mM to 800 mM, more preferred from 50 mM to 600 mM, further preferred from 250 to 400 mM.

[0124] In certain embodiments, the method of the disclosure further includes after method step b) and / or d) at least one washing step of the non-lysed cells or cell pellet. Preferably the non-lysed cell pellet is thereby resuspended in a suitable washing composition at room temperature, subsequently centrifuged, preferably at 15,0000xg for 5 minutes, and the supernatant is finally discarded.

[0125] In a preferred embodiment the washing composition is selected from the group consisting of water or the lysis solution, or any combination thereof.

[0126] In a preferred embodiment, the washing composition is applied at room temperature.

[0127] In a preferred embodiment the washing composition comprises a chaotropic agent having a concentration in the range of > 0,1 M to < 3,5 M, particularly preferably in the range of > 0,4 M to < 3 M based on the total volume of the buffer composition.

[0128] In an embodiment of the disclosure, lysis step e) is repeated once, twice or several times to ensure complete lysis of the sperm cells and sperm nuclei.

[0129] In an embodiment of the disclosure, first, second, third or any further lysis 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.

[0130] 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 first 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 or sperm nuclei, b) a second 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 sperm nuclei, wherein the second non- sperm cell lysis solution or the one or more non-sperm cell lysing agents are identical to the first non-sperm cell lysis solution or the one or more non-sperm cell lysing agents used in a), or wherein the second non-sperm cell lysis solution or the one or more non-sperm cell lysing agents are different from the first non-sperm cell lysis solution or the one or more non-sperm cell lysing agents used in a).

[0131] The present disclosure also provides a method for treatment of a biological sample comprising sperm cells and / or sperm nuclei and non-sperm cells, the method comprising: i. subjecting the biological sample comprising the sperm cells and / or sperm nuclei and the 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 first 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; ii. decomposing most or all the non-sperm DNA obtained from the lysate in step a) using an enzyme having a helicase and an exonuclease activity and / or having a size of above 50 kDA while not or not markedly decomposing sperm DNA; and iii. subjecting the non-lysed cells to a second lysis by applying a sperm cell and / or sperm nuclei lysis solution or one or more lysing agents to lyse sperm cells and / or sperm nuclei completely and to form a third or further lysate comprising sperm DNA,wherein 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 selected from a reduction agent, a sperm cell and / or sperm nuclei lysing enzyme, a chaotropic agent in a concentration suitable to lyse a sperm cell and / or sperm nuclei, and / or an alkaline agent in a concentration suitable to lyse sperm cells and or sperm nuclei or a combination thereof.

[0132] It has been shown that surprisingly an enzyme having a helicase and an exonuclease activity and / or having a size of above 50 kDA, which preferably is RecBCD, is suitable for completely or at least predominantly decomposing non-sperm DNA in a sample comprising non-sperm DNA and sperm DNA while not or only or only moderately, markedly or slightly decomposing sperm DNA which is still contained in the nuclei.

[0133] “Not markedly decomposing sperm DNA” is supposed to indicate that although desirable, it is not necessary that absolutely no sperm DNA is decomposed, but rather that it cannot be excluded that a small amount of sperm DNA may be decomposed also when using an enzyme like RecBCD.

[0134] In an embodiment, that no marked sperm DNA decomposition takes place may be determined by the DNA degradation ratio of the male DNA (as defined e.g. in the QIAGEN DNA Investigator Quantiplex Pro RGQ Kit). In an embodiment said ratio should be < 1.5, preferably < 1.4, preferably < 1.3, more preferably < 1.2, even more preferred < 1.1 and most preferred 1 or below. A further suitable indicator of a not marked decomposition of sperm DNA is the amount by which the sperm DNA is reduced. In an embodiment not more than 50% of the sperm DNA, preferably not more than 40%, more preferably not more than 30 %, even more preferred not more than 20%, in particular not more than 10%, preferably nor more than 5%, more preferred not more than 3% and most preferred not more than 1% of the sperm DNA is decomposed compared to the amount of sperm DNA contained in the sperm nuclei before the enzymatic digestion.

[0135] “Decomposing most of the non-sperm DNA” is supposed to indicate that the non-sperm DNA is reduced by at least 70%, preferably by at least 80%, more preferably by at least 85%, more preferred by at least 90%, even more preferred by atleast 95%, most preferred by at least 98% during the enzymatic digestion compared to the amount present before said digestion.

[0136] Smaller nucleases or nucleases having no combination of functionalities like a helicase and an exonuclease functionality, e.g. DNase I, are currently commonly used in order to decompose contaminating non-sperm DNA in samples comprising nonsperm DNA and sperm DNA, like in forensic samples and in particular samples obtained after sexual assault delicts. However, when using such nucleases like DNase I, these not only decompose non-sperm DNA released from cells in a lysis process, but they also decompose the target sperm DNA of such samples, even if the sperm DNA is still retained in the nucleus and not yet released and, thus, not directly accessible. Consequently, even though a selective lysis procedure is used to selectively lyse mainly non-sperm cells in order to allow a separation of excess non-sperm DNA from target sperm DNA, when trying to remove the contaminating non-sperm DNA by enzymatic decomposition the sperm DNA within the sperm nuclei is also decomposed, which tremendously reduces the sperm DNA yield obtained using such a procedure.

[0137] Consequently, while a procedure as described above in which a selective non- sperm cell lysis procedure is conducted at least twice is even superior, nevertheless when using the above specified enzymes with a combination of functionalities and / or a size above 50 kDa to decompose non-sperm DNA even with only conducting one non-sperm cell lysis procedure, the yield and quality of sperm DNA that is isolated after such a procedure is markedly better than when using the commonly used enzymes of the state of the art like DNase I under such conditions.

[0138] In one embodiment the one or more nucleases are selected from nucleases that have a large size, preferably of 50 kDa or above, such as e.g., of 60 kDa, 80 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa or preferably of 400 kDa or above, such as nucleases having a size of 70 kDa, 90 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa, or 500 kDa, or having any of the sizes in between.

[0139] In one embodiment the one or more nucleases are selected from nucleases that have a large size, of preferably over 50 kDa, such as EcoRl restriction endonuclease, Phosphodiesterase I, Phosphodiesterase II, Exonuclease I, Exonuclease V (RecBCD), Exonuclease VII, and Lactoferrin.

[0140] In a preferred embodiment the one or more nucleases are selected from nucleases that do not or only moderately digest or decompose sperm DNA when still being contained in sperm cells or sperm nuclei but predominantly digest and / or decompose non-sperm DNA, such as are typically present together in forensic samples.

[0141] In another embodiment the one or more nucleases are selected from nucleases that have multiple independent functions, such as a helicase activity and an exonuclease activity, optionally additionally an endonuclease activity. The nuclease can preferably also recognize and be altered by the Chi DNA sequence.

[0142] Preferably nucleases that have a helicase activity, an endonuclease activity and an exonuclease activity and in addition, the nuclease can also recognize and be altered by the Chi DNA sequence.

[0143] 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.

[0144] 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.

[0145] In an embodiment of the invention the nuclease has at least one or more of the subsequent features: i) it has a size of 50 kDa or above; ii) it has a size of 60 kDa, 80 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa or preferably of 400 kDa 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 exonuclease activity; ix) it has a helicase activity, an endonuclease activity and an exonuclease activity; x) the nuclease is chosen such that it does not get access to the packed DNA; xi) the nuclease is chosen such that it digests and / or decomposes non-sperm DNA but not sperm DNA; xii) the nuclease can recognize and be altered by the Chi DNA sequence.

[0146] More specifically, Exonuclease V (RecBCD) which has a helicase and exonuclease activity, does not digest and / or decompose sperm DNA while being present in the sperm nucleus, but digests and / or decomposes any other type of nonsperm DNA present in a sample. Exonuclease V (RecBCD) in particular can be used in methods, workflows, and kits for the treatment of forensic or sexual assault samplescomprising non-sperm cells and sperm cells, where selective isolation of sperm cell DNA that is essentially free from non-sperm cell DNA is paramount.

[0147] The present disclosure also provides the use of an enzyme having an exonuclease and helicase activity and / or a nuclease having a size of above 50 kDA for the decomposition of non-sperm DNA in a sample comprising a mixture of non-sperm DNA and sperm DNA, the sperm DNA being contained in sperm cells and / or sperm nuclei. Such a mixture of non-sperm DNA and sperm DNA, the sperm DNA being contained in sperm cells and / or sperm nuclei, is usually part of the treatment and analysis of a forensic sample, in particular a sample obtained after a sexual assault delict. Preferably, RecBCD is used for the decomposition of the non-sperm DNA in such mixtures.

[0148] In one embodiment the one or more nucleases are selected from nucleases that have a large size, preferably of 50 kDa or above, such as e.g., of 60 kDa, 80 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa or preferably of 400 kDa or above, such as nucleases having a size of 70 kDa, 90 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa, or 500 kDa, or having any of the sizes in between.

[0149] In one embodiment the one or more nucleases are selected from nucleases that have a large size, of preferably over 50 kDa, such as EcoRl restriction endonuclease, Phosphodiesterase I, Phosphodiesterase II, Exonuclease I, Exonuclease V (RecBCD), Exonuclease VII, and Lactoferrin.

[0150] In a preferred embodiment the one or more nucleases are selected from nucleases that do not or only moderately digest or decompose sperm DNA when still being contained in sperm cells or sperm nuclei but predominantly digest and / or decompose non-sperm DNA, such as are typically present together in forensic samples.

[0151] In another embodiment the one or more nucleases are selected from nucleases that have multiple independent functions, such as a helicase activity and anexonuclease activity, optionally additionally an endonuclease activity. The nuclease can preferably also recognize and be altered by the Chi DNA sequence.

[0152] Preferably nucleases that have a helicase activity, an endonuclease activity and an exonuclease activity and the nuclease in addition can also recognize and be altered by the Chi DNA sequence.

[0153] 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.

[0154] In an embodiment of the invention the nuclease has at least one of the subsequent features: i) it has a size of 50 kDa or above; ii) it has a size of 60 kDa, 80 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa or preferably of 400 kDa 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 exonuclease activity; ix) it has a helicase activity, an endonuclease activity and an exonuclease activity; x) the nuclease is chosen such that it does not get access to the packed DNA; xi) the nuclease is chosen such that it digests and / or decomposes non-sperm DNA but not sperm DNA; xii) the nuclease can recognize and be altered by the Chi DNA sequence.

[0155] More specifically, Exonuclease V (RecBCD) which has a helicase and exonuclease activity, does not digest and / or decompose sperm DNA while being present in the sperm nucleus, but digests and / or decomposes any other type of nonsperm DNA present in a sample. Exonuclease V (RecBCD) in particular can be used in methods, workflows, and kits for the treatment of forensic or sexual assault samples comprising non-sperm cells and sperm cells, where selective isolation of sperm cell DNA that is essentially free from non-sperm cell DNA is paramount.

[0156] For a further specification of the conditions and characteristics of the features specified in method steps i) to iii) and of the enzyme and / or nuclease in the indicated use, it is referred to the statements and information in the specification above which is applicable analogously, in particular the specification describing features a), b), d) and e) above.

[0157] 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 first 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 second and / or further non-sperm cell lysis solution or one or more nonsperm cell lysing agents capable of lysing non-sperm cells and not suitable for completely lysing sperm cells and / or sperm nuclei, wherein the second and / or further non-sperm cell lysis solution or the one or more non-sperm cell lysing agents is / are identical to the first non-sperm cell lysis solution or the one or more non-sperm cell lysing agents used in a), or wherein the second and / or further non-sperm cell lysis solution or the one or more non-sperm cell lysing agents is / are different from the first non-sperm cell lysis solution or the one or more non-sperm cell lysing agents used in a); c) optionally, one or more washing compositions; d) optionally one or more DNA digesting enzymes; e) a sperm cell and / or sperm nuclei lysing 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; or any combination thereof.

[0158] The first, second and / or further non-sperm lysis solutions or the one or more non-sperm cell lysing agents and / or the sperm cell and / or sperm nuclei lysis solutions or the one or more sperm cell and / or sperm nuclei lysing agents as well as the washing compositions used in the kits of the disclosure are described in detail above and apply to this aspect of the disclosure as well.

[0159] The first, second and or further lysis solution and / or the sperm cell and / or sperm nuclei lysis 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 by the user according to instructions present in thekit. The lysis solutions and / or their individual agents or compositions are provided in suitable containers.

[0160] In an embodiment, the kit further comprises suitable containers, collection tubes, sample tubes etc, spin columns or baskets and other material useful for sample processing according to the method of the disclosure. The kit also includes instructions for use.

[0161] 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.

[0162] 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.

[0163] In some embodiments, sperm DNA is obtained from the purified sperm fraction and further analysed. For example, sperm DNA may be quantified or genotyped.

[0164] 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.

[0165] 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.EXAMPLES:

[0166] Each example is provided by way of explanation of the disclosure and is not meant as a limitation of the disclosure.

[0167] Example 1

[0168] This example demonstrates that a considerable fraction of epithelial cells remains intact if subjected to only one lysis step. When subjected to a subsequent lysis step these intact epithelial cells are lysed and further DNA is set free. The yield of DNA isolated from epithelial cells after a first lysis and a subsequent second lysis is shown in Figure 1. An extract of the STR profiles of the isolated DNA is shown in Figure 2.

[0169] Vaginal swabs containing epithelial cells (but no sperm cells) were subjected to a first lysis step where the vaginal swab samples were mixed with 500 pL of a lysis buffer comprising 0.3% SDS and 25 pL Proteinase K (QIAGEN) and incubated at 56°C for an hour at 900rpm and then 80°C for lOmin. The samples were then transferred to a spin basket (e.g. QIAshredder-spin basket - QIAGEN) and subjected to centrifugation at full speed (15.000rpm) for about 5 minutes, whereby a pellet comprising the intact (non-lysed) cells is obtained. Most of the “supernatant” lysate was removed by pipetting and the DNA present in the “supernatant” fraction was purified using the EZ1&2 DNA Investigator Kit workflow (QIAGEN).

[0170] The cell pellet of intact / non-lysed cells and the remaining traces of the first lysate that couldn’t be removed without disturbing the pellet (about 30 pl) was subjected to DNase I digestion at 40°C for about 15 min to remove (digest) any DNA present in the remaining lysate and contaminating the cell pellet.

[0171] An aliquot of 50 pL of the liquid mixture after DNase digestion was removed and the DNA present in said “after DNase digestion” fraction was purified using the EZ1&2 DNA Investigator Kit workflow (QIAGEN).

[0172] In the remaining mixture after DNase digestion containing liquid and cells, the DNase enzyme was inactivated by subjecting said mixture to heat treatment at 80 °C for about 5 min.

[0173] After the enzyme was inactivated, said mixture comprising the intact (un-lysed) cells was subjected to a second lysis.

[0174] For the second lysis the mixture comprising the intact (un-lysed) cells was mixed with a lysis buffer comprising 0,5% SDS and 1% chaotropic salts and 25 pL Proteinase K (QIAGEN) and incubated at 56°C for about an hour. DNA present in the “2nd lysis” fraction was purified using the EZ1&2 DNA Investigator workflow (QIAGEN).

[0175] Following purification, DNA samples were quantified by qPCR on a PCR thermocycler (Applied Biosystems 7500 Real-Time Systems using the “Investigator Quantiplex Pro Kit” (QIAGEN Cat. No. / ID: 387216). A maximum of 15 pL eluate was used for the PCR of the QIAGEN Investigator 24plex QS Kit (Cat. No. / ID: 382415). The amplified DNA was analysed by capillary electrophoresis. For the analysis the samples are diluted to max. 500pg input and applied to the CE (ABI PRISM Genetic Analyzer 3500 XL).

[0176] Results are shown in Figures 1 and 2.

[0177] With reference to Figure 1, the three columns show the amount of DNA isolated from a female cell sample after the sample was subjected to a first lysis (“supernatant”), after DNase digestion and after second lysis (“2nd lysis”) of the sample. Parts A and B show the same data, but the DNA yield is either plotted in a logarithmic scale (A) or linear scale with broken Y axis (B). The linear scale better illustrates the tremendous reduction of the amount of autosomal DNA in the sample after digestion and 2ndlysis compared to the amount in the supernatant (from 1901.5 ng to 0.007 ng and 0.539 ng, respectively) which is more realistically shown in the absolute numbers of the linear scale than in the logarithmic scale. Autosomal DNA yield was calculated for 470 pL of the “supernatant” sample and for the 50 pL aliquots of the digested samples (“after DNase digestion”) and the twice lysed samples (“2nd lysis”).

[0178] Figure 1(A&B) shows that most of the vaginal cells in the sample were lysed already during the first lysis step. Accordingly, there is a high amount of DNA in the supernatant. The amount of DNA detected in the mixture after DNase digestion (whichis only slightly above the limit of detection of about 0,001 ng) shows that before the 2ndlysis takes place the mixture containing liquid and intact / non-lysed cells contains almost no DNA. Surprisingly one can see the after the 2ndlysis step a considerable amount of DNA could again be detected, although being lower than the amount of DNA contained in the supernatant after the first lysis.

[0179] The DNA quantified in the mixture of the “2nd lysis” are expected to result from cells that have not been lysed during the first lysis and which were obtained in addition to the “supernatant”. DNA digestion of the mixture remaining from the first lysis after having removed the supernatant removes free DNA that might be present in the remaining traces of the first lysate or cell debris. Only a minute DNA amount can be quantified in the supernatant after DNase digestion (see Fig. 1, plotted in logarithmic scale). DNA detected after second lysis is therefore assumed to come from epithelial cells that have not been lysed in the initial / first lysis of the cell sample but surprisingly remained intact.

[0180] This example shows that the first lysis step does not result in a complete lysis of the vaginal cells. Instead, enough cells remain that when not being removed in a second or further lysis procedure are supposed to contaminate other cells of interest, like sperm cells and sperm nuclei in sexual assault samples and will result in a contamination of sperm DNA with female cell DNA when the sperm cells and nuclei are lysed in the presence of the vaginal cells.

[0181] Results of the STR analysis are shown in Figure 2.

[0182] The DNA isolated from the three fractions obtained as explained above was amplified and the STR profiles of the DNA obtained from the “supernatant” fraction, the “after DNase digestion” fraction and the “2nd lysis” fraction. Each sample was diluted to a 500pg input and subjected to a capillary electrophoresis (CE) on an ABI PRISM Genetic Analyzer 3500 XL and the STR- Alleles of the violet channel were used for identification as outlined in Table 1.

[0183] Table 1 : STR-Allele (extract purple channel)

[0184] The “supernatant” graph shows all alleles of the reference DNA profile in the violet channel.

[0185] The “after DNase digestion” graph shows no alleles because DNA digestion of the mixture obtained from the first lysis after removing the supernatant was successful and any amount of free DNA remaining in the lysed sample was too low to be detected during STR analysis.

[0186] The “after 2nd lysis” graph shows the same allele profiles in the violet channel as the “supernatant” graph. This shows that the DNA was not a contamination of foreign DNA but came from the same person as the sample “supernatant”.

[0187] Example 2

[0188] This example shows the purity of the sperm-associated DNA isolated from mock samples (ejaculate spiked on vaginal swabs) if the non-sperm cells in the sample were subjected to lysis once (1st lysis) and twice (lst+2nd lysis). The yield of DNA isolated is shown in Figure 3a and 3b. The STR profiles are shown in Figure 4.

[0189] Workflow with 1 lysis step for the non-sperm cells:

[0190] Lysis of non-sperm cells

[0191] Vaginal swabs (treated under the same conditions and forming Sample 1 and Sample 2) containing epithelial cells were spiked with ejaculate (Sample 1 contained 0.0007% sperm, Sample 2 contained 0.0006% sperm) and subjected to a first lysis step under conditions whereby the non-sperm cells are lysed, but not the sperm cells or sperm nuclei. The samples were mixed with 500 pL of a lysis buffer comprising 0,1% SDS and 25 pL Proteinase K (QIAGEN) and incubated at 56°C for an hour at 900rpm. The samples were vortexed, transferred to a spin basket (e.g., QIAshredder-spin basket- QIAGEN) and subjected to centrifugation at full speed (15.000rpm) for about 5 minutes, whereby a pellet comprising the non-lysed (intact) cells is obtained. Most of the “supernatant” lysate was removed by pipetting and the DNA present in the “nonsperm” fraction was purified using the EZ1&2 DNA Investigator Kit workflow (QIAGEN). The amount of DNA recovered is shown in Figure 3a (non-sperm fraction- S ample 1 and Sample 2).

[0192] The remaining traces of the first lysate that couldn’t be removed without disturbing the pellet (about 30 pl) and the cell / nuclei pellet were then subjected to DNase I digestion at 40°C for about 15 min in order to remove any DNA present in the remaining lysate and contaminating the pellet.

[0193] Sperm lysis of the sperm fraction (SF)

[0194] For lysis of sperm cells and sperm nuclei the mixture after DNase digestion comprising liquid and still intact cells and sperm nuclei (referred to in the Figures 3b and 4 as “sperm fraction”) was first mixed with 408 pL of a 2,5M chaotropic buffer to stop the remaining DNase activity. Subsequently, 4,5 pL of a IM TCEP (tris(2- carboxyethyl) phosphine) solution was added to the resulting mixture containing the sperm cells and nuclei and any remaining non-sperm cells and incubated at 15min 25°C and 900rpm. Under these conditions the remaining cells and nuclei are completely lysed to obtain the sperm (or male) DNA and any contaminating non-sperm (female) DNA.

[0195] DNA (sperm DNA and residual non-sperm cell DNA) was then purified using the QIAGEN EZ2 DNA Investigator sexual Assault workflow (QIAGEN). The amount of DNA recovered is shown in Figure 3b (sperm fraction- Sample 1 and Sample 2).

[0196] Workflow with 1 st+ 2nd lysis of the non-sperm cells

[0197] 1stLysis of non-sperm cells

[0198] Vaginal swabs (treated under the same conditions and forming Sample 3 and Sample 4) containing epithelial cells were spiked with ejaculate (Sample 3 contained 0.0007% sperm, Sample 4 contained 0.0006% sperm) and subjected to a first lysis stepunder conditions whereby the non-sperm cells are lysed, but not the sperm cells or sperm nuclei. The samples were mixed with 500 pL of a lysis buffer comprising 0,1% SDS and 25 pL Proteinase K and incubated at 56°C for an hour at 900rpm. The samples were vortexed then transferred to a spin basket (e.g. QIAshredder-spin basket - QIAGEN) and subjected to centrifugation at full speed (15.000rpm) for about 5 minutes, whereby a pellet comprising the non-lysed (intact) cells is obtained.

[0199] Most of the “supernatant” lysate was removed by pipetting and DNA was purified using the EZ1&2 DNA Investigator Kit workflow (QIAGEN). The amount of DNA recovered is shown in Figure 3a (non-sperm fraction- Sample 3 and Sample 4) is).

[0200] 2ndLysis of non-sperm cells

[0201] The sample (remaining traces of the first lysate that couldn’t be removed without disturbing the pellet (about 30 pl) and intact cells) was subjected to a second lysis under conditions whereby non-sperm cells are lysed, but not sperm cells or sperm nuclei. This was achieved by mixing the sample with a lysis buffer comprising 1% Tween20 and 25 pL Proteinase K (QIAGEN) and incubated at 60°C for 25min at 900rpm and then 80°C for 5Min.

[0202] The mixture obtained from the second lysis containing the supernatant as well as the still intact cells and nuclei was then subjected to DNase I digestion at 40°C for about 15 min in order to remove any DNA present in the lysate.

[0203] Alternatively, the remaining non-sperm cell DNA can be digested by using an alternative nuclease such as Benzonase, all other members of DNase I family, Exonuclease V (RecBCD), DNaseMe, Masterase, Saltonase, a combination of at least one restriction endonucleases and Nuclease SI or Exonuclease III or both. Adjustments to the second lysis buffer might be necessary.

[0204] Sperm lysis

[0205] For lysis of the sperm cells and sperm nuclei, the sample (the mixture of the second lysis after DNase digestion comprising liquid and still intact cells) was first mixed with 408 pL of a 2,5M chaotropic buffer to stop the remaining DNase activity.Subsequently, 4,5 pL of a IM TCEP (tris(2-carboxyethyl) phosphine) solution was added to the sample fraction containing the sperm cells and nuclei and incubated at 15min 25°C and 900rpm. Under these conditions the sperm cells are lysed, and the resulting lysate contains sperm DNA.

[0206] DNA was purified using the QIAGEN EZ2 DNA Investigator sexual Assault workflow (QIAGEN). The amount of DNA recovered is shown in Figure 3b (sperm fraction- Sample 3 and Sample 4).

[0207] Quantification and STR analysis

[0208] DNA samples were quantified by qPCR on a PCR thermocycler (Applied Biosystems 7500 Real-Time Systems) using the “Investigator Quantiplex Pro Kit” (QIAGEN Cat. No. / ID: 387216). STR-PCR was conducted with the QIAGEN Investigator 24plex QS Kit (Cat. No. / ID: 382415). The amplified DNA was analysed by capillary electrophoresis. For the analysis the samples are diluted to max. 500pg input and applied to the CE (ABI PRISM Genetic Analyzer 3500 XL).

[0209] Results are shown in Figures 3a, 3b and 4.

[0210] Figure 3a shows the amount of autosomal DNA isolated from the non-sperm fraction (NSF) after the first non-sperm cell lysis of Sample 1 and Sample 3 (as well as Sample 2 and Sample 4). Starting material was calculated to have a sperm cell amount that is equivalent to 0.0007 % (0.0006 %, respectively) of sperm-associated DNA in relation to DNA coming from female vaginal swabs. Calculation of DNA was based on the amount of DNA and cells that were detached from swabs during the lysis. Figure 3a) shows that the amount of non-sperm (female) DNA in both workflows, (i.e. using only one non-sperm cell lysis step or two non-sperm cell lysis steps before lysing the sperm cells and nuclei) is very similar. Accordingly, all variations of sperm DNA amounts are expected to result from the chosen workflow and not from variations in the original sample material.

[0211] Figure 3b shows the amount of contaminating autosomal DNA compared to target male DNA isolated from the sperm fraction of a mixed epithelial and sperm containing sample after a single lysis (Sample 1 and Sample 2) compared to a 1stand a subsequent 2ndlysis of the non-sperm fraction (Sample 3 and Sample 4).

[0212] Figure 3b) clearly shows that after the first non-sperm cell lysis step a considerable amount of non-sperm cells remains intact and contaminates the also still intact sperm cells and nuclei even after a subsequent DNase digesting step. Thus, when the sperm cells and nuclei are lysed in the presence of the contaminating non-sperm cells a considerable amount of female DNA is contaminating the male DNA and makes the analysis of the DNA of a perpetrator more difficult (see the tremendously higher amount of autosomal female DNA in Sample 1 and Sample 2 compared to the relatively low amount of male DNA).

[0213] Yet, when conducting two subsequent lyses of the non-sperm fraction with removing the lysate supernatant of the first lysis before conducting the second lysis, the absolute amount of contaminating autosomal DNA in relation to the male DNA is tremendously lower. This facilitates the analysis of the male DNA, e.g., to identify a perpetrator, as the ratio of male DNA compared to contaminating autosomal female DNA is markedly higher (see the results for Sample 3 and Sample 4).

[0214] Fig. 4 shows STR DNA profiles of a non-sperm fraction (NSF) and a sperm fraction (SF) subjected to a 1stand 2ndlysis of the non-sperm fraction.

[0215] The samples were subjected to a capillary electrophoresis (CE) on a ABI PRISM Genetic Analyzer 3500 XL and the STR- Alleles of the yellow channel was used for identification (see Table 2). DNA isolated from the NSF and the SF was amplified and the STR profiles were generated. For the creation of the STR profiles, 500 pg DNA was used for the sample “non-sperm fraction” and 80 pg as the maximal DNA-input in 15 pL for “sperm fraction, lst+2nd lysis”, calculated according to figure 3b.

[0216] Table 2: STR- Allele (extract yellow channel) of male and female donor profiles

[0217] The sample “non-sperm fraction” shows alleles that are all concordant with the female donor. The genetic profile of the female donor is the major component and additional alleles of the male donor, occur only below 200 RFU.

[0218] The sample “sperm fraction, lst+2nd lysis” shows alleles that are all concordant with the male donor; alleles of the female donor do not occur over the threshold of 50 RFU.

[0219] These results show that by conducting at least two lyses procedures on the nonsperm cells before lysing the sperm cells and nuclei 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.

[0220] Example 3

[0221] In this example different lysis agents were used by following the same procedure as in Example 1. It was shown that the use of various detergents in the lysis buffer may also lead to various amounts of remaining intact cells remaining after only one lysis which makes a second lysis necessary.

[0222] Figure 5 (A&B) shows the amount of the remaining DNA isolated from female cell samples after the sample was subjected to a first lysis followed by a DNase digestion (“after DNase digestion”) and after second lysis (“2nd lysis”), wherein different lysis buffers were used. Parts A and B show the same data, but the DNA yield is either plotted in a logarithmic scale (A) or linear scale with broken Y axis (B). Here again the linear scale better illustrates the tremendous differences regarding the remaining amount of autosomal DNA after DNase and 2ndlysis between the various used lysing agents (between 85.926 ng and 0.004 ng), which is more realistically shown in the absolute numbers of the linear scale than in the logarithmic scale.

[0223] Vaginal swabs containing epithelial cells (but no sperm cells) were subjected to a first and second lysis, wherein the lysis solution comprises the different lysing agents listed in Table 3.Table 3: Different lysing agents*Vol% based on the total value of the lysis solution.

[0224] Workflow with 1 st+ 2nd lysis of the sample

[0225] Vaginal swabs containing epithelial cells (but no sperm cells) were subjected to a first lysis step where the vaginal swab samples were mixed with 500 pL of a first lysis solution comprising 25 pl Proteinase K and an agent as listed in Table 1. The samples were incubated at 56°C for an hour at 900rpm and then 80°C for lOmin.

[0226] The samples were then transferred to a spin basket (e.g. QIAshredder-spin basket - QIAGEN) and subjected to centrifugation at full speed (15.000 rpm) for about 5 minutes, whereby a pellet comprising the intact (non-lysed) cells is obtained.

[0227] After removing the supernatant (470 pl of 500 pl) the mixture containing the cell pellet of intact / non-lysed cells and the remaining first lysate that couldn’t be removed without disturbing the pellet (about 30 pl) were subjected to DNase I digestion at 40°C for about 15 min to remove (digest) any DNA present in the mixture.

[0228] An aliquot of 50 pL of the mixture after DNase digestion was removed and the DNA present in the “after DNase digestion” fraction was purified using the EZ1&2 DNA Investigator Kit workflow (QIAGEN).

[0229] The “after DNase digestion” results indicated in Figure 5 show that the DNase digestion was indeed successful as only autosomal DNA amounts slightly above the limit of detection could be detected.

[0230] In the larger amount of the remaining mixture from the DNase digest after the aliquot was removed, the DNase enzyme was inactivated by subjecting the sample to heat treatment at 80 °C for about 10 min.

[0231] After the enzyme was inactivated, the sample (mixture comprising the intact (un-lysed) cells and liquid from the digest and the DNase inactivation) was subjected to a second lysis.

[0232] For the second lysis said sample comprising the intact (un-lysed) cells was mixed with a lysis buffer containing 25 pL Proteinase K (QIAGEN) and the same agent listed in Table 1 that was used in the first lysis. The sample was incubated at 56°C for about an hour. DNA present in the “2nd lysis” fraction was purified using the EZ1&2 DNA Investigator workflow (QIAGEN).

[0233] The DNA detected in the samples “2nd lysis” was not accessible for the DNase and is therefore expected to originate from cells which were not lysed in the first lysis step.

[0234] The amount of DNA that could be detected after the second lysis is supposed to represent in indicator of the efficiency of the lysis agent to lyse non-sperm cells. It is expected that a lysis agent having a reduced efficiency for lysing non-sperm cells will result in a higher amount of non-sperm cells contained in the mixture resulting from the first lysis. Thereby, also a higher amount of DNA should be obtained after the second lysis. Consequently, the lower the amount of DNA detected in the second lysate, the more efficient the used lysis agent should be in lysing non-sperm cells.

[0235] Quantification was performed using the Investigator Quantiplex Pro Kit (QIAGEN). For the digested samples the DNA yield was calculated for 50 pl.

[0236] Example 4

[0237] Ejaculate containing sperm cells was subjected to a lysis step where the sperm samples were mixed with 495 pL of a Tris-based lysis buffer comprising 0.4% SDS, 20 mM EGTA and 25 pL Proteinase K (QIAGEN) and incubated at 56°C for an hour at 900rpm. The sample was then subjected to centrifugation at full speed (15.000rpm) for about 5 minutes, whereby a pellet comprising the intact (non-lysed) sperm cells was obtained. Most of the supernatant lysate was removed by pipetting. Afterwards, the obtained cell pellet was washed three times.

[0238] For the first washing step, 500 pL of 10 mM Tris buffer was added to the cell pellet of intact / non-lysed cells and the remaining traces of the first lysate that couldn’t be removed by pipetting without disturbing the pellet (about 30 pl). The samples were then subjected to centrifugation at full speed (15.000rpm) for about 5 minutes, whereby a pellet comprising the intact (non-lysed) cells was obtained. Most of the supernatant lysate was removed by pipetting.

[0239] For the second washing step, 500 pL of 10 mM Tris buffer was added to the cell pellet of intact / non-lysed cells and the remaining traces of the first lysate that couldn’t be removed by the first washing step. The sample was then subjected to centrifugation at full speed (15.000rpm) for about 5 minutes, whereby a pellet comprising the intact (non-lysed) cells was obtained. Most of the supernatant lysate was removed by pipetting.

[0240] For the third washing step, 500 pL of 10 mM Tris buffer was added to the cell pellet of intact / non-lysed cells and the remaining traces of the first lysate that couldn’t be removed by the first and second washing step. The sample was then subjected to centrifugation at full speed (15.000rpm) for about 5 minutes, whereby a pellet comprising the intact (non-lysed) cells was obtained. Most of the supernatant lysate was removed by pipetting. It was expected that after said third washing step no or only negligible amounts of non-sperm DNA from the lysate were still present.

[0241] 100 pL of 10 mL Tris buffer and 3 pg of purified gDNA of a female donor was added to the cell pellet of intact / non-lysed cells and the remaining traces of the washing buffers and, if at all, of the first lysate that couldn’t be removed by the three washingsteps. The sample was then divided into three aliquots and either subjected to a digestion step or were not subjected to a digestion as specified in the following.

[0242] Samples of Fig. 6 named “RecBCD digestion” were subjected to a RecBCD digestion at 37°C for about 60 min to remove (digest) any DNA present and contaminating the cell pellet (predominantly the spiked-in female gDNA).

[0243] Samples of Fig. 6 named “DNasel digestion” were subjected to a DNasel digestion at 21 °C for about 15 min to remove (digest) any DNA present in the and contaminating the cell pellet (predominantly the spiked-in female gDNA).

[0244] Samples of Fig. 6 named “no digestion” were kept at room temperature for 60 min and therefore were not subjected to a digestion.

[0245] Afterwards, 440 pL of QIAGEN MTL buffer (Cat. No. / ID: 19112) were added to all samples and mixed thoroughly in order to inactivate the digestion enzymes. All samples were subjected to a sperm lysis by adding 36 pL of DTT and incubation at 56°C for about one hour at 900 rpm. All samples were purified using the EZ1&2 DNA Investigator Kit workflow (QIAGEN).

[0246] Following purification, DNA samples were quantified by qPCR on a PCR thermocycler (QIAGEN RotorGene Q Real-Time Systems using the “Investigator Quantiplex Pro RGQ Kit” (QIAGEN Cat. No. / ID: 387316). A maximum of 15 pL eluate was used for the PCR of the QIAGEN Investigator 24plex QS Kit (Cat. No. / ID: 382415). The amplified DNA was analysed by capillary electrophoresis. For the analysis the samples are diluted to max. 500pg input and applied to the CE (ABI PRISM Genetic Analyzer 3500 XL).

[0247] Results are shown in Figures 6 and 7.

[0248] The yield of autosomal DNA and male DNA recovered from the sperm-cell fraction of a mixed gDNA and sperm-cell containing sample without digestion and after digestion either with RecBCD or after digestion with DNasel is shown in Figure 6a. The DNA degradation ratio of autosomal DNA and male DNA recovered from the sperm-cell fraction of a mixed gDNA and sperm-cell containing sample withoutdigestion and either after digestion with RecBCD or after digestion with DNasel is shown in Figure 6b.

[0249] With reference to Figure 6, the three columns show information about the quantity (Fig. 6a) and integrity (Fig. 6b) of DNA isolated from an ejaculate sample after the sample was subjected to a non-sperm cell lysis and further steps. The lysis solution was suitable to essentially lyse the non-sperm cells and to form a lysate comprising non-sperm DNA, sperm cells and / or sperm nuclei, but the lysis solution did not cause a complete lysis of sperm cells and / or sperm nuclei. The further steps included three washing steps of the remaining cell pellet, addition of 3 pg of purified gDNA of a female donor, digestion with either RecBCD (“RecBCD digestion”) or DNasel (“DNasel digestion”) or without digestion (“no digestion”), as well as digestion inactivation, sperm lysis and subsequent DNA purification.

[0250] Figure 6a shows for the “no digestion” sample that a mixed sample comprising male and female DNA has been used as starting material. The male DNA came from the ejaculate that was subjected to selective lysis that led to a lysis of all cells that were not sperm cells. The in comparison huge amount of autosomal DNA resulted from the addition of 3 pg of female DNA that was added after the three washing steps. Figure 6a shows that digestion with RecBCD as well as DNasel nearly completely eliminated female gDNA by digestion. Digestion with RecBCD did not result in a reduction or loss of the amount of male DNA coming from isolated sperm cells. In contrast, digestion with DNase I resulted in a tremendous reduction and, thereby, loss of the male DNA coming from sperm cells, indicating that DNase I somehow penetrated the sperm nuclei and decomposed the sperm DNA in the sperm nuclei while not inducing sperm lysis.

[0251] Figure 6b shows the DNA degradation ratio of autosomal DNA and male DNA, as well as the degradation ratio threshold of 1 (black line). Ratios below 1 and equal 1 indicate intact DNA while a ratio greater than 1 indicates DNA degradation. The greater the DNA degradation ratio, the greater the DNA degradation. Fig. 6b shows that the comparison experiment with “no digestion” as expected did not show any degradation, either of autosomal or of male DNA. A digestion with RecBCD also indicated an almost perfect undegraded DNA of about 1 for both, the autosomal aswell as the male DNA. However, a digestion with DNase I degraded both, autosomal as well as male DNA.

[0252] Figure 6 shows that RecBCD digestion nearly completely eliminated female DNA but did not change either the quantity or the integrity of the sperm DNA. In contrast, DNase I digestion led to near complete digestion of female gDNA as well as male DNA and at the same time also to a moderate degradation of the sperm DNA. Therefore, it seems that DNase I inflicted degradation on sperm DNA inside sperm nuclei and sperm cells. In contrast, RecBCD digestion did not inflict DNA degradation to sperm DNA inside sperm nuclei and sperm cells.

[0253] Results of the STR analysis are shown in Figure 7.

[0254] The DNA isolated from the three sample treatments obtained as explained above was amplified and the STR profiles of the DNA obtained from the “no digestion” samples, the “RecBCD digestion” samples and the “DNase I digestion” samples. A maximum of 15 pL eluate was used for the PCR of the QIAGEN Investigator 24plex QS Kit (Cat. No. / ID: 382415). The amplified DNA was analysed by capillary electrophoresis (CE) on an ABI PRISM Genetic Analyzer 3500 XL and the STR- Alleles of the yellow channel were used for identification as outlined in Table 4.Table 4: STR- Allele (extract yellow channel) of male donor profile and female gDNA profile

[0255] An extract of the STR profiles of the isolated DNA is shown in Figure 7.

[0256] The “no digestion” graph only showed all alleles of the female reference DNA profile in the yellow channel. No alleles of the male reference profile were detected.

[0257] The “RecBCD digestion” graph showed all alleles of the male reference profile as the major component, as well as additional, lower alleles of the female reference profile as a minor component.

[0258] The “DNase I digestion” graph showed all alleles of the male reference profile as the major component, as well as additional, lower alleles of the female reference profile as a minor component.

[0259] This example demonstrates that both enzymes, RecBCD and DNase I, caused extensive elimination of female DNA but only the RecBCD enzyme did not inflict degradation on the sperm DNA and therefore did not change the sperm DNA integrity.

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 the biological sample comprising the sperm cells and / or sperm nuclei and the non-sperm cells to a first 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 first 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) optionally separating and / or removing the non-lysed cells and / or sperm nuclei from the first lysate comprising non-sperm DNA and / or decomposing or separating or removing or separating and removing the non-sperm DNA from the first lysate comprising non-sperm DNA, sperm cells and / or sperm nuclei, preferably by subjecting the non-sperm DNA in the first lysate to a nuclease digestion; c) subjecting the non-lysed cells and / or sperm nuclei to at least a second lysis by applying one or more lysing agents or a lysis solution to the non-lysed cells and / or sperm nuclei and / or incubating the non-lysed cells and / or sperm nuclei 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 non-sperm cells and to form a second or further lysate comprising non-sperm DNA, wherein the one or more lysing agents or the lysis solution do / does not comprise 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; d) decomposing or separating or removing or separating and removing the non-sperm DNA from the second or further lysate comprising non-sperm DNA, preferably by subjecting the non-sperm DNA in the second or further lysate to a nuclease digestion; ande) subjecting the non-lysed cells to at least a third or further lysis by applying a sperm cell and / or sperm nuclei lysis solution or one or more lysing agents to lyse sperm cells and / or sperm nuclei completely and to form a third or further lysate comprising sperm DNA, wherein 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 selected from a reduction agent, a sperm cell and / or sperm nuclei lysing enzyme, a chaotropic agent in a concentration suitable to lyse a sperm cell and / or sperm nuclei, and / or an alkaline agent in a concentration suitable to lyse sperm cells and or sperm nuclei or a combination thereof.

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 and 2, wherein method step c) has at least one of the following characteristics: i) the non-lysed cells are incubated in the lysate, the one or more lysing agents or the lysis solution of step a); ii) a lysis solution is applied to the non-lysed cells, and the lysis solution comprises the same agents in the same concentration and / or amounts as the lysis solution in method step a); iii) the lysing agents are not applied to the non-lysed cells in the form of a pre-prepared solution but directly; iv) the non-lysed cells are re-suspended before or during the lysis solution or the one or more lysing agents are applied; v) the lysis solution or the one or more lysing agents are applied to the lysate obtained from step a) without a further separation and / or removing of the non-lysed cells in step b); vi) the lysis solution or the one or more lysing agents are applied to the non-lysed cells after their separation and / or removal from the lysate in step b); vii) the lysis solution or the one or more lysing agents are different to the lysis solution or the one or more lysing agents in method step a) in at least one agent and / or at least one agent concentration or amount. viii) the lysis solution or the one or more lysing agents are applied to the lysate obtained from step a) after the non-sperm DNA present in the first lysate is decomposed or separated or removed or separated and removed from the first lysate in step b).

4. The method of claims 1 to 3, wherein separating, removing, or separating and removing the non-lysed cells and / or sperm nuclei occurs through centrifugation, preferably by forming a pellet of the non-lysed cells and / or sperm nuclei, through filtration, through binding to a solid matrix, preferably to a membrane or beads, preferably to magnetic beads.

5. The method of claims 1 to 4, further comprising after method step b) and / or d) at least one washing step of the non-lysed cells and / or sperm nuclei and / or cell pellet.

6. The method of claims 1 to 5, wherein the lysis solution or the one or more lysing agents in steps a) and / or c) 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, nuclease, such as DNase, Benzonase(R), RNase, endonucleases, and exonucleases; or any combination thereof.

7. The method of claim 6, wherein the one or more surfactants 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 / or 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. vii) the anionic surfactant is selected from the group consisting of 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; viii) the non-ionic surfactant is selected from the group consisting of Triton X-100, Triton X-l 14, N-octylglucoside, Tween 20 (polysorbate 20, polyoxyethylene (20) sorbitan monolaurate), Tween 40, Tween 80, Span 80, Span 85, Ecosurf EH-6, Brij-35(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.

8. The method of claims 1 to 6, wherein the chaotropic agent is selected from the group consisting of sodium iodide, sodium perchlorate, guanidinium hydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate, urea and / or a mixture of two or more salts thereof.

9. The method of claims 1 to 8, wherein the lysis solution or the one or more lysing agents applied in method step c) are free of nuclease inhibiting agents, preferably free of chelating agents, such as ethylenediaminetetraacetic acid (EDTA), and / or free of anionic detergents, such as sodium dodecyl sulfate (SDS), sarkosyl, deoxycholate, sodium laureth sulfate (SLS), sodium octyl sulfate (SOS), sodium methyl cocoyl taurate and disodium coco sulfosuccinates, or mixtures thereof.

10. The method of claims 1 to 9, wherein the nuclease digestion in method step b) and / or d) is performed by one or more nucleases comprising 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, EndonucleaseG, 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.

11. The method of claim 10, wherein the nuclease has at least one of the subsequent features: i) it has a size of 50 kDa or above; ii) it has a size of 60 kDa, 80 kDa, 100 kDa, 150 kDa, 200 kDa, 25 OkDa, 300 kDa, 350 kDa or preferably of 400 kDa 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 selected such that it does not get access to the packed DNA. xiii) the nuclease is chosen such that it digests and / or decomposes non-sperm DNA but not sperm DNA; xiv) the nuclease can recognize and be altered by the Chi DNA sequence.

12. The method of claims 1 to 11, wherein the sperm lysing agent or lysing agents contain a reduction agent selected from the group consisting of dithiothreitol (DTT), Dithioerythrit, P-mercaptoethanol (BME), glutathione (GSH), Tris(2-carboxyethyl)phosphine (TCEP) andcombinations 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 sperm cells and or sperm nuclei; or a combination thereof.

13. The method of claim 1, further comprising purifying, isolating, detecting, analysing and / or quantifying the non-sperm DNA and / or the sperm -DNA.

14. The method of claim 13, 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.

15. The method of claim 1, wherein one or more steps are automated.

16. A kit comprising one or more of: a) a first 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 second and / or further non-sperm cell lysis solution or one or more non-sperm cell lysing agents capable of lysing non-sperm cells and not suitable for completely lysing sperm cells and / or sperm nuclei, wherein the second and / or further non-sperm cell lysis solution or the one or more non-sperm cell lysing agents is / are identical to the first non-sperm cell lysis solution or the one or more non-sperm cell lysing agents used in a), or wherein the second and / or further non-sperm cell lysis solution or the one or more non-sperm cell lysing agents is / are different from the first non-sperm cell lysis solution or the one or more non-sperm cell lysing agents used in a); c) optionally, one or more washing compositions; d) optionally one or more DNA digesting enzymes; 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 thesperm 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 sperm cells and / or sperm nuclei; or any combination thereof.

17. A method for treatment of a biological sample comprising sperm cells and / or sperm nuclei and non-sperm cells, the method comprising: i) subjecting the biological sample comprising the sperm cells and / or sperm nuclei and the 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 first 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; ii) decomposing most or all of the non-sperm DNA obtained from the lysate in step i) using an enzyme having a helicase and an exonuclease activity and / or having a size of above 50 kDA while not or not markedly decomposing sperm DNA; and iii) subjecting the non-lysed cells to a second lysis by applying a sperm cell and / or sperm nuclei lysis solution or one or more lysing agents to lyse sperm cells and / or sperm nuclei completely and to form a second or further lysate comprising sperm DNA, wherein 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 selected from a reduction agent, a sperm cell and / or sperm nuclei lysing enzyme, a chaotropic agent in a concentration suitable to lyse a sperm cell and / or sperm nuclei, and / or an alkaline agent in a concentration suitable to lyse sperm cells and or sperm nuclei or a combination thereof.

18. Use of a nuclease having an exonuclease and helicase activity and / or having a size of above 50 kDA for the decomposition of non-sperm DNA in a sample comprising a mixture of non-sperm DNA and sperm DNA, the sperm DNA being contained in sperm cells and / or sperm nuclei.

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