WO2004083819A2

WO2004083819A2 - Molecular forensic specimen marker

Info

Publication number: WO2004083819A2
Application number: PCT/US2004/008332
Authority: WO
Inventors: Jason Eshleman; Ripan Malhi
Original assignee: Trace Genetics, Inc
Priority date: 2003-03-17
Filing date: 2004-03-17
Publication date: 2004-09-30
Also published as: WO2004083819A3

Abstract

A molecular identification marker for biological samples taken, for example, at a crime scene to confirm the chain of custody and identity of the biological samples at the time of analysis in a laboratory and method of use. The molecular identification marker also acts as an indicator of cross-contamination between marked samples or between reference and unknown biological samples. One embodiment of the molecular identification marker is a segment of DNA with at least one mutagenic primer site configured to produce a unique variant after mutagenic PCR, a segment configured to amplify for a selected forensic locus; and a segment configured for use with a probe to quantify the molecular identification marker.

Description

TITLE OF THE INVENTION MOLECULAR FORENSIC SPECIMEN MARKER

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority from U.S. provisional application serial number 60/455,503 filed on March 17, 2003, incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not Applicable

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC [0003] Not Applicable

NOTICE OF MATERIAL SUBJECT TO COPYRIGHT PROTECTION [0004] A portion of the material in this patent document is subject to copyright protection under the copyright laws of the United States and of other countries. The owner of the copyright rights has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the United States Patent and Trademark Office publicly available file or records, but otherwise reserves all copyright rights whatsoever. The copyright owner does not hereby waive any of its rights to have this patent document maintained in secrecy, including without limitation its rights pursuant to 37 C.F.R. § 1.14.

BACKGROUND OF THE INVENTION [0005] 1. Field of the Invention [0006] This invention pertains generally to molecular markers, and more particularly to a molecular identification tag used as a forensic contamination marker for forensic samples and to verify the chain of custody. This invention also pertains to a method of use and a method of manufacture of molecular forensic identification tags. [0007] 2. Description of Related Art

[0008] A composite of pieces of forensic evidence permit a reliable reconstruction of a crime and the activities of the participants in the crime as well as the victim. Some of the most crucial pieces of evidence that are gathered during a criminal investigation include biological evidence from samples containing blood, fibers, hair, and semen. [0009] The analysis of samples of blood, semen, other body fluids and similar biological evidence has become an essential tool for law enforcement investigators who are attempting to identify an individual who has perpetrated a violent crime. Biological evidence may be the only evidence that ties a suspect to a particular crime or that clears an innocent suspect of a crime. [0010] For example, if a sample of blood or DNA collected from a crime scene matches that of a reference sample taken from a particular suspect, the sample can be used as one piece of evidence to directly link the suspect to the scene of the crime. Likewise, if the DNA or blood of the reference sample of the suspect does not match that taken from the crime scene then the suspect may be ruled out as a suspect. [0011] Evidence gathered during a criminal investigation must be properly collected, recorded, analyzed, stored and accounted for from the time of collection to the time of presentation to the court. The chain of custody is a legal standard that applies to the handling and documentation of specimens and testing to be admissible as evidence in a court of law. Analysis of specimens that have breaks in the chain of custody may not be admissible in court and excluded from review by the jury. [0012] Crime scene investigators may use a cotton swab or pad to collect body fluid evidence from the scene of a crime. Typically, swabs with collected biological evidence will be placed in a plastic tube or other container such as plastic bag for transportation and analysis at the lab. The container is usually marked or labeled to identify the evidence. However, the current methods of evidence collection do not adequately prevent tampering or contamination of samples or misidentification of a container. Care must be taken to prevent collected samples from coming into contact with each other during collection, transportation and laboratory analysis. Handling of containers by the investigators at the scene should be minimized to avoid contamination of the collected samples or new samples through cross contamination of lids, containers or evidence bags. Sample containers should also be stored and transported in single bags. [0013] Likewise, procedures need to be taken during sample analysis in the laboratory to avoid cross-contamination of the reference samples with the collected crime scene samples. Even with such precautions, inadvertent tampering and contamination of samples can occur leading to false positives or misidentification of evidence. [0014] In addition, the number of available analytical procedures for biological evidence is steadily increasing. For example, Polymerase Chain Reaction or PCR, is a powerful DNA replication system that allows the selective amplification of target DNA sequences. Target sequences can be replicated many times over in a period of a few hours to produce a significant quantity of material for analysis. PCR can be used to amplify very small sample quantities of DNA or degraded samples of DNA for analysis. For example, PCR has provided conclusive identifications of individuals in cases where conventional DNA typing was inconclusive or ineffective. [0015] PCR mimics the natural process of DNA replication in a test tube.

Generally, a small quantity of target DNA is placed in a test tube with a buffer with containing DNA polymerase, the four nucleotide bases, short single stranded primers and a cofactor such as MgCI₂. Standard PCR uses heat to separate the strands of the DNA molecule of the target sequence to form templates for replication. It has been observed that the hydrogen bonds holding the DNA double stranded helix together can be disrupted when the DNA is heated causing the helix to denature into single strands. [0016] Short single stranded DNA sequences are used as primers. The primers are used to bracket the target section of DNA that is to be amplified by the procedure. The first or forward primer is complimentary to one of the strands of the target DNA sequence and is placed at the beginning of the target sequence. The second or reverse primer is positioned at the end of the target sequence on the opposite strand of the DNA section. The polymerase adds nucleotides to the primer and eventually makes a complimentary copy of the single stranded target template. The process is repeated with each replication cycle copying the DNA exponentially using each denatured strand as a template. However, only the DNA between the primers is amplified exponentially and any other fungal or bacterial DNA that may be present in the sample is not when human specific primers are used. [0017] Although the ability of DNA to provide information for forensics and human identification is indeed powerful, modern DNA sample analysis techniques that allow for the amplification and analysis of minute amounts of DNA are prone to contamination and, consequently, misidentification. For example, contamination from even very small amounts of DNA that is left over from other amplifications can be re-amplified in the current PCR run. Two target DNA segments result and can lead to incorrect or inconclusive results. [0018] Furthermore, DNA used for identification of a criminal perpetrator must be compared against reference samples from one or more suspects. The reference samples are likely to be higher in concentration than the forensic unknowns. This presents the possibility that forensic unknowns could be inadvertently contaminated by reference samples even with strict protocols regarding the handling of the reference samples. If this contamination occurred before a sample was analyzed or early in the extraction process, the contamination would not necessarily be discovered and standard practices of verification though multiple analyses would produce the same erroneous results and therefore be positively misleading. Such false positives can lead to questions about the reliability of forensic examination and can put the examination procedures on trial rather than the defendant. [0019] Similarly, in cases involving paternity, missing persons or historical investigations, contamination of the subject sample by the reference sample may lead to an ultimately erroneous conclusion. [0020] Accordingly, it can be seen that present evidence gathering procedures do no more than identify and track the container rather than the specimen. A method for identifying an organic forensic sample rather than the container as well as a method for confirming the chain of custody is needed to maintain the integrity of a biological sample and ensure the reliability of forensic examination.

BRIEF SUMMARY OF THE INVENTION [0021] The invention generally comprises a molecular identification marker for labeling biological samples and a method for producing and using the marker in forensic evidence gathering or other investigations that rely on the integrity of biological evidence. The molecular identification marker is preferably a segment of double stranded DNA that is between approximately 300 and 500 base pairs in length that has a unique sequence that can specifically identify the marker. The marker is introduced to a forensic biological sample or a reference sample at the time of collection. In one embodiment, the molecular marker is applied to a collection container or swab that is used to collect forensic biological evidence and is therefore present when the evidence is obtained. The swab or collection container is preferably marked with an identifier such as a bar code or serial number so that the marker can be identified and this number is recorded when the sample is collected. [0022] During analysis of the biological sample, the marker is processed with the collected biological sample or reference sample and under normal circumstances the marker is not removed during processing. [0023] The presence of the molecular marker may be confirmed by the presence of abnormal peaks in or may be confirmed by probes that are preferably specific for the marker.

[0024] The molecular marker is preferably created with the use of a blank or template of double stranded DNA that has regions with a sequence that can be manipulated through the use of mutagenic PCR. The template preferably has a segment configured to amplify for a selected forensic locus and a segment configured for use with a probe to quantify the marker, and at least one mutagenic primer site configured to produce a unique variant after mutagenic PCR. It is also preferred that the template include restriction sites that will permit inclusion of the manipulated template into standard cloning vectors. [0025] An aspect of the invention is to provide a forensic marker that will mark the sample and not merely the container. Current methods of detecting contamination must utilize negative controls and reaction blanks that indicate when a proxy reaction has been contaminated. [0026] Another aspect of the invention is to provide a method of using a molecular forensic marker that will allow the sample to be traced and certified. [0027] Another aspect of the invention is to provide a method of producing a molecular marker that is efficient and simple to perform.

[0028] Further aspects of the invention will be brought out in the following portions of the specification, wherein the detailed description is for the purpose of fully disclosing preferred embodiments of the invention without placing limitations thereon. BRIEF DESCRIPTION OF THE SEVERAL VIEWS

OF THE DRAWING(S) [0029] The invention will be more fully understood by reference to the following drawings, which are for illustrative purposes only: [0030] FIG. 1 is a flow diagram of a method of use of a molecular marker for forensic tracking according to the invention.

[0031] FIG. 2 is a flow diagram of one embodiment of a method of producing a molecular marker according to the invention. [0032] FIG. 3 is a schematic diagram of one embodiment of a double stranded template for producing a molecular marker by PCR induced mutagenic insertion of random nucleotides according to the present invention.

[0033] FIG. 4 is a schematic diagram of one embodiment of a double stranded template for producing a molecular marker with targeted PCR mutagenesis. DETAILED DESCRIPTION OF THE INVENTION [0034] Referring more specifically to the drawings, for illustrative purposes the present invention is embodied in the methods generally shown in FIG. 1 through FIG. 4. It will be appreciated that the molecular marker may vary as to configuration and as to details of the parts, and that the method may vary as to the specific steps and sequence, without departing from the basic concepts as disclosed herein. [0035] Reliable identification of biological evidence and accurate DNA analysis are essential to forensic investigations as well as maternity or paternity determinations. Confidence of the legal system in the utility of DNA or other biological evidence to accurately identify familial relationships or the identity of the perpetrator of a crime depends on a stable and predictable system of DNA analysis and custody. While DNA analysis has become an increasingly powerful tool for forensic investigations, mishandling of samples either through inadvertent error or deliberate action is a substantial concern affecting the utility of current practices. Presently, collected forensic samples cannot be labeled directly. Instead, the containers that samples are collected in and stored are labeled to identify the sample. There is, however, no definitive way to determine if the contents of a labeled container have been altered through inadvertent action such as collection error, laboratory error or other contamination. The present invention provides a tag or label that cannot be removed or disassociated from the sample after collection. An appropriate label of the sample rather than the container alone can reduce the significance on the chain of possession of the sample. [0036] One method 100 for tracking samples and identifying the presence of contaminants or determining the source cross-contamination by marking the sample with a molecular tag according to the invention is shown in FIG. 1. At block 1 10, samples are collected. There are generally two types of samples that are collected in the typical investigation where biological evidence is relevant. The first type of sample is a sample that is collected at a crime scene, for example, and is called a forensic sample. Forensic unknowns that are collected at block 120 such as blood or semen may be a source of DNA that can be compared with DNA that is collected from an individual suspected of a crime or from relatives of a missing person or other person trying to identify a familial relationship with another person. This second type of sample is called a reference sample and is collected at block 130 of FIG. 1. It can be seen that the cross contamination of the forensic sample collected at block 120 by the reference sample collected at clock 130 will result in a false positive identification. Likewise, cross contamination of the reference sample with the forensic sample can lead to a false positive and will not only provide false evidence in a legal action but undermines the reliability of DNA evidence and confidence of such evidence in the legal system.

[0037] One embodiment of the method for avoiding the potential for misidentification is the use of a unique DNA segment that is applied to all reference samples in order to distinguish between reference and forensic unknowns that are collected at a crime scene. In another embodiment, a DNA molecular marker is provided to directly label forensic samples at the time of collection that cannot under normal circumstance be removed from the sample. In a third embodiment, both the forensic samples and the reference samples are labeled with a DNA marker so that each sample has a unique identifier associated with the sample. [0038] The molecular marker or tag that is used to label a sample for forensic tracking at block 140 of FIG. 1 is preferably a unique nucleotide sequence, which will provide a tag that is analogous to a serial number or product bar code. Rather than numbers in a series, the unique identifier is defined by the substitution of one of four nucleotides or bases in a stretch of DNA. Such DNA tags or markers could be made unique with a discrete sequence such that each reference or forensic sample could be identified by its own introduced molecular tag. For example, variation in a 10-nucleotide segment can be arranged to produce more than one million unique combinations, variation in 15 nucleotides can produce over a billion unique combinations. Consequently, minor incorporated variations in a sequence can make it uniquely identifiable with extreme precision. Therefore, any sample with the introduced molecular tag would be readily identifiable and any sample that has been cross contaminated with another would likewise be identifiable. [0039] Generally the introduced molecular tag or marker preferably comprises a segment of double stranded DNA that is approximately 300 to approximately

500 base pairs in length. A segment of approximately 400 base pairs in length is preferred. A segment within this range of lengths is of sufficient size to prevent easy separation of the molecular marker element from the collected reference sample as typical DNA extraction processes will extract the introduced element with all other DNA in this embodiment. Within the 400 base pairs are preferably several specific regions designed to facilitate detection. These unique elements may include a segment designed to amplify for a typical forensic locus such as the human D3S1358 STR region producing a fragment of known size without producing a fragment size that will confound genotyping. The marker may also include a segment that is designed to be used with a probe to quantify the introduced molecular marker and will also include a segment of DNA with a unique sequence that is specific to each introduced molecular marker. [0040] It can be seen that the molecular tag can be applied to a forensic or reference sample at block 140 in several ways. The preferred method is to treat collection swabs with the molecular tag of a known sequence. The used swab is usually kept in a container and often with a buffer or preservative after the biological sample is collected. In an alternative embodiment, the molecular tag can be placed in the container or buffer solution that receives the swab or other sample conveyor. [0041] At block 150 of FIG. 1 , the containers with the tagged samples are labeled with suitable indicia to associate collection information with the sample. In one embodiment, the molecular tag is given a number that is placed on the swab and the container. The labeling at block 150 preferably includes the date, time and location of the collection and the identity of the biological material that was collected. Conventional tracking systems that are used by investigators may also be used to identify the sample and the circumstances of the collection of the sample. Reliable tracking systems that are used in addition to the molecular tag of the present invention provide two indicators that the collected samples and the analysis of those samples are genuine and can be confidently admitted into evidence in a court of law. [0042] The biological samples are analyzed at block 160 of FIG. 1 using traditional methods of analysis. Techniques for the extraction, amplification and sequencing of DNA are well known in the art. While the present invention is particularly suited for labeling biological samples that include DNA that will be sequenced, it will be understood that the analysis does not require sequencing of DNA extracted from the biological sample. In one embodiment, the molecular marker can be separated and removed prior to analysis of the forensic sample or reference sample in the laboratory.

[0043] The presence of the molecular tag is confirmed at block 170 of FIG. 1 , preferably through the use of conventional DNA analysis and sequencing methods. For example, if reference samples were collected with cheek-swabs that had been treated with a unique DNA tag, the tag would be present in the sample along with DNA extracted from the individual sampled with the cheek swab. In one embodiment, the molecular tag contains a PCR primer binding site to test to see if the molecular tag was present and in what quantity. The molecular tag may also contain sites for specific probes to quantify the amount of the molecular tag that is present in a particular sample. The introduced molecular tag, though present in all extractions, would not interfere in any way with genotyping and conventional analyses of the sample, and provides a reliable label for the sample. [0044] If the presence of the molecular tag is confirmed at block 170 of FIG. 1 , and the tag corresponds with the sample label then the sample can be certified at block 180 of FIG. 1. If the molecular tag cannot be confirmed then the sample cannot be certified. Strict adherence to a policy of collecting samples with swabs treated with the molecular tag would mean that forensic samples that do not show the presence of an introduced molecular marker would indicate that the sample had been compromised or contaminated or inaccurately marked or have some other problem with the chain of possession of the sample. [0045] Since each introduced molecular marker has a unique sequence, the original source of each marker can be traced down to the specific swab used to collect the sample. It is preferred that records of the unique sequences be stored in a secure database before swabs are sent out for sample collection.

This system would guard against inadvertent swapping of reference samples prior to or during the extraction process as well as guarding against reference samples cross contaminating forensic samples and the like. Additionally, carry-over PCR from reference samples tainting forensic samples would be detectable, though not traceable to a specific swab. [0046] The detection of the introduced molecular marker is possible in several ways known in the art. In one embodiment, each molecular marker will preferably contain a section of DNA that is analogous to a human STR region utilized in standard forensic identification, amplification of a sample with primers for this STR region would also amplify this portion of the introduced element. For example, if the molecular marker element contained the flanking regions, but no copies of the tetra-nucleotide repeats, from D3S1358, amplification with available PCR kits would produce an approximately 67 base pair fragment if the sample contained the molecular marker. This 67 base pair fragment could not be confused with any natural human DNA as variation in humans produces amplified products ranging from 97 to 145 base pairs depending on the number of tetra-nucleotide repeats between the flanking regions. The presence of 67bp peak would thus not interfere with genotyping but would indicate the sample contained the element. If the sample were supposed to have contained the element (e.g. it was a reference sample collected with a treated swab), failure to see the 67bp peak would invalidate the authenticity of the sample. If there was no record that the sample had been collected with such a swab containing a molecular marker, this would indicate that the sample had been compromised with a foreign DNA, either by mixing samples, planting samples, or carry-over PCR from other investigations. This would positively guard against evidence being either errantly produced and against allegations that evidence was intentionally planted. [0047] If a sample was suspected to have been compromised by visualization of an anomalous peak (e.g. a 67bp peak for D3S1358), a molecular probe, designed to adhere to part of the introduced element, could be introduced to see if the introduced element was present in the DNA template or if the peak occurred as a result of carry-over PCR in the PCR reaction. It can be seen that such tests would greatly aid in detecting and controlling contamination in forensic laboratories and adding to quality assurance programs in such labs. [0048] While the presence of PCR products or probes would indicate if an introduced element were present in a sample, they would not be able to detect the exact source of the contamination in this embodiment. However, as each introduced element has a known unique sequence, the samples can be sequenced and compared against the database files created for each molecular marker element to verify which collection swab or other collection vessel the contaminating element had been initially applied. Accordingly, if a sample was found to have been compromised, the contamination could be traced back to a single collection swab sent to a single laboratory used on a single individual. A sample that has not been compromised could be further validated by showing that the introduced element matched that applied to the collection swab alleged to have been used to take the sample. This would greatly aid in verification of the chain of custody of samples. [0049] In another embodiment, the sequence of the unique portion of the marker for the introduced elements of both the forensic and reference samples are determined and validated through sequencing. [0050] If the presence of the molecular tag is not confirmed at block 170 of

FIG. 1 , then the presence of a different molecular marker is optionally determined at block 190 of FIG. 1. If a different marker is found to be present at block 190, then the source of contamination is preferably determined at block 210 of FIG. 1. [0051] Detection of a marker at block 170 of FIG. 1 , indicates conclusively that the sample DNA that is the subject of forensic analysis had been exposed to a marker at some point. Current methods of detecting contamination must utilize negative controls and reaction blanks, which only indicate if a proxy reaction has been contaminated. Sequencing of the marker allows precise identification of the source of the marker. If the sample was not collected with a molecular marker present, and the analysis results indicate the presence of a marker then the unique sequence can indicate the source of contamination.

If the marker is not detected in a sample known to have been collected with a molecular marker, the identity of the sample and the chain of custody are questionable and further investigation into the possibility of contamination is warranted. Furthermore, the original source of contamination can be determined in the event that the samples are switched or there is a recording error. [0052] Turning now to FIG. 2, one method 220 for producing a molecular marker according to the present invention is shown. At block 230 of FIG. 2, a blank or template of double stranded DNA that is approximately 300 to 500 base pairs long is provided as a base molecular marker for manipulation. It can be seen that the template strands can be engineered to include sequences that facilitate the engineering of the template, provide probe recognition sites or permit the easy amplification final marker and the like. Examples of preferred designs for a template DNA strand can be seen in FIG. 3 and FIG. 4. [0053] There are numerous methods that may be used to create unique DNA sequences known in the art and many of these methods are commercially available in kits or offered as custom oligonucleotide synthesis and gene synthesis services to order. For example, standard synthesis of oligonucleotides is now possible using any of a number of commercially available automated synthesizers. However, the oligonucleotides produced from such synthesizers are generally of limited length since it is difficult to accurately synthesize oligonucleotides much in excess of 100 base pairs and they are synthesized as single-stranded DNA. Commercial services may be useful in producing blanks or templates but the cost of such services make them unsuitable for use for producing forensic markers with unique sequences due to the number of different sequences that may be used with the method shown in FIG. 1. [0054] Referring now to block 240 of FIG. 2, a region of the marker is created that has a unique sequence that will serve as a distinguishing feature of the molecular marker. One method is to insert a short segment of DNA into the pre-designed DNA template that is provided at block 230 of FIG. 2. The short segment may be synthesized with a unique sequence and spliced into a section of the blank or template that is provided at block 230 of FIG. 2. [0055] A second method for creating a limited number of unique sites within the template strand is through selective mutation by PCR-induced mutagenesis. A wide variety of procedures for site-directed mutagenesis based on polymerase chain reaction (PCR) have been developed over the last decade. One popular PCR procedure is called "megaprimer" PCR that requires three primers and two rounds of PCR to generate a set of mutations. The first primer introduces a mutation in the sequence and the second and third primers flank the region with the first primer. One of the flanking primers is used with the mutated primer for the first round of PCR. The resulting double stranded product is purified and used as a "megaprimer" along with the third primer that was flanking region of interest in the second round of PCR. [0056] The preferred method for creating the molecular marker is to use only a single round of PCR-induced mutagenesis, utilizing degenerative primers to introduce the novel mutations into regions of the original template provided at block 220. In this embodiment, the use of degenerative primers has the advantage of requiring only a single round of PCR to generate the necessary variation. [0057] In one embodiment, utilizing this strategy, the original template strand has at least one restriction site and is first cleaved by a restriction digest to produce two fragments of double stranded DNA, fragment A and fragment B.

The digested products are preferably separated by electrophoresis or other suitable method for separation. A variant of megaprimer-extension PCR is then employed. One set of collected fragments, fragment A, is mixed with a conventional PCR reaction mix with three primers. The first or internal primer preferably includes a section that is complimentary to a section of the collected fragment A. The internal primer also includes a number of "N" bases, preferably synthesized with equal mola ty of all four nucleotides. These N bases are preferably located near the 3' end of the internal primer and are not complementary to the template strand. The number of N bases can vary creating greater or lesser degrees of variability in sequence between final products. The 3' end of the internal primer preferably contains a sequence that is complementary to fragment B of the template strand to facilitate annealing with fragment B. [0058] The internal primer with the degenerative sequence anneals with fragment A and the strand is extended from the primer to provide a double stranded fragment with "N" number of random nucleotides. Because of the degenerative nucleotide composition of the internal primer in the first mutagenic PCR reaction, there are limitations for exponential replication of the mutated A strand. In one embodiment, a third primer is included to facilitate exponential replication in the first PCR reaction. [0059] In another embodiment, the newly modified double stranded fragment A is isolated and mixed with fragment B, which is complimentary to one end of the fragment A, and suitable primers and reagents for extension of the megaprimer and exponential replication PCR. The resulting molecule is a modified template molecule with a region of sequence variability. The mutagenic PCR process thus produces a family of products differing at random based on the random composition of the "N" bases.

[0060] The inclusion of different number "N" bases in the mutagenic primers determines the relative number of variants in the "population" of mutagenic products. The number of possible combinations is a product of the number of N bases included, as four raised to the power of N possible unique sequences of nucleotides are produced (e.g. 10 N bases results in 1 ,048,576 possible nucleotide sequences for example). [0061] Additionally, the possibility exists for some recombination of un- mutated native template strands lacking any sequence variability. However, all mutagenic products lack the restriction site, a result of the targeted mutation designed to eliminate a restriction site designed into the original native strand.

To eliminate these recombinants, the PCR product is treated with the appropriate restriction enzyme and the digest is run on an electrophoretic gel in one embodiment. The gel separates the DNA by size, with digested, unmutated strands moving more rapidly through the gel medium. Strands that run slower on the gel did not digest and thus indicate mutagenesis has occurred. These strands can be cut out of the gel and purified for additional replication through PCR or immediate further processing to create a molecular marker. [0062] Referring further to block 240 of FIG. 2 and to FIG. 4, an alternative method for creating a region of unique sequence in a template marker in shown. The previously described method produces unique markers essentially through the insertion of random nucleotides. However, it is also possible to modify the native template strand in such a manner as to direct the changes specifically through a process of mutagenic PCR. [0063] Mutagenic PCR is often observed to have a comparatively low efficiency. However, by selectively targeting regions that will destroy restriction sites in the native template strand during PCR, mutated products can be separated from unmutated or partially mutated strands by exposing the DNA to specific restπction endonucleases that cleave fragments that have not been mutated and the separating the fragments with gel electrophoresis. After separation, the purified mutated products can be copied easily through standard PCR and subsequent restriction digests should confirm that mutagenesis has taken place. [0064] PCR generally requires specifically designed oligonucleotides that serve as internal primers to introduce a limited number of mutations (e.g. 3 or 4 base changes from the native strand) in a region of approximately 30 base pairs. Consequently, this requires a unique oligonucleotide. Alternatively, with common overlapping extension- PCR, two unique oligonucleotides that are partially complementary are used to produce characteristic mutations. Thus, a unique oligonucleotide would be required to introduce a unique mutation in any desired region of the native template strand. Accordingly, the production of 10 uniquely mutated products would require 10 separate mutagenic PCR cycles upon the native template strand in a desired region. However, each of these mutated products can be further mutated with subsequent reactions utilizing additional primers that mutate a second region of the template. Thus, 20 mutagenic primer pairs (10 in region one and 10 in region two) can be combined to produce 100 completely unique DNA sequences that can be used as markers. Likewise, fifty primers in each region can be paired such that 100 primers (50 in region one and 50 in region two) can produce 2500 unique sequences (50x50). Furthermore the use of 50 primers each in four distinct regions can produce 6,250,000 unique markers upon the same initial native template strand. [0065] Referring now to block 250 of FIG. 2, the final PCR products are preferably sub-cloned into an appropriate cloning vector to isolate and multiply the markers that each has a unique sequence. The PCR products may be sub-cloned with the use designed restrictions sites at each end of the marker or by commercially available cloning kits to provide cloned colonies. Each of the cloned colonies will contain the same created sequence. [0066] At block 260 of FIG. 2, the cloned fragments are separated from the colonies and the vector and preferably sequenced to confirm the sequence. Once sequenced and compared against a database of previously supplied markers to confirm uniqueness of the sequence, the colony can be used to provide a molecular marker to be used in collecting DNA samples. The confirmed marker is preferably placed into a pool of markers with known unique sequence. Any repetitive markers are preferably removed from the pool of markers. The variable sequence of each verified marker in the pool is preferably logged into a secure database. At block 270 of FIG. 2, an identifier such as a serial number is preferably assigned to the individual sequence and to the swab or container that receives the molecular identification marker. [0067] The verified markers from the pool can be applied to a swab, vacutainer, or other device utilized to collect a sample for DNA collection and packaged at block 280 of FIG. 2. The collection device or container containing the molecular marker is preferably assigned a serial number that is paired with the sequence information and logged into a secure database. The collection device or swab etc is preferably labeled with the serial number that has been assigned to the marker and placed in sterile packaging and shipped for use in the field. [0068] Referring also FIG. 3 and FIG. 4, preferred designs for the blank or template strands are schematically shown. FIG. 3 is configured for PCR induced mutagenic insertion of random nucleotides described at block 240 of FIG. 2. The marker template shown in FIG. 4 is a preferred design for targeted mutagenesis that is described as an alternative embodiment at block 240 of FIG. 2. The schematic representations of FIG. 3 and FIG. 4 are not intended to indicate scale or any particular sequence or position. It will be seen that there is flexibility in the design of the marker templates.

[0069] Since standard methods for analyzing forensic samples use amplification through PCR, primers specific to known regions of the human genome are typically used. Accordingly, the region 310 of the strand shown in FIG. 3 is a sequence that matches the flanking region of a forensic STR site. In standard STR analysis, the number of repeats of a repetitive nucleotide motif determines alleles. For example, in D3S1358, this motif is generally a GATA repeat. Natural human variation has a range of between 8 and 20 copies of this repeat. D3S1358 is used as a particular example because it is common to most forensic multiplex kits both in the United States and internationally. However, it will be understood that the flanking regions from any locus examined, minus the repetitive motifs, could be used should detection of the marker be desired. [0070] The flanking region will amplify with human STR primers but will produce a non-standard peak since the normal STR repeats are not included in region 310 of the template 300. Accordingly, the introduced DNA marker will not interfere with forensic analysis of the unknown or the reference samples. [0071] In one embodiment, the custom designed native strand is designed to have flanking regions that are complementary to primers in standard forensic "multiplex" kits. For example, by including regions containing primer-binding sites for STR primers used in standard forensic multiplex kits such as. D3S1358, a CODIS marker present in the Cofiler, Profiler Plus and Identifier AmpFlster kits sold by Applied Biosystems, Inc., the STR profiles would produce a non-standard peak for the locus, thereby facilitating the detection of the introduced marker though not identifying the actual sequence of the introduced marker. Therefore, the presence of the marker can be detected with any multiplex analysis but will not interfere with the genotyping of the sample itself. [0072] For the D3S1358 example, the sequence of the region of the genome is described in genebank accession number 11449919. This published sequence represents a variant with 18 copies of the repeat. A PCR product of 137 base pairs with this variant is produced by one available multiplex kit. In this example, the DNA from an individual with the variant allele with only 8 copies of the repeat, the minimum standard allele, would produce a PCR product of 97 base pairs in length. In this case, the PCR product contains 32 bases that are of the repetitive DNA motif and 65 bases flanking the region, split on either side of the repeat.

[0073] By incorporating a sequence that incorporates more than 65 bases upstream and downstream of the repeat, in this case a 133 base pair fragment, the multiplex kits will be able to amplify the sequence and produce a fragment 65 base pairs in length. This fragment is identifiable on standard instruments but too short to be confused with a natural human allele.

Consequently, any sample purposely labeled with the molecular marker would produce a 65 base pair fragment, in addition to the regular allele(s) in the sample, indicating conclusively that the sample had been labeled. [0074] In addition, the particular -130 bp fragment is not part of the region of the native template strand altered by mutagenesis to make each marker unique. Therefore detection of the 65bp fragment identifies that a molecular marker for forensic tracking is present. Subsequent sequencing of the template analyzed using primers specific for the entire molecular marker make it possible to check the sequence and determine the precise unique marker present that produced the 65 bp "allele."

[0075] Additionally, as the sequence of the entire molecular marker is known and the variation in sequence that makes each marker unique occurring only in a specific designed region, forensic multiplex kits themselves can be designed to include primer sequences. If these sequences are outside of the mutagenic regions, the PCR products could encompass the whole of the molecular marker and would thus include the unique mutated regions of the molecular marker. In this case, there would be no need to amplify the initial template used for forensic STR analysis as the PCR product from the multiplex kit analyzed could be sequenced directly to determine the sequence of the unique marker. [0076] It can be seen that the specific primer locations on the marker could easily be designed so that the PCR product would not be confused with a real genotype. Samples that showed such a peak would indicate that a marker was present. If a sample were not collected with a marker-labeled device, it would indicate that the integrity of the sample had been compromised with another sample collected with a marker. Conversely, a sample collected with, for example, a labeled swab, that did not produce the non-standard peak in one of the STR multiplex analyses would likewise indicate that the sample was not genuine. [0077] It is preferred that the template strand 300 have restriction sites 320 to facilitate cloning into most cloning vectors. The selection of the restriction site may be determined by the particular cloning vector that is used.

[0078] At least one set of forward and reverse replication and sequencing primer recognition sites 330A and 330B are provided to assist in the amplification of the molecular marker. Sequences can also be utilized for primers that are part of typical multiplex kits. Since majority of the sequence of the molecular marker template 300 is known, it is possible to design additional primers specific only to molecular markers that will selectively amplify the marker. Therefore, if such primers are included in PCR reaction mixes during standard analysis of an extract, they will amplify the marker and facilitate identification of the specific marker, traceable back to a specific swab, container or other collection device that was used.

[0079] It is also preferred that the template 300 have a region 340 with a sequence that permits probe recognition to detect and quantify the marker in samples. The sequence is preferably specific to the marker and not detected by human specific probes. In another embodiment, the molecular marker is identified with SNP probes for detection. This may be particularly useful with

SNP typing for forensic identification. [0080] Finally, the embodiment shown in FIG. 3 includes a restriction site 350 that separates the template into two fragments A and B for PCR induced mutagenic insertion of random nucleotides as described at block 230 of FIG. 2. The restriction site 350 should be different than the restriction site 320 selected to facilitate the use of a cloning vector. [0081] Turning now to FIG. 4, an alternative double stranded template 400 embodiment is shown schematically that is particularly suited for targeted PCR induced mutagenesis. In the embodiment shown, region 410 of segment 400 is approximately 140 base pairs in length and comprises a flanking region of a human STR site. A region of approximately 70 base pairs on both sides of the repeat region with the repeat deleted is preferred. The sequence of region 410 of segment 400 is preferably selected to be amplified for a typical forensic locus and produce a fragment of known size upon analysis that will not interfere with genotyping of the collected sample. [0082] Restriction sites 420 are placed near each end of the template strand to permit insertion of the modified template strand into a standard cloning vector. EcoRI restriction sites are preferred. The template 400 also preferably has at least one forward replication primer binding site 430A and at least one reverse replication primer binding site 430B or the like to facilitate the amplification of molecular marker. [0083] Optionally, a region 440 is provided in template 400 that has sequence that is a target for adhesion of a molecular probe to aid in detection of the marker in a sample. It is preferred that the sequence of region 440 of the template 400 be unreactive to human specific probes in the embodiment shown in FIG. 4. [0084] A plurality of regions 450 are provided that include a restriction site in mutagenic regions prior to PCR mutagenesis. Each region 450 is preferably approximately 60 bp to approximately 80 bp in length and contains a unique restriction site. In one embodiment, the regions 450 were Haelll restriction sites. In another embodiment, the regions 450 each had different restriction sites. It will be seen that mutagenesis will alter and remove the restriction sites. Although four mutagenic binding regions with restriction sites are shown, it will be understood that more or less than four regions may be used. Mutagenesis is preferably performed by overlapping extension PCR. [0085] As described previously, the process of creating the marker utilizes mutagenic PCR using successive mutagenesis at different locations 450 along the initial template 400. The recombination of different mutations at these different locations makes possible a sufficient variety of unique sequences for final markers with a limited number of oligonucleotides for mutagenic PCR. In addition, since the creation of each unique segment destroys (rather than creates) a restriction site, it is easy to separate out unique mutated DNA strands from un-mutated strands by restriction digests followed by size separation through electrophoresis.

[0086] It can be seen that the molecular marker that is produced from either template 300 or template 400 will provide an identifier that can directly mark the sample and will not interfere with the genetic analysis of the sample. It can also be seen that there is flexibility in the design of the molecular marker template to allow templates to be adapted to use many different sequences and elements. [0087] Accordingly, the present invention provides a molecular marker that will mark the sample that is easy to use. Detection of marker indicates conclusively that the template used for forensic analysis had been exposed to a marker at some point and sequencing will indicate the source of the marker.

In the event that the samples are switched, the markers can determine the original source of the contamination. [0088] Although the description above contains many details, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the presently preferred embodiments of this invention.

Therefore, it will be appreciated that the scope of the present invention fully encompasses other embodiments which may become obvious to those skilled in the art, and that the scope of the present invention is accordingly to be limited by nothing other than the appended claims, in which reference to an element in the singular is not intended to mean "one and only one" unless explicitly so stated, but rather "one or more." All structural, chemical, and functional equivalents to the elements of the above-described preferred embodiment that are known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the present claims. Moreover, it is not necessary for a device or method to address each and every problem sought to be solved by the present invention, for it to be encompassed by the present claims. Furthermore, no element, component, or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims. No claim element herein is to be construed under the provisions of 35 U.S.C. 112, sixth paragraph, unless the element is expressly recited using the phrase "means for."

Claims

CLAIMS What is claimed is:

1. A method for labeling a sample of unknown biological evidence, comprising: providing an unknown biological sample; and associating a molecular label with said biological sample.

2. A method as recited in claim 1 , further comprising: confirming the presence of the molecular label in the biological sample by analysis.

3. A method as recited in claim 2, further comprising: assigning a first reference identifier to said biological sample; assigning a second reference identifier to said molecular identification label; and matching said assigned reference identifier of said molecular identification label with said assigned reference identifier of said biological sample at the time of analysis of said biological sample.

4. A method as recited in claim 2, further comprising: recording said assigned first reference identifiers and said second reference identifiers.

5. A method as recited in claim 4, further comprising: maintaining a database of recorded reference identifiers of said molecular identification labels and said reference identifiers of said biological samples.

6. A method as recited in claim 2, wherein said introduction of marker comprises: applying a known quantity of a molecular identification label to a collection swab that is used to obtain said biological sample.

7. A method as recited in claim 6, further comprising: marking said collection swab with said reference identifier for said molecular identification label.

8. A method as recited in claim 2, wherein said introduction of marker comprises: applying a known quantity of a molecular identification marker to a collection vial that is used to store said collected biological sample.

9. A method as recited in claim 8, further comprising: marking said collection vial with said reference identifier for said molecular identification label.

10. A method for identifying cross-contamination in biological samples, comprising: collecting a plurality of biological samples for analysis; labeling each collected sample with a discrete molecular identification marker; and confirming the presence of said marker at the time of analysis of said biological sample.

11. A method as recited in claim 10, further comprising: assigning a reference identifier to said biological sample; assigning a reference number to said molecular identification marker; and matching said assigned reference number of said identification marker with said assigned reference identifier of said biological sample at the time of analysis of said biological sample.

12. A method as recited in claim 10, further comprising: sequencing said molecular identification marker of said biological sample at the time of analysis of said biological sample; and verifying the sequence of said molecular identification marker with a database of sequences of identification markers.

13. A method as recited in claim 10, further comprising: identifying the presence of an unassigned molecular marker in said biological sample; isolating said unassigned molecular marker; sequencing said unassigned molecular marker; and identifying the source of the unassigned molecular marker.

14. A method as recited in claim 10, wherein said presence of said marker is confirmed with a probe.

15. A method as recited in claim 10, further comprising: collecting a plurality of reference samples for analysis and comparison with said biological samples; labeling each collected reference sample with a discrete molecular identification marker; confirming the absence of said label of said reference sample at the time of analysis of said forensic biological samples; and. confirming the absence of said label of said biological sample at the time of analysis of said reference samples.

16. A method as recited in claim 15, further comprising: assigning a reference identifier to said reference sample; assigning a reference number to said molecular identification marker; and matching said assigned reference number of said identification marker with said assigned reference identifier of said reference sample at the time of analysis of said reference sample.

17. A method for identifying cross-contamination of biological samples and reference samples, comprising: collecting a plurality of reference samples for analysis and comparison with forensic unknown biological samples; labeling each collected reference sample with a discrete molecular identification marker; and confirming the absence of said label at the time of analysis of said forensic biological samples.

18. A method as recited in claim 17, further comprising: confirming the presence of said label at the time of analysis of said reference samples.

19. A method as recited in claim 18, wherein said presence of said marker is confirmed with a probe.

20. A method as recited in claim 17, further comprising: assigning a reference identifier to said reference sample; assigning a reference number to said molecular identification marker; and matching said assigned reference number of said identification marker with said assigned reference identifier of said reference sample at the time of analysis of said reference sample.

21. A method for collecting biological evidence samples, comprising: providing a biological sample collection swab; applying a quantity of a molecular marker to said collection swab; and using said collection swab to collect biological samples for analysis.

22. A method as recited in claim 21 , further comprising: assigning a first reference identifier to said biological sample collection swab; assigning a second reference identifier to said molecular marker; and matching said assigned reference identifier of said molecular identification marker with said assigned reference identifier of said biological sample collection swab at the time of analysis of a collected biological sample.

23. A method as recited in claim 22, further comprising: recording said assigned first reference identifier and said second reference identifier.

24. A method as recited in claim 23, further comprising: maintaining a database of recorded reference identifiers of said molecular identification labels and said reference identifiers of said biological samples.

25. A molecular identification marker, comprising: a segment of a nucleic acid with a characteristic sequence.

26. A molecular marker as recited in claim 25, further comprising: a sequence recognized by probes.

27. A molecular marker as recited in claim 26, wherein human specific probes do not detect said probe sequence.

28. A molecular marker as recited in claim 25, further comprising: a segment configured to amplify for a selected forensic locus;

29. A molecular marker as recited in claim 28, wherein said segment comprises: a sequence substantially matching a flanking region of a STR site.

30. A molecular marker as recited in claim 29, wherein said flanking region comprises an upstream region and a downstream region of a STR site.

31. A molecular marker as recited in claim 25, further comprising at least one pair of restriction sites configured to permit inclusion into a cloning vector.

32. A molecular marker as recited in claim 30, further comprising at third restriction site.

33. A molecular marker as recited in claim 25, further comprising at least one mutagenic primer site configured to produce a unique variant after mutagenic PCR.

34. A molecular marker as recited in claim 25, further comprising:

A plurality of primer recognition sites to permit amplification of said molecular marker.

35. A method for producing a molecular identification marker for labeling a sample of unknown biological evidence, comprising: providing a template marker of double stranded DNA; changing the sequence of at least one segment of said template marker to provide a unique sequence with mutagenic PCR; and cloning said changed template marker to provide a plurality of identical molecular identification markers with a specific unique sequence.

36. A method as recited in claim 35, wherein said provided template marker further comprises: a segment configured to amplify for a selected forensic locus; a segment configured for use with a probe to quantify the marker; and at least one mutagenic primer site configured to produce a unique variant after mutagenic PCR.

37. A method as recited in claim 36, wherein said mutagenic primer sites of said provided template are located at the same position on said template as a restriction site.