Systems and methods for rapid nucleic acid extraction, purification and analysis - Patents.com

Abstract

A method and kit for rapid nucleic acid extraction from nucleic acid-containing materials such as bone, tooth, or semen samples are disclosed. For bone and teeth, the method includes providing the nucleic acid-containing material in a form suitable for nucleic acid extraction, adding a lysis buffer to the nucleic acid-containing material to obtain a mixture, mixing the mixture for about 30 seconds or more by equivalent methods, and then separating the mixture by centrifugation to obtain a liquid supernatant. The liquid supernatant contains extracted nucleic acids that can be used for analysis, including STR profiling by conventional or rapid DNA analysis. For semen, the method and kit include applying an appropriate amount of a sperm disruption agent.

Description

[Technical field]

[0001] The present invention relates to systems and methods for nucleic acid extraction, purification and analysis. More particularly, in one embodiment, the present invention relates to the rapid purification and analysis of nucleic acids from samples, including bone, tooth and semen samples obtained from forensic, missing persons and mass casualty settings. [Background technology]

[0002] In forensic science, a primary objective is to identify sources of biological evidence such as bones, teeth, blood, semen, saliva, and hair found at scenes of crimes, terrorist activities and attacks, as well as mass disaster sites. DNA identification has provided the means to identify the source of such material with a very high degree of certainty. In the forensic science community today, most DNA identification methods are based on the amplification of small regions of the human genome that contain repeats of a class of DNA stretches known as short tandem repeats (STRs). The unit length of a given STR typically ranges from 2 to 10 base pairs, and STRs are generally found within non-coding and flanking sequences, and occasionally within coding regions. There are hundreds of thousands of STR loci in the human genome, which occur on average every 6 to 10 kb, and appear to be highly polymorphic. The number of repeats at a given STR site or locus in the genome is characteristic of an individual's cells, and by determining the number of repeats at several STR loci, a unique "DNA fingerprint" (also called a "STR profile" or "DNA ID") of an individual can be generated.

[0003] STR profiles are generated by a series of three basic methods. First, DNA is purified from cells on a sample collection device or substrate (e.g., a swab). This involves opening the cells to liberate the DNA, then removing proteins, other biomolecules, and cellular debris to generate a purified or partially purified DNA solution. Second, a set of STR loci is replicated (amplified) using a method known as polymerase chain reaction (PCR). Primers that bind to the target STR gene regions are labeled with multiple fluorescent dyes so that amplified fragments containing the primers can be detected by laser-induced fluorescence. A large number of dyes (over 50) are available for use in fluorescence excitation applications. These dyes include those from the fluorescein, rhodamine AlexaFluor, Biodipy, coumarin, and cyanine dye families. In addition, quenchers are also available to label oligonucleotide sequences to minimize background fluorescence. Dyes with emission maxima between 410 nm (Cascade Blue) and 775 nm (Alexa Fluor 750) are available and can be used. Dyes in the 500-700 nm range have the advantage of being in the visible spectrum and can be detected using conventional photomultiplier tubes. The wide range of available dyes allows the selection of a dye set with emission wavelengths spanning the detection range. Detection systems capable of discriminating between multiple dyes have been reported for flow cytometry applications (see Perfetto et al., Nat. Rev. Immunol. 2004, 4, 648-55; and Robinson et al., Proc of SPIE 2005, 5692, 359-365).

[0004] Fluorescent dyes have peak excitation wavelengths that are typically 20-50 nm blue-shifted from their peak emission wavelength. As a result, the use of dyes over a wide range of emission wavelengths may require the use of multiple excitation sources with excitation wavelengths that achieve efficient excitation of the dyes across the emission wavelength range. Alternatively, energy transfer dyes can be utilized to allow a single laser with a single emission wavelength to be used to excite all dyes of interest. This is accomplished by attaching an energy transfer moiety to the dye label. This moiety is typically another fluorescent dye with an absorption wavelength that is compatible with the excitation wavelength of the light source (e.g., laser). The placement of this absorber in close proximity to the emitter allows the absorbed energy to be transferred from the absorber to the emitter, allowing for more efficient excitation of the longer wavelength dyes (Ju et al., Proc Natl Acad Sci USA 1995, 92, 4347-51). A fourth dye is used as a standard marker for sizing STR fragments.

[0005] Third, the size of the replicated STR fragments is determined by electrophoresis. The STR fragments are carried by an electric current through a gel-like material, with smaller fragments moving faster through the gel than larger fragments. A laser is used to excite four fluorescent dyes at the detection window. The dyes emit light, which is then detected and used to determine the size of each STR fragment. An expert system, either automated or manually operated, analyzes the size patterns and generates a final STR profile--the DNA fingerprint of the individual. Note that the basic three-step DNA fingerprint generation can be modified; for example, cells in the sample may be lysed and the resulting cell extract may be subjected to amplification (without DNA purification). Similarly, the amplified DNA fragments can be identified by methods including mass spectrometry.

[0006] STR analysis was first applied in the early 1990s and has since become a major tool in the forensic science facility, with a growing set of applications including law enforcement, parentage, mass casualty, immigration and human identification in sexual assault cases, unclaimed minor (child) vindication and human trafficking. In the United States, STR profiles generated from selected human samples are collected in the Combined DNA Index System (CODIS). This electronic database was established by the FBI in 1997 and standardized to a set of 13 tetramer STR loci for data submission, and in January 2017, the number of core loci available in the FBI database was expanded to 20. Other STR databases outside the United States contain overlapping subsets of the CODIS loci. To generate a DNA profile, the first and crucial step is obtaining DNA from the sample of interest.

[0007] Forensic samples present unique challenges. The invention presented herein provides a solution for bone, tooth and semen samples, which are among the most difficult to process in a forensic laboratory. For bone and teeth, there is no universal protocol that allows extraction of DNA from materials at different stages of degradation. The efficiency of DNA extraction, the level of desalting, the issue of low content of DNA in the sample, and the degree of contamination with inhibitors all pose significant problems. At least two types of semen samples are relevant. First, semen stains containing a relatively pure population of sperm can be found, for example, on clothing items or exposed skin of rape victims. Second, Sexual Assault Kit samples (SAKs) often include vaginal or cervical swabs - such swabs can contain a mixture of vaginal and cervical cells (including vaginal and cervical epithelial cells) as well as sperm. In addition, both semen sample types can contain cells from more than one man, whether from a consensual partner or an additional sexual assailant. Also, separation of male and female cells and deconvolution of the mixture presents challenges.

[0008] A related challenge concerns the development and use of processing protocols, including processing protocols for rapid DNA identification. Processing protocols are critical in many different fields. Processing protocols are created and followed to ensure that similar sample types are processed in a similar manner. The use of such protocols helps provide consistent processing results and ensures that, if the results are inconsistent, the differences are not due to variations caused by the processing itself. For STR analysis, certain protocols must be followed to remain within compliance with federal regulations as well as a matter of sheer scientific principle. Protocols developed for manual processing or not for field-forward use or rapid processing may not be fully suitable for forensic use, including field-forward forensics. The inventors herein present novel inventive protocols that solve the unique challenges presented by the need for automated processes, fast protocols, and forensic samples.

[0009] A major challenge remains in the efficient purification of nucleic acids from certain biological evidence such as bone. Bones are a good source of nucleic acids under scenarios where the biological evidence is exposed to various environmental conditions because the bone matrix protects the nucleic acids from degradation. Current methods of nucleic acid extraction from bone are generally cumbersome, lengthy, and inefficient in that they require hours or even days to obtain nucleic acids of suitable quality for analysis. Bone and tooth purification protocols are based on the extraction and purification of DNA from bone or tooth powder comminuted by cryo-grinding or blending cup (Loreille et al., FSIG 1 (2007), 191-195; Johnston and Stephenson, JFS 61 (2016), 898-902; Turingan et al., Inv. Genetics 7 (2016), bone slices generated using a rotary dental saw or its equivalent (Kitayama et al., Legal Med 12 (2010), 84-89) and related protocols. The need to start the process with a powder requires cumbersome equipment and limits the technique to sophisticated laboratories. In addition, powdering devices and tooth-cutting disks can cause the generation of airborne biological samples, with attendant health risks to the operators as well as the attendant risks of inter-sample contamination. Furthermore, extensive demineralization of bone powder or bone slices often requires overnight demineralization at high temperatures including agitation (e.g., Lee et al., 2013). Forensic Science International: Genetics, 4 (2010) 275-280), is time-consuming, and requires sophisticated equipment. Finally, the prior art protocols rely on a large volume of desalting buffer relative to the amount of bone powder to be desalted and rely on a complex reagent mix. The large volume of desalting buffer requires concentration (often using a concentration membrane) to obtain a suitable volume for subsequent processing (e.g., purification) and analysis. Concentration is yet another step that adds time and complexity to the overall method.Taken together, it is no wonder why the time to generation of STR profiles from bone is a major issue in law enforcement forensic laboratories, medical examiner offices, and coroner's offices.

[0010] To date, STR analysis of bone and tooth powders has been performed in conventional laboratories. The investment in labor, machine use, and time has dramatically limited the utility of this approach. Many forensic laboratories send bone and tooth processing to highly specialized laboratories, which can take a year or more to return DNA fingerprint results, and have limited the ability to do even that. As a result, it is estimated that 40-80,000 unidentified bodies are now in the hands of medical examiners and coroners, many of which will be buried or cremated before they can be identified. Similarly, even in relatively minor disasters such as airplane crashes, it can take years for body parts to be identified by forensic laboratories. In the case of large mass disasters, bodies may remain unidentified for years or even decades. The 2004 Indian Ocean earthquake and tsunami is an example of a disaster - thousands of victims remain unidentified after more than a decade.

[0011] Both conventional and rapid DNA analysis systems would benefit greatly from faster, simpler and more efficient processing of samples of interest. The method and kit in the present invention are surprising in the following aspects. First, in the present invention, the need to generate bone or tooth powder is eliminated. Unexpectedly, a few blows of a hammer or mortar and pestle or brick (or similar material) break the sample into small pieces enough for further processing. The surface area generated for demineralization by hammering creates a sufficient surface area to allow efficient demineralization while surprisingly avoiding the generation of airborne bone particles. Second, the process of demineralization of bone samples takes less than 60 minutes, with 10 minutes being generally applicable in many cases, and only 1 minute being sufficient. For teeth, the process of demineralization takes about 180 minutes, with 10 minutes being sufficient in many cases. Third, prior art protocols require overnight demineralization to extract nucleic acids from bone powders and sections, and often include a heating step that can damage DNA. See Higgins et al., Forensic Sci Med Pathol 2014, 10:56-61; Amory et al., Forensic Science International: Genetics 6 (2012) 398-406; Loreille et al., Forensic Science International: Genetics 1 (2007) 191-195; and Lee et al., Forensic Science International: Genetics 4 (2010) 275-280. In the Loreille method, total lysis of bone powder using a large volume of buffer was required for analysis, and sample concentration was required followed by organic extraction for purification of DNA. Thus, the Loreille method requires excessive sample handling and time, both of which limit the process to sophisticated and well-equipped laboratories, dramatically limiting laboratory throughput. Higgins is a modification of the Loreille method that uses a lower volume assay, but still requires overnight incubation and laborious purification.In addition, the protocol requires the use of an additional reagent, carrier RNA, and includes additional processing steps. Carrier RNA is taught to be added to increase DNA yield, however, its addition does not result in enhanced DNA recovery. The Lee method uses a larger number of reagents and is more complicated than either Loreille or Higgins. Fourth, the standard analytical method uses large volumes of desalting buffer, which makes the processing of the solution and subsequent DNA concentration and purification more challenging for conventional rapid DNA analysis. Taken together, none of these methods are suitable for field-forward rapid DNA identification.

[0012] And fourth, the method of the present invention uses specialized reagents, including desalting reagents. In the present invention, the desalting solution contains only a minimal EDTA buffer volume and includes rapid desalting prior to downstream purification from bone and STR profile generation. The inventors show unexpected results herein, including that the desalting solution, which may contain lysis buffer / detergent and proteinase K, does not need to contain a high initial volume (approximately 15 ml for complete lysis of 1 g of sample). Prior art methods using lysis buffer / detergent and high initial volumes of EDTA teach the use of long incubation steps followed by DNA purification after sample concentration. The inventors show the unexpected discovery that these steps are not necessary. Similarly, prior art protocols for DNA extraction from desalted bone and teeth include organic extraction methods. Prior art protocols for isolating nucleic acids from semen samples require the use of complex reagents and long processing steps necessary to isolate and lyse cells from the mucus environment in which they are normally found, including centrifugation, washing and incubation steps. The inventors herein present new and unexpected protocols for isolating nucleic acids from bone, tooth and semen samples that dispel conventional knowledge.

[0013] The simplified solutions and protocols of the present invention not only reduce reagent and raw material costs, but also substantially reduce processing times and the potential for sample loss during extensive manipulation. Indeed, the present invention is based, in part, on the unexpected discovery that there is no need for complete dissolution of the sample for total desalting involving longer incubations and high buffer volumes to generate a callable STR profile. The solutions and protocols of the present invention are easily adapted for use in integrated biochip and instrument systems capable of sample-in to result-out processing.

[0014] The solutions and protocols of the present invention can extract nucleic acids in sufficient quantities to generate STR profiles that can be uploaded to the CODIS database. For example, from 15 fresh bone samples using the protocols of the present invention, the inventors obtained nucleic acid yields of 4.2ng to 108.1ng per mg of bone. In addition, from 12 fresh tooth samples, the inventors obtained nucleic acid yields of 0.1ng to 2.7ng per mg of tooth root. Finally, the solutions and protocols of the present invention can be adapted for use in high and low DNA content integrated Rapid DNA Identification systems (using A-chip and I-chip, respectively), including sample-in to result-out processing. Also, automated data processing and automated expert system analysis can be incorporated into the rapid DNA identification systems.

[0015] Regarding protocols for DNA extraction from semen, faster, simpler, and more efficient methods have been developed that can be utilized for conventional rapid DNA analysis systems. Standard DNA purification methods for somatic cells are ineffective in lysing sperm cells due to their high degree of nuclear compaction and the presence of abundant protective membranes on disulfide bonds. Modifications to standard lysis methods include the addition of strong reducing agents to disrupt these disulfide bonds that impede lysis. These alternative methods rely primarily on lengthy chaotropic digestion with proteinase K at high temperatures and incubation with reducing agents such as dithiothreitol (DTT) to allow for the consequent isolation and purification of DNA. A rapid method of mammalian sperm DNA isolation has also been performed using steel beads and the addition of tris(2-carboxyethyl)phosphine (TCEP) instead of DTT for 5 minutes of homogenization at room temperature in the presence of a chaotropic lysis buffer (Wu et al. Biotechniques. 2014; 58(6): 293-300). This method substantially reduces time and complexity by eliminating heating and slow incubation, and uses an odorless sperm disruption agent, but mechanical bead homogenization techniques are not always available and still require highly skilled personnel, sophisticated equipment and cleanup / bead isolation for downstream processing. In short, a laboratory environment. In the present invention, semen samples are collected on swabs (prepared from neat semen, dried semen on fabric, carpet, skin, tile or other carriers, or isolated sperm from sexual assault kits), and then a sperm disruption agent (e.g., 150 mM DTT or 50 mM TCEP) is simply added. Incubation of the semen on the swab at room temperature for 10 minutes, and in many instances as little as 1 minute, was sufficient to generate a DNA fingerprint using the ANDE Rapid DNA System.

[0016] In sexual assault kits, vaginal swabs often contain cells from more than one contributor. Often the swab will contain cells from the female victim and the male perpetrator. Therefore, to avoid "mixed" DNA IDs (where DNA IDs from more than one individual are present), sperm may be separated from the vaginal epithelial cells; processing to achieve this separation is necessary prior to DNA extraction and / or purification steps. The differential lysis method was described by Gill and colleagues in 1985 and relies on separating intact sperm from the DNA of lysed epithelial cells. Although some modifications to the method have been made over the years to reduce time and improve efficiency, the basic concept and method have remained unchanged. Differential extraction is time consuming, laborious, and requires sophisticated laboratories and highly skilled personnel. Physical separation of intact sperm cells from the lysed female fraction is achieved by centrifugation and repeated washing of pelleted sperm cells. An alternative method eliminates the time-consuming wash steps by selective degradation of soluble DNA using nuclease enzymes (Garvin et al. Investigative Genetics 2012 3:25). A commercially available kit, Sperm Erase (Paternity Testing Corporation), utilizes the latter approach. Processing of SAK using prior art methods requires 2-5 hours from initial epithelial cell lysis to generation of the sperm fraction.

[0017] The present invention unexpectedly provides a rapid method for lysing epithelial cells that can be performed in 10 seconds at room temperature; previous approaches require at least 1 hour and typically require heating. Furthermore, the present invention provides a method that efficiently combines both physical separation and chemical degradation techniques to allow for effective isolation and purification of sperm cell DNA while reducing overall processing time.

[0018] In one method of the present invention, the sperm pellet is washed once with a wash buffer optimized for nuclease activity. The elimination of repeated washing steps not only improves time but also reduces potential sperm cell loss. The wash buffer is stable at room temperature and prepares the reaction for the addition of nuclease, thus resulting in fewer reagents in the SAK processing kit. The nuclease degrades any soluble DNA, and then a stop solution containing 0.5M EDTA is added to inactivate the enzyme. To allow lysis of the sperm cells, a sperm disrupting agent (with a final concentration of about 150 mM DTT or 50 mM TCEP) is added to the mixture prior to collecting the liquid with a swab for rapid DNA analysis. The generation of the male fraction was completed in 32 minutes. The reduction in the centrifugation time for the initial separation and the reduction in the time to quench the activity of the nuclease allowed the generation of the sperm fraction for analysis in about 22 minutes.

[0019] Furthermore, the method steps have been simplified so that they can be performed by non-specialized personnel. In fact, Sexual Assault Nurse Examiners, Forensic nurses, or other hospital personnel can perform the work outside of the forensic laboratory. If the work is performed in the hospital (even in the hospital laboratory), the transportation time to the police station / forensic laboratory is eliminated. Taken together, the teachings of the present invention result in the rapid generation of all actionable DNA ID results. By generating results quickly, law enforcement investigations can proceed more quickly, leading to doubts about the apprehension and exoneration of innocent people. Since many criminals are repeat offenders (e.g., a typical sexual assaulter commits more than 10 rapes before being arrested), it can be expected that the teachings herein will dramatically reduce crime and improve public safety. Summary of the Invention

[0020] In a first embodiment, the inventors provide a method for extracting nucleic acids from a bone or tooth sample, the method comprising: providing a sample in a form suitable for nucleic acid extraction; adding a desalting buffer to the nucleic acid-containing material to obtain a mixture; vigorously mixing the mixture; and then separating the mixture to obtain a liquid supernatant; wherein the liquid supernatant contains the extracted nucleic acids. In a related embodiment, the separation step does not require concentrating the nucleic acids and does not result in complete dissolution of the bone sample as a result of desalting.

[0021] In another embodiment, the inventors provide a method for nucleic acid extraction from a bone or tooth sample, the method comprising: providing a sample in a form suitable for nucleic acid extraction; adding less than 500 μl of 0.5 M EDTA or an equivalent desalting buffer to the nucleic acid containing material to obtain a mixture; vigorously mixing the mixture; incubating the mixture for at least 1 minute without applying heat; and separating the mixture to obtain a liquid supernatant without concentration, wherein the liquid supernatant is characterized as containing extracted nucleic acid.

[0022] In yet another embodiment, the inventors provide a method for determining a STR profile of nucleic acids from a nucleic acid-containing material, the method comprising: providing the nucleic acid-containing material in a form suitable for nucleic acid extraction; adding a desalting buffer to the nucleic acid-containing material to obtain a mixture; vigorously mixing the mixture; separating the mixture to obtain a liquid supernatant, wherein the liquid supernatant contains the extracted nucleic acids; and then subjecting a portion of the liquid supernatant to nucleic acid analysis to determine the STR profile of nucleic acids from the nucleic acid-containing material.

[0023] In another embodiment, the inventors provide a method and kit for determining the STR profile of nucleic acid from a semen sample, said method and kit comprising: collecting the semen sample on a transport medium; and applying an appropriate amount of a sperm disrupting agent onto the transport medium. In a related embodiment, the transport medium can be swabbed into a rapid DNA analysis system or processed for STR analysis.

[0024] In another embodiment, a method and kit for isolating female epithelial cells from sperm cells in a post-coital vaginal swab is characterized in that it includes lysing the epithelial cells from the transport medium; physical separation of intact sperm cells from the lysed epithelial cells; removal of the female aqueous fraction from the intact sperm cells; washing the pelleted intact sperm cells with a wash buffer; degrading any soluble DNA by nuclease treatment; inactivating the nuclease, lysing the sperm cells with a sperm disrupting agent (e.g., DTT or TCEP, other chemicals or physical methods); and collecting the lysed sperm fraction.

[0025] In yet another embodiment, the inventors provide a method for extracting nucleic acids from a sample suspected of containing sperm, the method comprising: inserting the sample into a sample container containing a first lysis solution and agitating to create a first mixture; centrifuging the first mixture; washing the pellet in the first container with a buffer; subjecting the first container to a second centrifugation to repellet any sperm present in the sample; transferring at least a portion of the sperm to a second container containing a nuclease in an amount and under conditions sufficient to degrade DNA; adding a sperm disruption agent; and recovering a disrupted sperm fraction for further processing.

[0026] As will be apparent to one of skill in the art, the inventive methods of nucleic acid extraction, purification and analysis result in an output suitable for further analysis, including, but not limited to, downstream processing in an integrated biochip. The specific examples mentioned in this Summary are not intended to limit the scope of the claims of this application or any related or unrelated application. Other aspects, embodiments, modifications to the claimed invention and features of that invention will be apparent to one of skill in the art in view of the disclosure herein. [Brief description of the drawings]

[0027] [Figure 1] Figure 1 shows the STR profile of all 16 loci of nucleic acid purified from 200 mg of frozen ground bone powder of a fresh femur sample. The sample was desalted for 1 min and analyzed on an A-chip. [Diagram 2] Figure 2 shows the STR profile of all 16 loci of nucleic acid purified from 10 mg of frozen ground bone powder of a fresh femur sample. The sample was desalted for 1 min and analyzed on an I-chip. [Diagram 3] Figure 3 shows the STR profile of all 16 loci of nucleic acid purified from 500 mg of frozen ground bone powder from an artificially aged femur sample. The bone was immersed in 84°F salt water for 4 days, cleaned and dried, demineralized for 1 min, and then analyzed on an I-chip. [Figure 4] Figure 4 shows the STR profile of all 16 loci of nucleic acid purified from 200 mg of frozen ground bone powder from an artificially aged femur sample. The bone was immersed in 84°F salt water for 4 days, cleaned and dried, demineralized for 1 min, and then analyzed on an I-chip. [Diagram 5] Figure 5 shows the STR profile of all 16 loci from nucleic acid purified from 500 mg of crushed bone fragments of an artificially aged and degraded femur sample. The bone was immersed in 84°F salt water for 4 days, cleaned and dried, demineralized for 1 minute, and then analyzed on an I-chip. [Figure 6] Figure 6 shows the STR profile of all 16 loci of nucleic acid purified from 200 mg of crushed bone fragments of an artificially aged and degraded femur sample. The bone was immersed in 84°F salt water for 4 days, cleaned and dried, demineralized for 1 minute, and then analyzed on an I-chip. [Figure 7] Figure 7 shows the STR profile of all 16 loci of nucleic acid purified from 500 mg of frozen ground bone powder of an artificially aged femur sample using the nucleic acid extraction method reported by Loreille et al. Forensic Science International: Genetics 1 (2007) 191-195. The bone was immersed in 84°F salt water for 4 days, cleaned and dried, then analyzed on an I-chip. [Figure 8]Figure 8 shows the STR profile of all 16 loci of nucleic acid purified from 200 mg of freeze-ground root of a fresh molar sample. The sample was desalted for 10 minutes and analyzed on an I-chip. [Figure 9] FIG. 9 shows the STR profile of all 16 loci of nucleic acid purified from dried semen stains on denim and analyzed on an I-chip. [Figure 10] FIG. 10 shows the STR profile of all 16 loci of nucleic acid purified from dried semen on cotton and analyzed on an I-chip. [Figure 11] Figure 11 shows the STR profile of all 27 loci of nucleic acid purified from 5 mg of frozen ground bone powder of a fresh femur sample. The sample was desalted for 1 min and analyzed on an A-chip. [Figure 12] Figure 12 shows the STR profile of all 27 loci of nucleic acid purified from a 5 mg crushed bone fragment of a fresh femur sample. The sample was desalted for 1 min and analyzed on an A-chip. [Figure 13] Figure 13 shows the STR profile of all 27 loci of nucleic acids purified from 5 mg of frozen ground bone powder of a fresh femur sample and using only 15 μl of the 120 μl bone demineralized solution for analysis in the I-chip. The sample injection is theoretically equivalent to 0.6 mg of sample. The sample was demineralized for 1 min. [Figure 14] Figure 14 shows the STR profile of all 27 loci of nucleic acid purified from 5 mg crushed bone fragments of a fresh femur sample and using only 15 μl of 120 μl bone demineralized solution for analysis in the I-chip. The sample input is theoretically equivalent to 0.6 mg of sample. The sample was desalted for 1 min. [Figure 15] Figure 15 shows the STR profile of all 27 loci of nucleic acid purified from 5 mg of crushed fresh distal phalanx sample and using only 15 μl of 120 μl of bone demineralized solution for analysis on an I-chip. The sample injection is theoretically equivalent to 0.6 mg of sample. The sample was desalted for 1 min. [Figure 16]Figure 16 shows the STR profile of all 16 loci of nucleic acid purified from 200 mg of freeze-ground fresh tooth root (not subjected to any form of degradation). The tooth was desalted for 1 min and analyzed on an I-chip. [Figure 17] Figure 17 shows the STR profile of all 16 loci of nucleic acid purified from 200 mg of freeze-ground root of an artificially aged molar sample. The tooth was immersed in 84°F salt water for one month, cleaned, then dried, after which the root was separated from the crown, demineralized for 10 minutes, and then analyzed on an I-chip. [Figure 18] Figure 18 shows the STR profile of all 16 loci of nucleic acid purified from 200 mg of pulverized root of a forensic molar sample. The tooth was found to have been buried in soil for at least 2 years, and after cleaning and drying, the root was separated from the crown, demineralized for 180 minutes, and then analyzed on an I-chip. [Figure 19] Figure 19 shows the STR profile of all 27 loci of nucleic acid purified from 10 mg of crushed root pieces of fresh molar samples. Samples were desalted for 10 min and analyzed on an I-chip. [Figure 20] Figure 20 shows the STR profile of all 27 loci of nucleic acid purified from 10 mg of crushed root fragments of a fresh incisor sample. Samples were desalted for 10 minutes and analyzed on an I-chip. [Figure 21] Figure 21 shows a partial STR profile (24 of 27 loci) of nucleic acid purified from 200 mg of crushed root pieces of a timed molar sample. The tooth was removed from the body, placed on field ground for 120 days, cleaned, dried, and the root was separated from the crown, demineralized for 180 minutes, and then analyzed on an I-chip. [Figure 22]Figure 22 is a plot showing the average amount of DNA (in ng) purified as a function of the volume (in microliters) of desalting buffer added to a 500 mg bone powder sample. Data are from quadruplicate determinations and error bars represent 1 STDEV. This data led the inventors to consider that less buffer could be used with less bone (or tooth) as starting material. [Diagram 23] Figure 23 is a time course data showing bone demineralization as measured by purified DNA (in ng) as a function of time. The data shows that overnight demineralization is not necessary for STR analysis. One minute of demineralization is sufficient for STR analysis using the inventive compositions and methods of the present invention. [Figure 24] Figure 24 is a plot showing the average amount of DNA (in ng) purified using EDTA with and without detergent. This data is a comparison of simplified desalting buffers that can be used in the present invention. The data also shows that a simple 0.5M EDTA buffer is sufficient to desalt bone and obtain sufficient amounts of DNA for STR analysis. [Diagram 25] FIG. 25 shows the 32-minute protocol developed for processing a sexual assault kit. [Figure 26] Figure 26 shows a 22 minute protocol developed for processing sexual assault kits. The centrifugation time to separate intact sperm cells from the lysed epithelial fraction was reduced from 5 minutes to 2 minutes at 20,000 x g, and the incubation time for the stop solution to inactivate nucleases was reduced from 10 minutes to 3 minutes at 56C. [Figure 27] FIG. 27 lists the components of the SAK kit based on the protocol developed. [Figure 28]Figure 28 shows male STR profiles for all 27 loci of nucleic acid purified from the male fraction isolated from vaginal swabs collected 24 hours post-coitus (donor couple A / B) using the 32 minute protocol. Male genotypes are indicated by blue arrows based on processing of the male buccal reference swab. Female genotypes are indicated by red arrows based on processing of the female buccal reference swab. [Figure 29] Figure 29 shows male STR profiles for all 27 loci of nucleic acid purified from the male fraction isolated from vaginal swabs collected 48 hours post-coitus (donor couple A / B) using the 32 minute protocol. Male genotypes are indicated by blue arrows based on processing of the male buccal reference swab. Female genotypes are indicated by red arrows based on processing of the female buccal reference swab. [Diagram 30] Figure 30 shows male STR profiles for all 27 loci of nucleic acid purified from the male fraction isolated from vaginal swabs collected 72 hours post-coitus (donor couple A / B) using the 32 minute protocol. Male genotypes are indicated by blue arrows based on processing of the male buccal reference swab. Female genotypes are indicated by red arrows based on processing of the female buccal reference swab. [Diagram 31] Figure 31 shows a partial male STR profile (24 of 27) of nucleic acid purified from the male fraction isolated from vaginal swabs (donor couple C / D) collected 72 hours post-coitus using the 32 minute protocol. The STR profile is missing alleles for D2S441, D10S1248 and TPOX. Male genotypes are indicated by blue arrows based on processing of the male buccal reference swab. Female genotypes are indicated by red arrows based on processing of the female buccal reference swab. [Diagram 32]Figure 32 shows a partial 27 locus male and female mix (female less) STR profile of nucleic acid purified from male fraction isolated from vaginal swabs (donor couple E / F) collected 72 hours post-coitus using the 32 minute protocol. The male STR profile is missing alleles for vWA. Male genotypes are indicated by blue arrows based on processing of male buccal reference swabs. Female genotypes are indicated by red arrows based on processing of female buccal reference swabs. [Diagram 33] Figure 33 shows a partial 27 locus male and female mixture (female predominant) STR profile of nucleic acid purified from male fraction isolated from vaginal swabs collected 72 hours post-coitus (donor couple G / H) using the 32 minute protocol. The male STR profile also lacks alleles for D10S1248, PentaE, D5S818, CSF1PO and SE33. Male genotypes are indicated by blue arrows based on processing of male buccal reference swabs. Female genotypes are indicated by red arrows based on processing of female buccal reference swabs. [Diagram 34] Figure 34 shows the STR profile of a 1:1 male and female mixture of all 27 loci purified from the male fraction isolated from vaginal swabs collected 72 hours post-coitus (donor couple I / J) using the 32 min protocol. Male genotypes are indicated by blue arrows based on processing of the male buccal reference swab. Female genotypes are indicated by red arrows based on processing of the female buccal reference swab. [Diagram 35] Figure 35 shows male STR profiles for all 27 loci of nucleic acid purified from the male fraction isolated from vaginal swabs collected 72 hours post-coitus (donor couple C / D) using the 22 minute protocol. Male genotypes are indicated by blue arrows based on processing of the male buccal reference swab. Female genotypes are indicated by red arrows based on processing of the female buccal reference swab. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] DETAILED DESCRIPTION OF THE PRESENT EMBODIMENT The inventors have discovered an easier and faster method for extracting and purifying nucleic acids from nucleic acid-containing materials, particularly bones, teeth and semen (including vaginal swabs from sexual assault kits). For bones and teeth, the simple fragments generated by a hammer are suitable for processing, and bone / tooth powder is unnecessary. In one embodiment, the present invention is directed to a simplified and fast method for extracting nucleic acids from nucleic acid-containing materials. In another embodiment, the present invention is a kit for carrying out the simplified and fast method for extracting nucleic acids from nucleic acid-containing materials. In yet another embodiment, the present invention is an integrated method for rapid nucleic acid analysis, e.g., rapid DNA identification based on STR profiling, in which sample processing, DNA extraction, purification, amplification, separation, detection and analysis occur in a fast, efficient and fully integrated manner.

[0029] The invention described herein is further described and enabled, and may be used alone or in combination with other nucleic acid sequencing and sizing tools, biochips and methods, and is disclosed in the following patents: US Pat. Nos. 9,889,449 and 9,366,631, entitled "Integrated systems for the multiplexed amplification and detection of six and greater dye labeled fragments"; US Pat. Nos. 9,797,841 and 9,310,304, entitled "Methods and Compositions for Rapid multiplex amplification of STR loci"; US Pat. Nos. 9,797,841 and 9,310,304, entitled "Ruggedized Apparatus for Analysis of Nucleic Acid and Protein Analysis"; Nos. 9,606,083, entitled "Methods for Forensic DNA Quantitation," 9,523,656, 8,206,974, 8,173,417; No. 9,550,985, entitled "Methods for Forensic DNA Quantitation," 9,494,519 and 8,425,861, entitled "Methods for Rapid Multiplexed Amplification of Target Nucleic Acids," 8,018,593, entitled "Integrated Nucleic Acid Analysis," and "Unitary Biochip Providing Sample-In to Results-Out Processing and Methods of Manufacturing." Nos. 9,354,199; 9,314,795; and 8,720,036, all of which are entitled "Manufacture";Nos. 9,174,210 and 9,012,208, entitled "Nucleic Acid Purification," and 8,961,765 and 8,858,770, entitled "Plastic Microfluidic Separation and Detection Platforms," ​​each of which is hereby incorporated by reference;

[0030] Methods for Extracting Nucleic Acid from Bones and Teeth In one embodiment, the invention is a simplified and rapid method for extracting nucleic acids from bone or teeth, said method comprising the steps of providing a sample of nucleic acid-containing material in a form suitable for nucleic acid extraction, vigorously mixing the sample in a desalting buffer, e.g., 0.5 M EDTA, and separating the mixture to obtain a liquid portion of the mixture containing the extracted nucleic acids.

[0031] In some embodiments, the bone and teeth samples can be fresh samples from donors with a PMI of less than 8 hours, or relatively fresh samples from donors with a PMI of 2-6 days. In other embodiments, the bone and teeth can be from casework, disaster, missing persons, or related samples, such samples obtained decomposed, as in the present case, for example, decaying bodies in fresh water, salt water, or soil, or after a fire or explosion. Bone and teeth samples can be years, decades, or centuries old, and often exposed to the elements. This ability to generate STR profiles from such aged bones is of great importance in mass disasters, disappearance cases, terrorist and criminal cases, and atrocities committed by dictatorial regimes.

[0032] In some embodiments, the sample is first cleaned and then ground to a suitable form for nucleic acid extraction. For example, the sample is washed with water, scrubbed to remove debris, immersed in a 10% bleach solution, then washed again with sterile water, followed by a final wash with 70% ethanol. After the final wash with ethanol, the sample can be dried at room temperature and ground, for example, by freeze milling to give a powdered sample, or crushed to give a fragmented or granulated sample. Although not absolutely necessary, cleaning is advantageous in that it can remove a large amount of microbial contaminants as well as, importantly, human DNA introduced by human handling or mixtures from two or more sample donors (if cleaning is not utilized, potential mixture profiles can be deconvolved manually or using software). As described below, grinding is not necessary when the work is performed outside the laboratory, but is an important consideration.

[0033] In some embodiments, bone or tooth samples can be comminuted or powdered. For example, a piece of bone can be comminuted using a number of techniques, such as a SPEX Sample Prep 6870 Large Freezer / Mill® (SPEX Sample Prep LLC, Metuchen, NJ) at a rate of 10 cycles per second with a 3-cycle program, 10 minutes pre-cooling, 2 minutes run-in time, and 2 minutes cooling time. Aged and dried bone samples can be completely comminuted after this protocol, but when using fresh bone, a few smaller fragments may remain. For fresh bone, the comminuted sample can be collected for further processing after the completion of the comminuted cycle. The powdered bone or tooth sample can be transferred to a sterile tube container.

[0034] In other embodiments, bone or tooth samples can be hammered into fragments. Coarse fracture of bone or tooth can also be effective. For example, a piece of bone or tooth can be wrapped in a DurX® 770 (Berkshire Corporation, Great Barrington, MA) wipe (a nonwoven sheet of polyester / cellulose material) or equivalent. With a clean hammer or similar hard blunt instrument, such as a rock (cleaned and wiped clean with an ethanol wipe, if available), the bone or tooth can be struck several times until the piece breaks into smaller pieces. Striking the bone or tooth sample at different angles with the hammer can facilitate the breaking of the sample into smaller fragments. When the bone is broken, small bone marrow pieces can be collected immediately. Harder bone bits (those that resist the initial break) can be broken into smaller pieces again by wrapping the bone piece in a new DurX® 770 wipe and striking it several times. The ideal size of bone particles for processing is preferably about 1 / 8 inch or less. Generally, the effect of comminuting or crushing is to produce smaller fragments and increase the exposed surface area of ​​the bone accessible for further processing (thereby enhancing subsequent extraction and purification steps).

[0035] The ground or crushed bone or tooth sample may be vigorously mixed in a desalting buffer to desalt the bone matrix and consequently release the DNA. In some embodiments, the desalting buffer may simply be an EDTA solution with an EDTA concentration of 0.1M to 1M, preferably 0.3 to 0.7M, more preferably 0.5M, without any other chemicals. In other embodiments, the desalting buffer, e.g., 0.5M EDTA, may further comprise the lysis buffer DTT (0.02M) and proteinase K. In still other embodiments, the desalting buffer, e.g., 0.5M EDTA, may further comprise a detergent, e.g., 0.5% SDS or 1% lauroyl sarcosinate or equivalent, proteinase K (3-4 mg), or both.

[0036] The volume of desalting buffer added to the sample may vary depending on the amount of sample being processed, its size and texture (crushed bone tends to absorb less than finely ground bone; aged, dried bone tends to absorb more than fresher, red marrow-containing samples). Figure 20 shows the average amount of purified DNA (in ng) as a function of the volume of desalting buffer added to 500 mg bone powder samples. Data are from quadruplicate measurements and error bars represent one standard deviation. This data shows that sufficient desalting can be achieved with much less buffer than used by prior art methods. For ≦10 mg of finely ground or crushed bone, 120 μl of buffer is sufficient. For 200 mg of bone, 200-220 μl of buffer is sufficient. For 500 mg of bone, 320-350 μl of buffer is sufficient. Prior art methods were based on the inaccurate assumption that complete dissolution of bone required DNA to be trapped in cells and bound to hydroxyapetite in free forensic samples and available for STR processing. The inventors show herein that this is not the case and that partially demineralized bone using a simple demineralization solution allows for the generation of excellent STR profiles. Figure 21 shows the time course of demineralization. DNA yield (in ng) / 200 mg bone powder is plotted on the Y-axis as a function of time on the X-axis. The data shows that overnight demineralization is not required to obtain sufficient DNA for STR analysis. The data in the figure are from six measurements and the error bars represent one standard deviation.

[0037] The present invention can be designed to eliminate the lengthy process of sample concentration and maximize the volume of liquid analyzed by conventional laboratory or rapid DNA analysis methods without compromising pretreatment efficiency. In some embodiments, 300-350 μl of desalting buffer, e.g., 0.5 M EDTA, may be used for every 500 mg of bone or tooth; 150-220 μl of desalting buffer, e.g., 0.5 M EDTA, may be used for every 100-200 mg of bone or tooth; and 100-150 μl of desalting buffer, e.g., 0.5 M EDTA, may be used for less than 100 mg of bone or tooth.

[0038] The present invention can be practiced with several desalting buffers, including but not limited to 1% sarcosine, 0.002% SDS, 0.5% SDS and others, with 0.5M EDTA being preferred. Prior art methods suggest that 0.5% EDTA requires the use of detergents and proteinase K to obtain sufficient demineralization of bone to be useful for STR analysis. Figure 22 shows that this is not the case. Figure 22 shows that the simplified desalting buffer of the present invention results in sufficient amounts of purified DNA for use in STR analysis.

[0039] The amount of pulverized or crushed sample can be 1 mg to 1 g, preferably 200 to 500 mg for aged / degraded samples on an I-chip. For fresh or intact bone samples in an I-chip, about 5 to 10 mg of sample can be enough to obtain nucleic acid that will give rise to a full STR profile, and less than 5 mg can generate a full or useful partial STR profile. The actual DNA content of the sample will affect the results. However, a degraded bone sample without intact DNA will not generate a profile regardless of how much sample is utilized. A full STR profile or a useful partial profile of a person means that the profile is sufficient to identify and match the person when submitted to a database or utilized in a kinship algorithm. The inventors report herein that STR profiles have been generated using 16 locus and 27 locus assays, the latter showing superior sensitivity. Since small samples cannot be measured using standard analytical scales, the buffer volume can be adjusted to load the swab for analysis without signal saturation. The sample input for a 27 locus STR profile theoretically corresponds to 0.6 mg of sample.

[0040] Depending on the amount of genomic material expected to be present in the sample (high or low), the type of rapid DNA chip most suitable for processing can be selected. In the ANDE system, if several hundred ng or more is expected to be present in the sample, the A chip is used, while if less than several hundred ng of DNA is expected to be present, the I chip is used. For crushed bones and teeth, for example, the I chip will typically be used to process with the ANDE rapid DNA system. The same selection process can be used with other rapid DNA systems. In the event that too much or too little sample is used (which can be determined by examining the signal intensity-peak height of the resulting electropherogram), repeated rapid DNA trials can be performed using more or less starting material. In addition, an adaptive expert system can be used to expand the dynamic range of the rapid DNA discrimination system.

[0041] The amount of DNA present in bone can be affected by several factors, including the time of collection of the donor (fresh vs. decomposed remains), the presence or absence of red bone marrow, the bone type of the body, and the age and health of the donor. However, the choice of chip type can be straightforward: relatively fresh bones can likely be effectively processed in high DNA content chips, and older bones can likely be effectively processed in high DNA content chips.

[0042] Simple and rapid desalting can be facilitated by vortex mixing of the mixture for 30 seconds to 1 minute, for example, on a benchtop vortex mixer (Vortex-Genie 2, Scientific Industries Model #G560) set at the maximum setting (scale 10). This desalting process in the presence of EDTA increases the efficiency of nucleic acid purification. In some embodiments, for example outside the laboratory, this vortex mixing can be replaced by vigorous shaking by hand for at least 1 minute and tapping the tube several times to homogenize the contents.

[0043] In some embodiments, the liquid sample or desalted sample solution can be allowed to settle under the influence of gravity until a supernatant forms. In other embodiments, sedimentation can be promoted by rotating the tube by hand. In still other embodiments, the liquid sample or desalted sample solution can be obtained from the mixture, for example, by centrifugation. Centrifugation can be for 2 minutes at 20,000×g. The supernatant (in the case of a pulverized sample) or sample liquid (in the case of a ground sample) contains the extracted nucleic acid.

[0044] In some embodiments, the supernatant containing the extracted nucleic acids can be used directly for further analysis without purification in a rapid DNA analysis system, in other embodiments, the nucleic acids in the supernatant can be used for further analysis after further purification.

[0045] Purification of nucleic acids from the supernatant can be accomplished by various methods known in the art, such as organic extraction using phenol / chloroform / isoamyl alcohol (25:24:1) or silica purification using a filter such as that from the MagAttract® (Quiagen, NV, Venlo, NL) DNA mini M48 kit.

[0046] In one embodiment, a small portion of the supernatant can be directly transferred onto a transport medium (e.g., a swab, pipette, or filter paper, etc.), which can then be inserted into a rapid DNA analysis system for nucleic acid analysis. The portion of the supernatant can be about 100-150 μl. The invention can be practiced with a rapid DNA analysis system capable of processing a small portion or the supernatant, such as the ANDE™ system available from ANDE, Corporation, Waltham, MA. See, e.g., Tan et al., Investig Genet. 2013; 4: 16 and Turingan et al., Investig Genet. 2016; 7: 2 and Grover et al., Int J Legal Med (2017) 131:1489-1501, which are incorporated herein by reference in their entirety.

[0047] Nucleic Acid Extraction Kit In another embodiment, the invention is a kit for carrying out a simplified and rapid method for extracting nucleic acids from nucleic acid-containing material, such as bone or teeth.

[0048] In one embodiment, a kit for extracting nucleic acids from bone and teeth includes a buffer for demineralizing bone matrix, and optionally, a brochure detailing a rapid nucleic acid extraction method. In another embodiment, the demineralizing buffer may contain only 0.5 M EDTA. In some embodiments, 0.5 M EDTA may be combined with lysis buffer DTT (0.02 M) and proteinase K. In other embodiments, 0.5 M EDTA may be combined with detergent (0.5% SDS or 1% lauroyl sarcosinate or equivalent) and proteinase K (3-4 mg).

[0049] In some embodiments, the booklet may include instructions on how to perform nucleic acid extraction using lysis buffer from bone and tooth samples. In some instances, the booklet may further describe how to pre-treat bone and tooth samples. In still some instances, the booklet may describe how to analyze extracted nucleic acid, for example, using swabs for analysis of extracted nucleic acid in HDC BCS and / or LDC BCS systems. In still some further instances, the booklet may describe the methods of nucleic acid extraction described herein above.

[0050] In other embodiments, the kits may further include solutions for washing and cleaning the nucleic acid-containing material, hi yet other embodiments, the kits may further include swabs for analyzing extracted nucleic acids in the HDC and / or LDC BCS.

[0051] Method for determining STR profiles of nucleic acids from bone or tooth samples - Patents.com In yet another embodiment, the invention is a method for determining a STR profile of nucleic acids from a bone or tooth sample. In some embodiments, the method includes providing nucleic acid-containing material in a form suitable for nucleic acid extraction; adding EDTA and desalting the nucleic acid-containing material to obtain a mixture; vigorously mixing the mixture; separating the mixture to obtain a liquid sample containing extracted nucleic acids; and subjecting a portion, if not all, of the liquid supernatant to a fully integrated nucleic acid purification and generation of the STR profile.

[0052] In some embodiments, the bone and tooth samples may be fresh samples from donors with a postmortem interval (PMI) of less than 8 hours or relatively fresh samples from donors with a PMI of 2-6 days. In other embodiments, the bone and teeth may be from casework, disaster, missing persons or related samples. Such samples may be obtained, for example, in fresh water, salt water or soil, or as decomposed as a decaying body after a fire or explosion.

[0053] In some embodiments, the sample is first cleaned and then ground so that the sample is in a suitable form for nucleic acid extraction. For example, the sample may be washed with water, scrubbed to remove debris, immersed in a 10% bleach solution, then washed again with sterile water, followed by a final wash with 70% ethanol. After the final wash with ethanol, the sample may be dried in saline and ground, for example, by creating a powdered sample, or by crushing to create a fragmented or granular sample. Although not absolutely required, cleaning is advantageous in that it can remove large amounts of microbial contaminants, and, very importantly, human DNA that may have been introduced by human handling or mixing from two or more sample donors. Also, fine grinding is advantageous, but is not a necessary consideration if the work is to be performed outside of a laboratory.

[0054] In some embodiments, bone or tooth samples can be comminuted and powdered. For example, a piece of bone can be comminuted using multiple techniques, such as a SPEX Sample Prep 6870 Large Freezer / Mill®, with a 3-cycle program, 10 minutes pre-cooling, 2 minutes run-in time, 2 minutes cooling time, and at a rate of 10 cycles per second. Aged and dried bone samples can be completely comminuted after this protocol, but when using fresh bone, a few smaller fragments may remain. For fresh bone, the available comminuted sample can be collected for further processing after the completion of the comminuted cycle. The powdered bone or tooth sample can be transferred to a sterile tube container.

[0055] In other embodiments, the bone or tooth sample may be crushed into fragments. Coarse breakage of the bone or tooth may also be effective. For example, a piece of bone or tooth may be wrapped in a DurX® 770 (Berkshire Corporation, Great Barrington, MA) wipe (a nonwoven sheet of polyester / cellulose material) or equivalent. With a clean hammer or similar hard blunt instrument, such as a rock (cleaned and wiped clean with an ethanol wipe, if available), the bone or tooth may be struck several times until the sample piece breaks into smaller pieces. Smashing the bone or tooth sample at different angles with the hammer may facilitate the breaking of the sample into smaller fragments. When the bone is crushed, small bone marrow pieces may be collected immediately. Harder bone bits (those that resist the initial crushing) may be crushed again into smaller pieces by wrapping the bone piece in a new DurX® 770 wipe and striking it several times. The ideal size of the bone pieces for processing is preferably about 1 / 8 inch or less. Generally, the effect of comminuting or crushing is to generate smaller fragments and increase the exposed surface area of ​​bone that is accessible to facilitate further processing (thereby enhancing subsequent extraction and purification steps).

[0056] Ground or crushed bone or tooth samples should be vigorously mixed in 0.5 M EDTA to demineralize the bone matrix. In some embodiments, 0.5 M EDTA may be combined with lysis buffer and DTT (0.02 M) and proteinase K (10 mg). In other embodiments, 0.5 M EDTA may be combined with detergent (0.5% SDS or 1% lauroyl sarcosinate or equivalent) and proteinase K (3-4 mg).

[0057] The volume of desalting buffer added to the sample may vary depending on the amount of sample being processed, its size and texture (crushed bones tend to absorb less than pulverized bones; aged and dried bones tend to absorb more than fresher, red marrow-containing samples). The present invention may be designed to eliminate the lengthy process of sample concentration without compromising pretreatment efficiency, and to maximize the volume of liquid loaded onto the swab for analysis without oversaturation. In some embodiments, 300-350 μl of 0.5 M EDTA may be used for every 500 mg of bone or tooth; 150-220 μl of 0.5 M EDTA may be used for every 100-200 mg of bone or tooth; and 100-150 μl of 0.5 M EDTA may be used for less than 100 mg of bone or tooth. Tooth samples may require longer incubation than bone. In most cases, a full STR profile can be generated from bone with a 1 minute incubation, while a full STR profile from teeth can be generated with a 10 minute incubation.

[0058] The amount of pulverized or crushed sample can be 1 mg to 1 g or more for aged / degraded samples in the I-chip, preferably 200 to 500 mg. For fresh or intact bone samples in the I-chip, about 5 to 10 mg of sample can be enough to obtain nucleic acid that will give rise to a full STR profile, and less than 5 mg can generate a full or useful partial STR profile. The actual DNA content of the sample will determine the outcome. However, a degraded bone sample that does not contain intact DNA will not generate a profile regardless of how much sample is utilized. A full STR profile or a useful partial profile of a person means that the profile is sufficient to identify and match a person when submitted to a database or utilized in a kinship algorithm.

[0059] Depending on the amount of genomic material expected to be present in the sample (high or low), one can choose the type of chip, A-chip or I-chip, that is better suited for processing. The amount of DNA present in bone can be influenced by several factors, including collection time (fresh vs. decomposed remains), the presence or absence of red bone marrow, bone type of the body, and age and health of the donor. However, the choice of chip type can be straightforward: relatively fresh bones can probably be effectively processed in high DNA content chips / A-chips, and older bones can probably be effectively processed in low DNA content chips / I-chips.

[0060] Simple and rapid desalting can be facilitated by, for example, vortex mixing of the mixture for 30 seconds to 1 minute on a benchtop vortex mixer set at the maximum setting (scale 10). This desalting step in the presence of EDTA can help increase nucleic acid yields, as a decrease in nucleic acid yield is observed without treatment with EDTA. In some embodiments, for example outside of the laboratory, this vortex mixing can be replaced by vigorous shaking by hand for at least 1 minute and tapping the tube several times to homogenize the contents.

[0061] In some embodiments, the liquid sample or desalted sample solution can be allowed to settle under gravity until a supernatant forms. In other embodiments, sedimentation can be promoted by rotating the tube by hand. In still other embodiments, the liquid sample or desalted sample solution can be obtained from the mixture, for example, by centrifugation. Centrifugation can be for 2 minutes at 20,000×g. The supernatant (in the case of a pulverized sample) or sample liquid (in the case of a ground sample) contains the extracted nucleic acid.

[0062] In some embodiments, the supernatant containing the extracted nucleic acids may be used directly for further analysis without purification, while in other embodiments, the nucleic acids in the supernatant may be further purified before being used for further analysis.

[0063] Purification of nucleic acids from the supernatant can be accomplished by various methods known in the art, such as organic extraction using phenol / chloroform / isoamyl alcohol (25:24:1) or silica purification using a filter such as that from the MagAttract® (Quiagen, NV, Venlo, NL) DNA mini M48 kit.

[0064] Kit for determining STR profiles of nucleic acids from bone or tooth samples In yet another embodiment, the invention is a kit for determining the STR profile of nucleic acid from a bone or tooth sample. In some embodiments, the kit may include 0.5M EDTA as a desalting buffer; a booklet detailing a rapid nucleic acid extraction method; and a swab for analyzing the extracted nucleic acid in the LDC BCS.

[0065] In some embodiments, the kit further includes a solution for cleaning the bone and tooth samples to remove adhering tissue debris, dirt and extraneous DNA on the surface using water followed by a 10% bleach solution, then rinsing again with water, and finally rinsing with 70% ethanol.

[0066] In some embodiments, the kit is for profiling a STR multiplex including the following loci: D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S17, D7S820, D16D539, CSF1PO, Penta D, Amelogenin, vWA, D8S1179, TPOX and FGA (Powerplex 16 chemistry). In other embodiments, the kits include kits for detecting the following loci: D3S1358, D19S433, D2S1338, D22S1045, Penta B, TH01, D18S51, D1S1656, D10S1248, D2S441, Penta C, D16S539, vWA, D21S11, D12S391, amelogenin, Penta D, D5S818, D13S317, D7S820, TPOX, CSF1PO, Penta E, for profiling an STR multiplex including D8S1179, FGA, SE33, DYS391, D6S1043, DYS439, DYS389II, DYS19, DYS392, DYS393, DYS389I, DYS390, DYS385a, DYS385b, DYS437 and DYS438. In still other embodiments, the STR multiplex will include the following loci: 23 autosomal loci (D1S1656, D2S1338, D2S441, D3S1358, D5S81, D6S1043, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D22S1045, FGA, CSF1PO, Penta E, TH01, vWA, TPOX, SE33), 3 Y chromosome loci (DYS391, DYS576 and DYS570) and amelogenin. For all sample types described herein, there is no limitation on the set of STR multiplexes utilized for the generation of DNA ID. Typically, at least seven STR loci should be used to generate a DNA sample because the Random Match Probability with this number is low enough (1 in tens of thousands to 1 in millions) to allow for a positive DNA sample.The specific STRs utilized and multiplexed are the basis of the present application (e.g., searching very large DNA databases [over 10 million samples, such as those in the US, UK and China databases] to match DNA from potential sexual assailants; or searching smaller databases [hundreds to thousands of samples for aircraft crash victim identification]).

[0067] A simplified and rapid method for semen DNA analysis In another embodiment, the present invention is a method for analyzing nucleic acid from a semen sample. The semen sample can be neat semen or a semen stain on a fabric. Alternatively, the semen sample can be collected directly or indirectly from an individual. In some instances, the semen sample can be a forensic semen sample, e.g., a casework semen sample. Even though the morphology of the semen sample can change, the present invention is equally applicable as long as the semen sample can be collected on a swab head.

[0068] In some embodiments, a method for determining a STR profile of nucleic acid from a semen sample includes collecting a semen sample on a swab, applying an appropriate amount of dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution onto the swab head; and inserting the swab into a rapid nucleic acid analysis system for STR analysis. The inventive method herein may be used with any panel of STR loci. In some embodiments, the kit is for profiling an STR multiplex including the following loci: D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S17, D7S820, D16D539, CSF1PO, Penta D, Am, vWA, D8S1179, TPOX, and FGA. In other embodiments, the kit includes a kit for detecting the following loci: D3S1358, D19S433, D2S1338, D22S1045, Penta B, TH01, D18S51, D1S1656, D10S1248, D2S441, Penta C, D16S539, vWFA31, D21S11, D12S391, amelogenin, Penta D, D5S818, D13S317, D7S820, TPOX, CSF1PO, Penta E, for profiling an STR multiplex including D8S1179, FGA, SE33, DYS391, D6S1043, DYS439, DYS389II, DYS19, DYS392, DYS393, DYS389I, DYS390, DYS385a, DYS385b, DYS437 and DYS438. In yet other embodiments, the STR multiplex will include the following loci: 23 autosomal loci (D1S1656, D2S1338, D2S441, D3S1358, D5S81, D6S1043, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D22S1045, FGA, CSF1PO, Penta E, TH01, vWA, TPOX, SE33), 3 Y chromosome loci (DYS391, DYS576 and DYS570) and amelogenin.

[0069] In other embodiments, the semen sample may be neat semen or a dried semen sample on a piece of fabric, and the method includes collecting a sample of nucleic acid-containing material on a swab, applying DTT or TCEP to the swab, and applying the swab to an ANDE system, e.g., an I-tip, for analysis. In some examples, the semen sample may be a dried semen stain on fabric, and the method may include scraping the semen sample from the fabric with a scraping tool, such as a knife, razor blade, or scalpel. In some examples, the semen stain on the fabric may be moistened with sterile water and a swab may be rubbed against the fabric.

[0070] In some embodiments, the collecting step may be performed by rubbing the fabric with a tool such as a knife, razor blade, or scalpel. The collecting step may be to collect the fabric fibers on which the semen sample has been dried. In a preferred embodiment, the semen sample is collected on a swab. The swab may be used to collect the semen sample after being moistened with water. In some instances, the swab may be moistened with a few drops, preferably 1-2 drops, of molecular grade water from a water dropper. In other instances, the swab may be moistened with more water due to an increase in the size of the swab head. In other instances, the stain on the fabric may be moistened with a few drops, preferably 1-2 drops, of molecular grade water from a water dropper. Wetting the swab head and the fabric stain may facilitate the collection of the semen sample.

[0071] In some embodiments, the moistened swab may be used to wipe the surface of the scrubbed fabric material. In other embodiments, the swab may be further used to collect clumps of fabric fibers previously scraped off the fabric material. After fixing the clumps of fabric fibers on the swab head, it is preferred to further process the swab.

[0072] In some embodiments, the swab head may apply 50 μl of 150 mM DTT or 50 μl of 50 mM TCEP. The presence of DTT or TCEP may facilitate the breakdown of disulfide bonds on the sperm nuclear membrane in the semen sample. The application of 150 mM DTT or 50 mM TCEP is preferably applied in such a way that the DTT / TCEP covers the entire swab head to facilitate the recovery of nucleic acids.

[0073] In some embodiments, nucleic acid from the swabs can be extracted and analyzed by using an I-chip in the ANDE Rapid DNA Analysis System. The I-chip was developed by maximizing the efficiency of the purification module and incorporating a post-purification DNA concentration module. These modifications allowed the system to generate STR profiles from samples containing a few nanograms of DNA or less.

[0074] Kit for determining STR profiles of nucleic acids from semen samples In yet another embodiment, the present invention is a kit for determining the STR profile of nucleic acid from a semen sample.In one embodiment, the kit includes a solution of DTT or TCEP, a booklet detailing the rapid nucleic acid extraction method from a semen sample as disclosed herein, and a swab used to collect and analyze the extracted nucleic acid in an I-chip.In some examples, the DTT solution has a DTT concentration equal to or close to 150 mM, and the TCEP solution has a TCEP concentration equal to or close to 50 mM.If the semen sample is a semen stain on a fabric, the kit may further include a tool, such as a knife, a razor blade, a scalpel, to collect the semen sample from the fabric.The kit also includes a dropper bottle containing sterile water or an ampoule containing sterile water. In some embodiments, the kit can be used to generate profiles for the following STRs: D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S17, D7S820, D16D539, CSF1PO, PentaD, Am, vWA, D8S1179, TPOX and FGA.

[0075] Further Analysis of Nucleic Acids Extracted Using the Present Invention The extracted nucleic acid may be analyzed with a variety of nucleic acid analysis systems, such as the ANDE™ Rapid DNA Analysis System (ANDE, Inc.). The ANDE™ system is a fully automated system with on-board expert system software for automated data analysis. The system can generate what are called STR profiles from high DNA content samples (e.g., buccal swabs) in approximately 90 minutes and from casework, sensitive site exploitation, and mass casualty samples (e.g., blood stains, chewing gum, drink containers, clothing, touched objects such as steering wheels, door handles, cell phones, and keyboards, liver, lung, brain, bone, and teeth) in 105 minutes. The system is designed for use with high and low DNA content chips (referred to herein as "A-chips" and "I-chips"). The A-chip and I-chip consumables are "all-in-one" disposable cassettes pre-packaged at the factory with all reagents needed for STR analysis. The A-tip is designed for samples that contain large amounts of DNA, such as buccal swabs, and the I-tip is designed for samples that typically contain smaller amounts of DNA, such as small blood stains, cigarette butts, or steering wheel swabs. Both of these consumables are suitable for processing bone, tooth, semen, and other samples prepared using the teachings herein. The amount of DNA present in bone can be affected by several factors, including the PMI until collection, the condition of the body (e.g., decomposed body, burned), the presence or absence of bone marrow, the type of bone from the body, and the age and health of the donor. It appears that relatively fresh and intact bones may contain more DNA than older ones that have been exposed to the environment or damage.

[0076] In one embodiment, a small portion of the supernatant can be transferred directly onto a swab, which can then be inserted into a rapid DNA analysis system, such as either an A-chip or an I-chip, for nucleic acid analysis. The portion of the supernatant can be about 100-150 μl. The swab can be an ANDE® swab or a similar swab fitted with a swab cap and swab chamber for rapid DNA analysis systems. The A-chip is described in Tan et al., Inv. Genetics 4 2013 and Grover et al., Intg J Legal Med 2017; the I-chip is described in Turingan et al., Inv Genetics 7 2016, both of which are incorporated herein by reference in their entirety.

[0077] Example 1. Extraction, purification and analysis of nucleic acids from bone. In this example, we performed rapid nucleic acid extraction from bone samples and analyzed the extracted nucleic acids using swabs and I-chips in the ANDE system.

[0078] Bone samples were artificially decomposed to mimic casework and / or disaster victim identification during aircraft crashes (e.g., Indonesia AirAsia Flight 8501). Simulated decomposition of bone samples (femur and humerus fragments) was performed by immersing the fragments in Atlantic coastal saltwater at 84°F (mean temperature of the Java Sea) for 4 days. The bone samples were then washed with water, bleached, washed again with water and then with ethanol. After drying, the bones were either ground by a cryo-grinding machine to obtain powdered samples or simply crushed to obtain fragmented or granular samples. The ground bone samples were placed in four separate 2 ml centrifuge tubes containing 200 mg of crushed bone, 500 mg of crushed bone, 200 g of frozen-ground bone, and 500 g of frozen-ground bone, respectively.

[0079] We then added 200-350 μl of 0.5 M EDTA to each tube. EDTA was added to help demineralize the bone samples and release DNA. The tubes were then vortex mixed for 1 min using a benchtop vortex mixer at speed setting "10". The tubes were then centrifuged (Centrifuge 5417R; Eppendorf North America, Hauppauge, NY, USA) for 2 min at 20,000 rcf to separate the bone particulates from the desalted supernatant. Approximately 150 μl of each supernatant was pipetted onto an ANDE swab for sample insertion into an I-chip for analysis of STR profiles. The STRs were D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S17, D7S820, D16D539, CSF1PO, Penta D, Am, vWA, D8S1179, TPOX and FGA. As a result, the inventors were able to obtain STR profiles for each of the four test samples. See Figures 1-4, nucleic acids were extracted from each of the four test samples. All STR profiles (PP16) were generated from pulverized or crushed samples and were reduced in bone input from 500mg to 200mg.

[0080] As a positive control, we performed nucleic acid extraction from 500 mg of frozen ground bone from the same source following the global desalting method reported in Loreille et al., Forensic Science International: Genetics 1 (2007) 191-195. Specifically, bone powder was incubated overnight in 7.5 ml of extraction buffer (EDTA 0.5 M and 1% lauroyl sarcosinate) and 200 μl of 20 mg / mL proteinase K in a rotary shaker at 56° C. The supernatant was transferred to an Amicon 30 centrifugal filter unit (Millipore) for concentration to about 150 μl and loaded onto a fully integrated trial NetBio swab in an I-chip. As a result, we were able to obtain STR profiles from the purified DNA. See FIG. 8. All STR profiles were generated using a lengthy process of total desalting. The overall signal intensity was comparable to profiles generated from a simplified and rapid bone analysis method.

[0081] In the traditional method, the desalting buffer contains EDTA, detergent and proteinase K, a high initial buffer volume is used (7.5 ml for 500 mg powder), desalting requires overnight incubation at 56° C. with agitation in a rotary shaker, and the method requires sample concentration and washing using a centrifuge unit.

[0082] In contrast, in the present invention, the desalting solution is simply an EDTA solution that does not contain any detergent or proteinase K. In addition, there is no need for long-term incubation and heating. In fact, the desalting method takes less than 5 minutes, including sample processing and solution transfer. The resulting supernatant contains the extracted nucleic acids, which can be directly used to generate profiles using our HDC or LDC BCS and ANDE systems.

[0083] Example 2. Extraction and analysis of nucleic acids from teeth. In this example, we performed rapid nucleic acid extraction from tooth samples and analyzed the extracted nucleic acid in swab and LDC BCS system. The procedure is a modification of Example 1. After vortex mixing for 1 minute, the tooth samples were incubated at 56°C for 10 minutes to enhance demineralization. As a result, we were able to obtain the STR profile of the nucleic acid extracted from the tooth samples. See Figure 9, which shows the STR profile of the nucleic acid extracted from 200 mg of frozen ground tooth powder of tooth root.

[0084] Our results show that the 1 minute demineralization method on teeth was insufficient to obtain a full or good partial profile. This is likely due to the more compact matrix of teeth compared to bone, and longer demineralization is required for DNA release. The root of a tooth sample has been reported to contain more DNA than the crown or other parts of the tooth. We note that when processing the crown relative to the root tissue from the same tooth sample, full STR profiles can be obtained more consistently from the root rather than the crown tissue. For fresh or digested teeth, the root is preferred over the crown. If the crown is available for processing, more material can be done (e.g., 500 mg instead of 200 mg), and fine comminution is preferred over crushing.

[0085] Figure 17 is a profile of all 16 loci for a 200 mg root (undegraded); Figure 18 is a 16 loci profile from a root aged for 1 month in 84F saline (200 mg); Figure 19 is an artificially aged molar that was cut / comminuted after cleaning in bleach and drying.

[0086] Example 3. Extraction and analysis of nucleic acid from casework dental samples. Casework tooth samples were obtained from bodies that had been buried and burned for at least two years. Tooth samples were washed with running water and antibacterial soap while scrubbing the surface with a cloth and bleach. The samples were then subjected to a bleach-sterile water-ethanol rinse and allowed to dry for 10 minutes. The roots were separated from the crowns using a dremel. The roots were then cryo-fractured and 200 mg demineralized.

[0087] Example 4. Extraction and analysis of nucleic acid from neat semen. Typically, semen samples contain approximately 50 million cells per mL. After thawing on ice, the tubes containing the semen samples were carefully opened and the samples were gently mixed by pipetting up and down to ensure sample homogeneity prior to sample retrieval. 50 μl of semen diluted 10-100 times in 1× PBS buffer was pipetted onto the ANDE swab. A final concentration of 150 mM DL-dithiothreitol (DTT) (Sigma Aldrich; Catalog #43816) or 50 μl of 50 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma Aldrich; Catalog #646547) was added to the web and incubated for 1-10 min at room temperature prior to analysis in the I-chip.

[0088] Example 5. Extraction and analysis of nucleic acid from semen dried on fabric. In this example, we performed rapid nucleic acid extraction from semen samples and analyzed the extracted nucleic acids in swabs using an I-chip, where we used mock semen samples prepared as described below in step 1. For the casework semen samples, we skipped this semen sample preparation step (step 1).

[0089] Step 1: Preparation of simulated semen-soiled clothing Approximately 100 μl of neat semen was transferred onto cotton fabric and / or denim. Since the dried stain is not visible to the naked eye, the adjacent area surrounding the semen stain was marked with a magic marker while the semen stain was still wet to ensure that the biological material was collected from the correct location. The semen stain on the fabric was allowed to dry overnight at room temperature.

[0090] Step 2: Collection of dried semen samples from clothing This process was performed to collect DNA-containing semen samples from semen-stained fabric pieces. The fabric pieces were placed on a sterile work space. We thoroughly scrubbed all marked soiled fabrics using a clean razor blade or a sterile disposable scalpel until a cluster of fabric fibers was formed. DNA-containing semen samples were collected using a 30 mL drop-dispenser bottle pre-moistened with 2-3 drops of sterile water (VWR; Cat. #16354-421).

[0091] As shown in FIG. 23, to collect the DNA-containing semen sample, the inventors moved a group of fibers to one side of the soiled fabric piece and swabbed the surface of the previously scrubbed material by moving the swab head back and forth to collect the remaining cellular material. Next, a drop of molecular biology grade water was placed on the swab head and the group of fibers was collected. In some cases, an additional 1-2 drops of molecular biology grade water were placed on the group of fibers to ensure that they were firmly attached to the swab head. The inventors then smoothed the surface of the swab head with a razor blade or a sterile scalpel to further immobilize the fibers. In addition, the inventors also wiped the used razor blade or sterile scalpel and rubbed the article of clothing on the dry portion of the swab head. Typically, the dry portion of the swab head was adjacent to a plastic shaft. Finally, we loaded the fabric bundle with 50 μl of 150 mM DL-dithiothreitol (DTT) (Sigma Aldrich; Catalog #43816) or 50 μl of 50 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma Aldrich; Catalog #646547), then rotated the swab to ensure coverage of the entire swab, and placed the swab containing the fiber bundle back into the clear plastic tube. DTT or TCEP was used to break down disulfide bonds in the sperm nuclear membrane.

[0092] Step 3: DNA analysis by performing a rapid DNA run with the ANDE system The inventors used the ANDE I-chip to process and analyze the nucleic acid on the swab. Specifically, the inventors removed the protective plastic seal from the first swab chamber (found on the top of the I-chip), selected "Perform Run" on the ANDE instrument, then, using only the swab cap, screened to scan the swab RFID, entered the sample ID, and then inserted the swab into the swab chamber by pressing down on the swab until the swab cap clicked securely into place.

[0093] As a result, we successfully generated a full STR profile of nucleic acid from dried semen on clothing. The generated signal intensity may vary depending on the donor and fabric material. See Figures 24 and 26 for full 16 locus and 27 locus STR profiles of nucleic acid purified from dried semen sample on denim, respectively, and full 16 locus STR profile of nucleic acid purified from dried semen sample on cotton.

[0094] Example 6. Extraction and analysis of nucleic acid from timed post-coital vaginal swabs and Sexual Assault Kit (SAK) swabs. Vaginal swabs from several donor pairs at intervals of 24, 48 and 72 hours after intercourse were processed and analyzed. The SAK protocol is summarized in Figure 27. The swabs were placed in tube 1 containing ANDE's proteinase K and lysis solution. The samples were then vortex mixed for 10 seconds and flashspinned. The swabs were manually transferred to a rotating basket inserted in tube 1 and centrifuged for 5 minutes. This centrifugation time can be reduced to 2 minutes. Centrifugation removes the swabs from any residual liquid that may contain biological samples. The rotating basket containing the dried swabs was then removed and discarded. The aqueous phase is essentially the female fraction containing lysed epithelial cells. The female fraction was carefully pipetted and transferred to another microcentrifuge tube, tube 2. For analysis of the female fraction in the I-tip, 5-10 μl of that fraction was placed on the ANDE swab. Sperm cells, if present, are pelleted at the bottom of tube 1. It is therefore important not to disturb the pellet or accidentally remove it. The pipette tip should not touch the bottom of the tube and can retain approximately 50 μl of liquid to avoid sperm cell sample loss. The pellet with the least liquid from the female fraction was then washed by adding wash buffer containing MgCl2 and CaCl2 up to the 1 milliliter line of tube 1. The mixture was then vortex mixed for 5 seconds and centrifuged at 20,000 x g for 2 minutes to re-pellet the sperm. Approximately 50 μl of the aqueous phase was retained and the rest was discarded. The homogenized sperm was then transferred to tube 3 containing 2000 U to 10000 U of nuclease; 2000 U is a sufficient amount to degrade soluble DNA for this work with a 10 minute incubation at 37C. The reaction is quenched by adding 20 μl of stop solution containing 0.5 M EDTA followed by incubation at 56° C. for 10 min; further reduction at 56° C. for up to 3 min is sufficient to inactivate the nuclease. The nuclease used in this invention is recombinant DNAse I (Sigma Aldrich; Catalog #04536282001).Finally, DL-dithiothreitol (DTT) (Sigma Aldrich; Catalog #43816) or tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma Aldrich; Catalog #646547) was added to final concentrations equal to or close to 150 mM DTT and 50 mM TCEP, respectively, and vortex mixed for 5 seconds. The resulting solution is the male fraction and can be collected with an ANDE swab for analysis in an I-chip.

[0095] It is noted that the teachings herein are applicable to a wide range of other samples. Also, for example, methods for vaginal swabbing can be applied to separate male cells from female cells in multiple cases, or in penile-oral and penile-anal penetrations. Although the examples herein discuss the male sexual assailant and female victim settings, male sexual assailant-male victim and female sexual assailant-male victim cases are also found and these methods are effective in such cases.

[0096] Although the present invention has been disclosed with respect to certain embodiments, numerous modifications, alterations and variations to the described embodiments are possible without departing from the sphere and scope of the present invention, as defined in the appended claims. It is therefore intended that the present invention not be limited to the described embodiments, but rather that it shall have the full scope defined by the language of the following claims and equivalents thereof.

Claims

1. 1. A method for nucleic acid extraction from a bone or tooth sample, comprising: a. providing a sample ground into a powder or crushed into pieces; b. adding a desalting buffer to the nucleic acid-containing material to obtain a mixture, wherein said desalting buffer is a 0.3-0.7M EDTA solution without any other chemicals; c. vigorously mixing the mixture; and then d. separating the mixture to obtain a liquid supernatant, wherein the liquid supernatant only contains extracted nucleic acids; Demineralization of bone samples was performed in less than 60 minutes, demineralization of tooth samples was performed in less than 180 minutes, The bone and tooth samples are optionally washed and cleaned with a solution prior to step a. The method, wherein a portion of the liquid supernatant is optionally subjected to nucleic acid analysis.

2. The method of claim 1 , wherein the separating step does not include concentrating the nucleic acid.

3. The method of claim 1 , wherein the sample is a decomposed bone or tooth sample.

4. 1. A method for nucleic acid extraction from a bone or tooth sample, comprising: a. providing a bone or tooth sample that has been ground into a powder or crushed into fragments; b. adding less than 500 μl of 0.3-0.7 M EDTA solution or equivalent desalting buffer, free of any other chemicals, to the nucleic acid-containing material to obtain a mixture; c. vigorously mixing the mixture; d. subjecting the mixture to heat or incubating said mixture without heat for at least 1 minute; e. Separating the mixture to obtain a liquid supernatant without concentration, wherein the liquid supernatant contains the extracted nucleic acids. It consists only of Demineralization of bone samples was performed in less than 60 minutes, demineralization of tooth samples was performed in less than 180 minutes, The bone and tooth samples are optionally washed and cleaned with a solution prior to step a. The method, wherein a portion of the liquid supernatant is optionally subjected to nucleic acid analysis.

5. The method of claim 4, wherein the sample has a weight of less than 10 mg.

6. The method of claim 4, wherein the sample is a decomposed bone or tooth sample.

7. 10. The method of claim 1, wherein the step of separating the mixture to obtain a liquid supernatant is accomplished by centrifugation.

8. 8. The method of claim 7, wherein the centrifugation is at a speed of 20,000 rcf or greater for 2 minutes or greater.

9. 2. The method of claim 1, wherein said nucleic acid analysis comprises transferring a portion of the liquid supernatant to a rapid DNA processing system.

10. 2. The method of claim 1, wherein the step of vigorously mixing the mixture is carried out by vortexing the mixture for about 30 seconds or more.

11. A kit for rapid nucleic acid extraction from nucleic acid-containing material for use in the method of claim 1, comprising: a. The kit includes a desalting buffer that is a 0.3-0.7M EDTA solution without any other chemicals.

12. 12. The kit of claim 11, further comprising a medium for transporting the extracted nucleic acid to a rapid DNA analysis system.

13. 12. The kit of claim 11, further comprising a solution for washing and cleaning the bone and tooth samples prior to step a of claim 1.

14. 14. The kit of claim 13, wherein the solution is 70% ethanol and 10% bleach.

15. 12. The kit of claim 11, wherein the desalting buffer is 0.5 M EDTA.

16. 1. A method for determining the STR profile of nucleic acids from a bone or tooth sample, comprising: a. providing bone or teeth that have been ground into a powder or crushed into fragments; b. adding a desalting buffer to the bone or tooth to obtain a mixture, wherein said desalting buffer is a 0.3-0.7M EDTA solution without any other chemicals; c. vigorously mixing the mixture; d. separating the mixture to obtain a liquid supernatant, wherein the liquid supernatant contains the extracted nucleic acids; and then e. A portion of the liquid supernatant is subjected to nucleic acid analysis to determine the STR profile of the nucleic acid from the bone or tooth. It consists only of Demineralization of bone samples was performed in less than 60 minutes, demineralization of tooth samples was performed in less than 180 minutes, The method, wherein the bone and tooth samples are optionally washed and cleaned with a solution prior to step a.

17. 17. The method of claim 16, wherein the sample is a decomposed bone or tooth sample.

18. 17. The method of claim 16, wherein the step of separating the mixture to obtain a liquid supernatant is accomplished by centrifugation.

19. 20. The method of claim 18, wherein the centrifugation is at a speed of 20,000 rcf or greater for 2 minutes or greater.

20. 17. The method of claim 16, wherein the step of vigorously mixing the mixture is carried out by vortexing the mixture for about 30 seconds or more.

21. 17. The method of claim 16, wherein the nucleic acid analysis comprises transferring a portion of the liquid supernatant to a rapid DNA processing system.

22. 17. The method of claim 16, wherein the desalting buffer is 0.5 M EDTA.

23. A kit for determining the STR profile of nucleic acids from a bone or tooth sample for use in the method of claim 16, comprising: a. a desalting buffer that is a 0.3-0.7M EDTA solution without any other chemicals; and b. Swabs for analyzing extracted nucleic acids in a rapid DNA analysis system The kit comprising:

24. 24. The kit of claim 23, further comprising a solution for washing and cleaning the bone or tooth sample prior to step a of claim 16.

25. 24. The kit of claim 23, wherein the solution is 70% ethanol and 10% bleach.

26. 24. The kit of claim 23, wherein the desalting solution is 0.5 M EDTA.

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