TO CONFIRM PATIENT IDENTIFICATION

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/467,164, filed March 24, 2011, which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

[0001] The present invention relates to the field of genetic testing, and more specifically to comparison of genetic material between samples taken from the same individual for identification and confirmation of medical diagnoses.

Description of Related Art

[0002] Millions of biopsies are taken from patients in the United States each year for diagnosis of diseases such as cancer. For example, it is estimated that approximately 1.6 million new incidences of cancer are diagnosed in the U.S. every year (American Cancer Society, Cancer Facts and Figures, 2011). These biopsies are labeled and examined, and results are provided to the doctor, who makes treatment decisions based thereon. However, the procedures for obtaining a biopsy, subsequently testing the sample, and reporting results to the doctor who informs the patient and forms a treatment strategy are numerous and complex, and errors are common.

[0003] Errors in the above process may occur at any stage, but often occur in one of three areas: specimen transposition, foreign cell contamination, and patient misidentification. It is Icnown that errors occur; however, it is unknown how common such errors are in the practice of surgical biopsies. Some studies have indicated that as many as five percent of biopsies are defective. Considering the significant number of biopsies taken each year, the total number of potential errors is staggering.

[0004] By verifying identification of the patient and the appropriate biopsy sample with a high degree of accuracy, a significant number of errors may be avoided, resulting in a decrease in unnecessary surgeries, savings in the healthcare system, and a reduced number of medical malpractice suits based on such errors.

[0005] Current solutions for this problem are inadequate. As such, there exists a need in the art to provide rapid and accurate identification of biopsy samples for confirmation of genetic identity with specific patients.

SUMMARY OF THE INVENTION

[0006] Provided herein are methods of verifying the identity of a biological sample by obtaining a biological sample and a biological reference sample from a patient. Genetic material is extracted from the biological sample and the biological reference sample. The genetic material is amplified to produce biological amplicons and biological reference amplicons. The amplicons are then compared in such a way that a match between the biological amplicon and biological reference amplicon indicates that the biological sample and the biological reference sample are from the patient.

[0007] Also provided herein are methods of verifying the identity of a biological sample by obtaining a biopsy sample and a reference sample from a patient. Genetic material is extracted and isolated from the biopsy sample and the reference sample. From each sample a hypervariable region of mitochondrial DNA is amplified to produce a first plurality of amplicons corresponding to the biopsy sample and a second plurality of amplicons corresponding to the reference sample. The first and second plurality of amplicons are then compared in such a way that a match between the first plurality of amplicons corresponding to the biopsy sample and the second plurality of amplicons corresponding to the reference indicates that the biopsy sample and the reference sample are from the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 is a flow diagram of a process of verifying the identity of a biological sample according to one non-limiting embodiment of the present invention;

[0009] FIG. 2 is a flow diagram of a process of verifying the identity of a biological sample according to another non-limiting embodiment of the present invention; and

[0010] FIG. 3 is a flow diagram of a process of verifying the identity of a biological sample according to a further non-limiting embodiment of the present invention. DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The following description is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses. While this disclosure is described as having exemplary steps and applications, the present disclosure can be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations of the disclosure using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice of those skilled in the art to which this disclosure pertains and which fall within the limits of the appended claims.

Definitions

[0012] The following brief explanations of terms are provided:

[0013] Amplifying: To increase the number of copies of a nucleic acid molecule. The resulting amplification products are called amplicons. Amplification of a nucleic acid molecule refers to use of a technique that increases the number of copies of a nucleic acid molecule in a sample. An example of amplification is the polymerase chain reaction (PCR), described more thoroughly below. The product of amplification can be characterized by such techniques as electrophoresis and sequencing, also described below.

[0014] DNA: Deoxyribonucleic acid (DNA) is a long chain polymer which comprises the genetic material of most living organisms. The repeating units in DNA polymers are four different nucleotides, each of which comprises one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached. Unless otherwise specified, any reference to a DNA molecule is intended to include the reverse complement of that DNA molecule. The term "DNA", as used herein, encompasses both single and double-stranded DNA molecules. Thus, a reference to DNA, or a fragment thereof, encompasses both the sense strand and its reverse complement.

[0015] DNA sequencing: The process of determining the nucleotide order of a given molecule of nucleic acid. Specific types of sequencing are explained in more detail below.

[0016] Mass spectrometry: An analytical technique utilized to obtain the molecular mass of a molecule based on the charge-to-mass ratio of the molecule. In a general method, a sample is vaporized, ionized, and separated on the basis of mass-to-charge ratio in a plurality of electromagnetic fields. Ions are detected, generating a signal, and the signal is processed into spectra.

[0017] Mitochondrial DNA: Non-chromosomal DNA located within mitochondria. Mitochondrial DNA contains coding and non-coding regions, included in the non-coding region is a control region known as the hypervariable region. Within the hypervariable region are hypervariable region I and hypervariable region II, which are unique for the high level of variability seen between individuals.

[0018] Primer: Primers are short nucleic acid molecules, for example DNA oligonucleotides 10 nucleotides or more in length, which are annealed to a complementary target nucleic acid molecule in a PCR protocol. A primer can be extended along the target nucleic acid molecule by a polymerase enzyme, for example, Taq polymerase. Therefore, primers can be used to amplify a target nucleic acid molecule. The specificity of a primer increases with, length. Thus, for example, a primer that includes 30 consecutive nucleotides will anneal to a target sequence with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, to obtain greater specificity, probes and primers can be selected that include a greater number of nucleotides.

[0019] RNA: Ribonucleic acid (RNA) is a long chain polymer of nucleotides. The repeating units in RNA polymers differ from those of DNA, in that they are adenine, guanine, cytosine, and uracil (rather than thymine) bound to a ribose sugar to which a phosphate group is attached. While RNA in the majority of cases adopts only a single-stranded structure, the term should not be construed to exclude double-stranded RNA. RNA may refer to messenger RNA (mRNA), ribosomal RNA (rRNA), or transfer RNA (tRNA).

[0020] Sample: A sample as utilized herein may include any type of biological sample from which genetic material may be extracted and isolated such as, without limitation, blood, saliva, hair, buccal swab, tissue sample, tumor sample, or the like.

[0021] Sequence identity: The comparison step of the present methods described below may utilize comparison of sequences, thus a comparison of sequence identity. Sequence identity may be described as the similarity between two or more nucleic acid sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Methods of alignment of sequences for comparison are well known in the art. Altschul et al., Basic local alignment search tool. J. Mol. Biol. 215(3): 403-10 (1990), presents a detailed consideration of sequence alignment methods. [0022] Other terms utilized frequently in the below description are defined therein. It is understood that many terms, reagents, and techniques are familiar to those skilled in the art, and as such, have not been specifically defined.

[0023] The present invention provides methods of verifying the identity of a biological sample by obtaining a biological sample and a biological reference sample from a patient. Genetic material is extracted and isolated from the biological sample and the biological reference sample. The genetic material is amplified to produce biological amplicons and biological reference amplicons. The amplicons are then compared in such a way that a match between the biological amplicon and biological reference amplicon indicates that the biological sample and the biological reference sample are from the patient.

[0024] With reference to Fig. 1, in typical usage, a biological sample is obtained from a patient 110. The biological sample may be of any type in which identification and matching of the sample to a patient is important. In non-limiting embodiments, the biological sample is a biopsy sample, obtained in a manner known to those sldlled in the art. For example, and without limitation, a biopsy sample may be obtained by use of a biopsy needle. In a preferred, non- limiting embodiment, the biopsy sample is a tumor biopsy sample, and may be a biopsy obtained from any type of tumor. Without limitation, the tumor may be benign or malignant, and may correspond to breast cancer, ductal carcinoma, lobular carcinoma, colon cancer, colorectal cancer, adenocarcinoma, lung cancer, stomach cancer, liver cancer, pancreatic cancer, skin cancer, cervical cancer, uterine cancer, ovarian cancer, prostate cancer, testicular cancer, leukemia, lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, melanoma, head and neck cancer, thyroid cancer, bladder cancer, benign or metastatic brain tumor, glioblastoma, oligodendroglial tumor, astrocytic tumor, pineal gland tumor, and/or pituitary tumor. In further non-limiting embodiments, the biological sample is a blood sample. In non-limiting embodiments, a unique bar code corresponding to the patient is applied to the biological sample, and the bar code is applied to each container or receptacle in which subsequent reactions or analyses (described below) are conducted, so that the sample may be verified as originating from the patient.

[0025] With further reference to Fig. 1, a biological reference sample is also obtained from the patient 120, to obtain a genetic reference for the patient. The genetic identity of the biological sample will be compared to the genetic identity of the biological reference sample to determine whether the biological sample, for example a biopsy sample, originated from the patient. In non- limiting embodiments, the same unique bar code applied to the biological sample is applied to the biological reference sample, so that the reference sample may be verified as originating from the patient.

[0026] In non-limiting embodiments, the biological reference sample is a buccal sample, which may be obtained in any usual manner known to those skilled in the art. In non-limiting embodiments, the buccal sample is obtained by a buccal swab. In other non-limiting embodiments, the biological reference sample is a blood sample or a saliva sample.

[0027] The biological reference sample may be obtained at the same time, and in the same place, that the biological sample is obtained. In non-limiting embodiments, the biological reference sample is obtained at a different time than the biological sample. In other non-limiting embodiments, the biological reference sample is obtained at another geographic location. For example, in a non-limiting embodiment in which the biological sample is a biopsy sample, obtained at a physician's office or hospital, the biological reference sample may be obtained at the physician's office or hospital at the time that the biological sample is taken. Additionally, in non-limiting embodiments, the biological reference sample may be obtained at the physician's office or hospital at a later time than the biopsy sample is obtained. In further non-limiting embodiments, the biological reference sample is obtained at a different location, for example at the patient's home.

[0028] Once the patient's biological sample and biological reference sample have been obtained, genetic material is extracted and isolated 130 from the respective samples. In non- limiting embodiments, the genetic material is DNA. hi a preferred, non-limiting embodiment, the isolated genetic material is mitochondrial DNA (mtDNA). In other non-limiting embodiments the isolated genetic material is RNA. In further non-limiting embodiments, the isolated genetic material is mRNA.

[0029] Extraction of genetic material may be carried out by any manner known to those skilled in the art. In non-limiting embodiments, the process includes lysing cells, removing membrane lipids, and precipitating the genetic material. Each step may be carried out in any manner known to those skilled in the art. For example, cells may be lysed through use of mechanical grinding, sonication, or through use of solutions such as lysis buffers or detergents, and membrane lipids may be removed by use of detergents or surfactants. Isolation kits are common in the art, and often contain a single solution for extraction. For example and without limitation, a typical kit may contain a lysis solution containing 200 nM sodium chloride, 10 mM Tris, 20 mM EDTA, sodium dodecyl sulfate (SDS) or Triton X-100, and 100 μ^πύ Proteinase K. Genetic material may then be isolated by precipitating the sample, for example DNA, with cold ethanol or isopropanol, followed by centrifugation. In non-limiting embodiments in which DNA or mitochondrial DNA is the genetic material, enzymes which degrade RNA may also be added, such as RNase. Those of skill in the art will understand that the protocol described here may be modified to achieve the extraction and isolation.

[0030] In non-limiting embodiments in which mitochondrial DNA is the genetic material, a separate isolation kit may be used. For example, and without limitation, the Mitochondrial DNA Isolation Kit sold by Abeam ^® (Cambridge, MA) may be utilized to isolate that material of interest. In other non-limiting embodiments, mitochondrial DNA may be extracted and isolated utilizing the Hirt Extraction method, which in non-limiting embodiments may utilize a lysis buffer of 0.6% SDS and 10 mM EDTA at a pH of 7.5, 5 M sodium chloride, 2 mg/ml of RNase, Phenol saturated with 0.1 M Tris at pH 7.4, and 5 M sodium acetate at pH 6.0. Cells are incubated in lysis buffer for 0-20 minutes at room temperature, lysate is separated and immersed in the sodium chloride, stored at 4 degrees Celsius for 8-20 hours, and centrifuged at 18,000 rpm for 40 minutes. The heavier chromosomal material will be in the pellet, and the lighter, extra- chromosomal material will be present in the supernatant, which is incubated with RNase at 37 degrees Celsius for 1 hour. Phenol extraction is conducted at least once, followed by ether extraction, and sodium acetate and isopropanol are added to the resultant material for overnight storage at -20 degrees Celsius. The solution is then centrifuged at 10,000 rpm for 30 minutes, the pellet is washed with cold 70% ethanol, and dried, leaving isolated mitochondrial DNA. Those of skill in the art will understand that the protocol described here may be modified to achieve the extraction and isolation.

[0031] In non-limiting embodiments in which RNA is the genetic material, a separate isolation kit may be used, For example, and without limitation, the RNeasy Mini Kit sold by Qiagen (Valencia, CA) may be utilized to isolate that material of interest. In further non-limiting embodiments, RNA may be extracted and isolated according to the following protocol. A lysis buffer comprising 0.1 M sodium chloride, 10 mM Tris at pH 8.0, 2 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and 1% beta-mercaptoethanol is applied to the sample. The sample is homogenized by any suitable means, for example by vortexing or repeated pipetting. The sample is then centrifuged at 5000 g for 10 minutes. Supernatant is transferred and 20% SDS (to 0.5%) and Proteinase K are added. The mixture is incubated at 45 degrees Celsius for 30-60 minutes. The mixture is then extracted with phenol/chloroform/isoamyl alcohol (25:24: 1) and centrifuged. Aqueous phase is transferred and extracted again with chloroform/isoamyl alcohol (24:1), if necessary. After centrifugation, the aqueous phase is again transferred and 3 M sodium acetate at pH 5.5 (to 0.15 M) and absolute cold ethanol are added to the liquid. The mixture is incubated at -20 degrees Celsius for 2-24 hours, after which the mixture is centrifuged to pellet RNA. Those of skill in the art will understand that the protocol described here may be modified to achieve the extraction and isolation.

[0032] With further reference to Fig. 1, following extraction and isolation of the genetic material from the biological sample and the biological reference sample, the isolated genetic material may be amplified 140 to produce amplicons. As used herein, the term "amplicon" refers to a segment of a polynucleotide which is amplified in an amplification reaction, such as that utilized in a PCR, or other similar method known to those skilled in the art for amplifying genetic material. The PCR reaction has been utilized in the field for nearly 30 years, and is well understood by those of ordinary skill in the art. (Saiki et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487 (1988)). Other suitable methods compatible with the present methods may be ligase chain reaction (LCR) and strand displacement amplification (SDA), though these examples should not be considered limiting.

[0033] Any suitable type of PCR may be utilized i the present methods, depending on. the desired sensitivity, specificity, starting material, and cost. For example, and without limitation, Reverse Transcription PCR (RT-PCR), Quantitative PCR (Q-PCR), Quantitative Real Time PCR (QRT-PCR), Multiplex PCR, and other amplification methods known to those skilled in the art may be utilized in the methods of the present invention. It is also to be understood that an amplification step may not be required in the methods of the present invention, based on the amount of starting material available, and as such, while amplification utilizing PCR or the like is a preferred in a non-limiting embodiment, it is not required in a non-limiting embodiment.

[0034] A typical PCR reaction includes multiple amplification steps, or cycles that selectively amplify a target nucleic acid species. Because detection of transcripts is necessary, the PCR reaction is coupled with a reverse transcription step. A typical PCR reaction includes three steps: (1) a denaturing step in which a target nucleic acid is denatured; (2) an annealing step in which a set of PCR primers anneal to complementary DNA strands; and (3) an elongation step in which a DNA polymerase elongates the primers. As used herein, the term "primer" defines a molecule comprised of at least three deoxyribonucleotides or ribonucleotides. Primers may be designed according to techniques known to those skilled in the art, or may be acquired from known vendors.

[0035] In non-limiting embodiments, the deoxyribonucleotide primers are specific for mitochondrial DNA. In further non-limiting embodiments, the deoxyribonucleotide primers are specific for at least one of hypervariable region I or II of mitochondrial DNA. The hypervariable regions of mitochondrial DNA are approximately 342 base pair (bp) (Region I) and approximately 268 bp (Region II) non-coding, control regions of mitochondrial DNA. The hypervariable region of mitochondrial DNA is an optimal region for the methods provided herein because of the high degree of variability between individuals, reducing the likelihood of a given strand of mitochondrial DNA being identical between individuals. In non-limiting embodiments, specific primers for the hypervariable region(s) of mitochondrial DNA may be purchased from, for example, Applied Biosystems (Carlsbad, CA), sold as mitoCR™ and including nine pairs of primers including hypervariable regions I and II

[0036] A typical PCR reaction may be carried out according to the following non-limiting protocol. 85.5 μΐ of extracted and isolated genetic material (described above) is added to a sample tube after resuspension in sterile water. Also added to the tube are 0.5 μΐ of polymerase (for example, and without limitation, Taq polymerase), 1 μΐ of each of at least two primers, 10 mM deoxynuleoside triphosphates (dNTPs), and 10 μΐ of an appropriate PCR buffer. Tubes are then inserted into a thermocycler, heated to 94 degrees Celsius, and temperature is then cycled according to, without limitation, the following program: 94 degrees Celsius for 1 minute, 55 degrees Celsius for 1 minute, and 72 degrees Celsius for 1-3 min, depending on the length of the genetic material added to the tube. 30 cycles of the preceding program may be run, followed by a final stage at 72 degrees Celsius for 7 minutes. Those of skill in the art will understand that the protocol described here may be modified to achieve the amplification.

[0037] By repeating the elongation step multiple times, a DNA fragment is amplified to produce an amplicon, corresponding to the target DNA or RNA sequence. Typical PCR reactions include 30 or more cycles of denaturation, annealing and elongation. In many cases, the annealing and elongation steps can be performed concurrently, that is at the same temperature, in which case the cycle contains only two steps. In non-limiting embodiments, the amplicon is an amplified mitochondrial DNA sequence. In further non-limiting embodiments, the amplicon is an amplified mitochondrial sequence comprising at least a portion of hypervariable region I or II.

[0038] The length of the denaturation, annealing and elongation steps may be any desirable length of time. However, the steps may be shortened for rapid turnaround of sample identification when a biopsy is conducted in close temporal proximity to a planned surgery, be it for health purposes or facility availability, or a combination of the those factors, or any other factor that increases the importance of rapid turnaround. The denaturation step may, for example, be conducted for times of one second or less. The annealing and elongation steps may also be less than 10 seconds each, and when conducted at the same temperature, the combination annealing/elongation step may be less than 10 seconds. Use of recently developed amplification techniques can dramatically shorten the PCR reaction time beyond these time limits (Krishnan et al., PCR in a Rayleigh-Benard convection cell. Science 298: 793 (2002); Braun et al., Exponential DNA Replication by Lominar Convection. Physical Review Letters, 91(15): 158103 (2003)).

[0039] Higher concentrations of primers may be used to shorten the necessary PCR cycle time. High concentrations typically indicate those greater than about 400nM, and often greater than about 800nM. Apart from speed and timing, specificity of a PCR reaction is of utmost importance, particularly when employed in the present methods, where matching of transcripts or amplicons is utilized for identification of biological samples. The specificity of any given PCR reaction relies heavily on the identity of the primer sets. The primer sets are pairs of forward and reverse oligonucleotide primers that anneal to a target DNA sequence to permit amplification of the target sequence, thereby producing a target sequence-specific amplicon. PCR primer sets may include two primers internal to the target sequence, or one primer internal to the target sequence and one specific to a target sequence that is ligated to the DNA or cDNA target, using a technique known as "ligation- anchored PCR" (Troutt et al. Ligation-anchored PCR: A Simple Amplification Technique with Single-sided Specificity. Proc. Natl. Acad. Sci. USA, 89: 9823- 9825 (2002)). [0040] The progress of PCR reactions typically is monitored by determining the relative rates of amplicon production for each PCR primer set. Equipment and software are readily available for monitoring amplicon accumulation in PCR, including the Smart Cycler, commercially available from Cepheid (Sunnyvale, CA), the ABI Prism 7700 Sequence Detection System (TaqMan), commercially available from Applied Biosystems (Carlsbad, CA). A cartridge-based sample preparation system (GenXpert) combines a thermal cycler and fluorescent detection device having the capabilities of the Smart Cycler product with fluid circuits and processing elements capable of automatically extracting specific nucleic acids from a tissue sample. The system uses disposable cartridges that can be configured and pre-loaded with a broad variety of reagents.

[0041] In non-limiting embodiments, the genetic material is mitochondrial DNA. In preferred non-limiting embodiments, the mitochondrial DNA comprises at least a portion of a hypervariable region. In further preferred non-limiting embodiments, at least a portion of the hypervariable region comprises hypervariable region I and/or hypervariable region II. In such non-limiting embodiments, specific primers for the hypervariable region(s) of interest are utilized in the amplification step. Such primers are available commercially, for example, from Applied Biosystems (Carlsbad, CA), sold as mitoCR™ and including nine pairs of primers including hypervariable regions I and II. Amplification protocols for the hypervariable region are similar to standard PCR protocols and are known in the art, but may differ in some respects. For example and without limitation, such protocols utilize mitochondrial DNA-specific primers and may call for heat activation at 96 degrees Celsius for 5 minutes, followed by 40 cycles of 94 degrees Celsius for 30 seconds, 60 degrees Celsius for 45 seconds, and 72 degrees Celsius for 45 seconds, followed by a final extension of 72 degrees Celsius for 10 minutes. Those of skill in the art will understand that the protocol described here may be modified to achieve the amplification.

[0042] In other non-limiting embodiments, the genetic material is RNA, and may be amplified by, without limitation the AccessQuick™ RT-PCR System sold by Promega (Madison, WI). In such procedures, the RNA is reverse transcribed to produce cDNA. The cDNA is then amplified by PCR, as described above.

[0043] With continuing reference to Fig. 1, biological sample amplicons and biological reference sample amplicons are produced through amplification 140. The amplicons of the biological sample are then compared 150 to the amplicons of the biological reference sample to determine whether the sample, in non-limiting examples a biopsy sample, is the particular patient's sample. If a positive match 160 is determined, and the biological sample is determined to have originated from the same individual from whom the biological reference sample originated, the result is recorded and transmitted to the health care provider, hospital, physician, or other medical professional for consideration of treatment design, confirmation of prior identification, or both. Results may be transmitted through electronic medical records (EMR) or by paper transmittal, and may include graphical representation of the results and a statistical analysis of the results of the comparison. In this way, a physician or other medical professional or health care provider may themselves have visual confirmation of the results, and communicate said results to the patient.

[0044] If a negative match 170 is determined (the biological sample and biological reference sample did not originate from the same individual, here the patient), then the result is again transmitted to the physician, medical professional, or other health care provider so that any treatment decisions may be delayed while additional testing consistent with the methods provided herein are undertaken.

[0045] In non-limiting embodiments, the amplicons may be compared through gel electrophoresis and detection of the amplicons by one of a variety of methods, such as, without limitation ethidium bromide staining, Southern blotting and hybridization to probes, or by incorporating fluorescent or radioactive moieties into the amplicons and subsequently viewing the product on a gel.

[0046] In non-limiting embodiments, the amplicons are exposed to restriction enzymes and subjected to restriction fragment length polymorphism analysis. This may be conducted in either a horizontal or vertical electrophoresis agarose gel setup. Briefly, amplicons are incubated with restriction digest enzymes, for example in non-limiting embodiments EcoRI or Hindlll. Other suitable digestion enzymes are known to those skilled in the art and may be utilized in the present methods. In a typical, non-limiting protocol, the genetic material is digested with the appropriate enzyme for at least five hours, incubated in sodium acetate and ethanol. A pellet is precipitated by centrifugation at 10,000 rpm for 20 min at 0 degrees Celsius, and supernatant is removed. 70% ethanol is added to the pellet, which is then centrifuged again at 10,000 rpm for 10 minutes at 0 degrees Celsius, and the removal of supernatant and centrifugation steps are repeated at least one more time. Following the final centrifugation step, supernatant is removed and the pellet is dried at room temperature for no longer than 1 hour. The pellet is then resuspended in a buffer comprising 8mM Tris, 4 mM sodium chloride, 1 mM EDTA, 10% glycerol, and 0.03% bromophenol blue, at pH 7.4. The mixture is heated at 55 degrees Celsius for five minutes and cooled to room temperature, and then is loaded into the agarose gel. Standard molecular weight markers are added to a single lane for analysis and authentication, and to aid in identification. Those of skill in the art will understand that the protocol described here may be modified to achieve the separation.

[0047] In non-limiting embodiments, and particularly useful when the hypervariable region of mitochondrial DNA is the genetic material, the amplicons of the hypervariable region may be separated on a polyacrylamide gel. The gel may be from 4-10% acrylamide. In non-limiting embodiments, the gel is 4% acrylamide.

[0048] When gel electrophoresis is employed, the gels are imaged, for example and without limitation, with products sold commercially by Syngene (Frederick, MD) or the Molecular Imager ^® Gel Doc™ XR+ System, sold by Bio-Rad (Hercules, CA). Briefly, a gel is stained, or labeled amplicons are imaged to produce graphical representations of the amplicons and the standard weight markers. Comparisons are then made between lanes to confirm or deny a match between the samples, and thus, between the biological sample and the biological reference sample.

[0049] In non-limiting embodiments, at least 90% similarity between lanes of the gel is needed to confirm identity between a biological sample and a biological reference sample. In non-limiting embodiments, at least 95% similarity is needed for confirmation of identity. In a preferred, non-limiting embodiment, at least 99% similarity is needed to confirm identity between a biological sample and a biological reference sample.

[0050] With reference to Fig. 2, a biological sample and biological reference sample are obtained 210, 220. The samples may be obtained as described previously. Genetic material is then extracted and isolated 230 from the respective samples, and the genetic material (DNA, mtDNA, RNA) is amplified 240 using known techniques discussed previously. In non-limiting embodiments, the amplicons are sequenced 245 after being amplified. Sequencing may be accomplished utilizing Maxam-Gilbert sequencing, Sanger sequencing, high throughput sequencing methods, or any other sequencing method known to those of skill in the art. In non- limiting embodiments, the amplicons are sequenced utilizing the sequencers that are commercially available, such as the GS series sold by 454 Life Sciences (Branford, CT), the MiSeq system sold by Illumina ^® (San Diego, CA), and the SOLiD™ system sold by Applied Biosystems (Carslbad, CA). While a possible embodiment within the scope of the present methods, Maxam-Gilbert sequencing is not desired due to the requirement of radioactive labeling. Sequences are compared 250 on the basis of sequence identity, with at least 90% sequence identity needed to confirm identity between a biological sample and a biological reference sample. In non-limiting embodiments, sequence identity of at least 95% is needed for confirmation of identity. In a preferred, non-limiting embodiment, sequence identity of at least 99% is needed to confirm identity between a biological sample and a biological reference sample. If a positive match 260 is determined, and the biological sample is determined to have originated from the same individual from whom the biological reference sample originated, the result is recorded and transmitted to the health care provider, hospital, physician, or other medical professional for consideration of treatment design, confirmation of prior identification, or both. Results may be transmitted through EMR or by paper transmittal, and may include graphical representation of the results and a statistical analysis of the results of the comparison. In this way, a physician or other medical professional or health care provider may themselves have visual confirmation of the results, and communicate said results to the patient.

[0051] If a negative match 270 is determined (the biological sample and biological reference sample did not originate from the same individual, here the patient), then the result is again transmitted to the physician, medical professional, or other health care provider so that any treatment decisions may be delayed while additional testing consistent with the methods provided herein are undertaken.

[0052] Sequencing may be conducted, without limitation, in a standard Sanger manner including denaturing, annealing, and elongation, as in PCR protocols described previously. Instead of a mixture of only normal nucleotides (dNTPs), however, in Sanger based sequencing a small percentage of dideoxynucleotides, for example and without limitation thymine, are included in the mixture. These modified nucleotides lack a hydroxyl group at the 3' position, and as such, when a dideoxythymine is added to the end of a strand of DNA by DNA polymerase, the strand cannot be further elongated. Due to the presence of the dideoxythymine, elongation of each replicant strand will be halted randomly upon addition of a dideoxythymine nucleotide. Addition of a fluorescent tag to the nucleotide allows for imaging, and gel electrophoresis (described below) may be performed to separate replicant strands based on size. Addition of a differentially labeled, dideoxy form of each nucleotide allows for simultaneous imaging of the fragments and analysis of the sequence of the starting strand of genetic material.

[0053] Sequencing may also be earned out using so-called pyrosequencing, which makes use of DNA annealed to beads and amplified utilizing EmPCR ^® techniques commercialized by 454 Life Sciences (Branford, CT). In this method, the amplification produces adenosine triphosphate (ATP), which is consumed by light-emitting enzymes, the emission of light captured by any suitable imaging method.

[0054] In non-limiting embodiments, in which the genetic material comprises the hypervariable region of mitochondrial DNA, sequencing may be accomplished by using the mitoSEQr™ system sold by Applied Biosystems (Carslbad, CA), or other equipment known to those skilled in the art.

[0055] In non-limiting embodiments, sequencing may be performed utilizing nanopore-based sequencing, for example and without limitation utilizing the GridlON or MinlON systems sold by Oxford Nanopore Technologies (Oxford, UK). In further non-limiting embodiments, the extraction, amplification, and sequencing steps may be performed by a single machine, to consolidate processes and reduce the possibility of contamination and error. In non-limiting embodiments, an integrated microfluidic sample preparation card and instrument, such as manufactured by Ibis Biosciences (Abbott Park, IL), may be utilized in the methods of the present invention.

[0056] With reference to Fig. 3, a biological sample and biological reference sample are obtained 310, 320. The samples may be obtained as described previously. Genetic material is then extracted and isolated 330 from the respective samples, and the genetic material (DNA, mtDNA, RNA) is amplified 340 using known techniques discussed previously. In non-limiting embodiments, the molecular mass of the amplicons is determined 345, and the step of comparing biological sample amplicons to biological reference sample amplicons includes comparing molecular mass of the amplicons 350. In non-limiting embodiments, the molecular mass of the biological sample must be within 90-110% of the molecular mass of the biological reference sample to confirm identity between a biological sample and a biological reference sample. In non-limiting embodiments, the molecular masses of each sample must be within 95-105% for confirmation of identity. In a preferred, non-limiting embodiment, the molecular masses of each sample must be within 99-101% to confirm identity between a biological sample and a biological reference sample.

[0057] If a positive match 360 is determined, and the biological sample is determined to have originated from the same individual from whom the biological reference sample originated, the result is recorded and transmitted to the health care provider, hospital, physician, or other medical professional for consideration of treatment design, confirmation of prior identification, or both. Results may be transmitted through EMR or by paper transmittal, and may include graphical representation of the results and a statistical analysis of the results of the comparison. In this way, a physician or other medical professional or health care provider may themselves have visual confirmation of the results, and communicate said results to the patient.

[0058] If a negative match 370 is determined (the biological sample and biological reference sample did not originate from the same individual, here the patient), then the result is again transmitted to the physician, medical professional, or other health care provider so that any treatment decisions may be delayed while additional testing consistent with the methods provided herein are undertaken.

[0059] In non-limiting embodiments, molecular mass is determined by mass spectrometry, a technique that measures the mass-to-charge ratio of ionized particles. Briefly, mass spectrometry is advantageous because there is no need for use of radioactive or fluorescent labels to be integrated into the genetic material, and there is no need to sequence the genetic material. In a general method, amplicons are vaporized, ionized, and separated on the basis of mass-to-charge ratio in a plurality of electromagnetic fields. Any suitable mass spectrometry methods may be utilized in the methods of the present invention, including, without limitation, tandem mass spectrometry, infrared multiphoton dissociation mass spectrometry, electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry, fast atom bombardment mass spectrometry, and pyrolytic gas chromatography mass spectrometry.

[0060] In non-limiting embodiments, mass spectrometry may be carried out utilizing commercially available mass spectrometers, such as those sold by Agilent Technologies (Santa Clara, CA). In further non-limiting embodiments, the steps of determining molecular mass and comparing the molecular mass of the amplicons are conducted using the PLEX-ID mtDNA 2.0 Analyzer and associated reagents and assays, sold by Ibis Biosciences (Abbott Park, IL). [0061] At the conclusion of comparing the biological sample to the biological reference sample, common identity of the biological sample and the biological reference sample is confirmed or denied. In the event that there is common identity between the biological reference sample and the biological reference sample, a physician may be assured that the sample, for example and without limitation the biopsy sample, was taken from the patient in question, and may make treatment decisions based thereon. If the biological sample does not match the biological reference sample, a physician is then alerted and treatment is postponed until proper identity can be confirmed.

[0062] In further non limiting embodiments, provided herein are methods of verifying the identity of a biological sample by obtaining a biopsy sample and a reference sample from a patient. Genetic material is extracted and isolated from the biopsy sample and the reference sample. From each sample a hypervariable region of mitochondrial DNA is amplified to produce a first plurality of amplicons corresponding to the biopsy sample and a second plurality of amplicons corresponding to the reference sample. The first and second plurality of amplicons are then compared in such a way that a match between the first plurality of amplicons corresponding to the biopsy sample and the second plurality of amplicons corresponding to the reference indicates that the biopsy sample and the reference sample are from the patient.

[0063] In a preferred, non-limiting embodiment, the biopsy sample is a tumor biopsy sample, and may be a biopsy obtained from any type of tumor. For example and without limitation, the tumor may be benign or malignant, and may correspond to breast cancer, ductal carcinoma, lobular carcinoma, colon cancer, colorectal cancer, adenocarcinoma, lung cancer, stomach cancer, liver cancer, pancreatic cancer, skin cancer, cervical cancer, uterine cancer, ovarian cancer, prostate cancer, testicular cancer, leukemia, lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, melanoma, head and neck cancer, thyroid cancer, squamous cell cancer, bladder cancer, benign or metastatic brain tumor, glioblastoma, oligodendroglial tumor, astrocytic tumor, pineal gland tumor, and/or pituitary tumor.

[0064] Owing to the sensitivity of the analyses and the need to reduce or eliminate cross- contamination and other errors that may compromise the results, care should be taken in the preparation and execution of the above methods, including, without limitation, use of prepackaged, sterile equipment and reagents, aerosol-resistant pipette tips, gloves, masks, lab coats, and, when applicable, separated, dedicated areas for amplification, sequencing, and comparison/analysis.

[0065] In non-limiting embodiments in which a biological reference sample is acquired at home by a patient through, without limitation, a buccal swab, a pre-packaged, sterile kit may be provided. The kit may include instructions for acquiring, storing, and transmitting the sample in a sterile manner, pre-packaged sterile swabs, sterile elongated tubes for isolation of the swab after acquisition of the reference sample, sterile gloves (nitrile and latex), and a pre-packaged sterile volume of a DNA stabilizing solution for application to the buccal swab in the sterile elongated tube. The swabs, tubes, gloves, and DNA stabilizing solution may be any variety known to those of skill in the art appropriate for sterile collection and storage of genetic material. The kit may also include a pre-addressed sterile envelope. In a further non-limiting embodiment, the kit and the pre-addressed sterile envelope are labeled with a bar code corresponding to the bar code applied to identify the biological sample throughout its processing steps.

[0066] In a further non-limiting embodiment, the biological reference sample is acquired at a physician's office, hospital, or other healthcare site, and may be, without limitation, a buccal swab, a saliva sample, or a blood sample, and a sterile kit may be provided. The kit may include instructions for acquiring, storing, and transmitting the sample in a sterile manner, pre-packaged sterile swabs, sterile elongated tubes for isolation of the swab after acquisition of the reference sample, sterile tubes for acquisition and storage of a saliva sample, sterile tubes for acquisition and storage of blood sample, sterile gloves (nitrile and latex), and a pre-packaged sterile volume of a DNA stabilizing solution for application to the buccal swab in the sterile elongated tube. The swabs, tubes, gloves, and DNA stabilizing solution may be any variety known to those of skill in the art appropriate for sterile collection and storage of genetic material. The kit may also include a pre-addressed sterile envelope or shipping container. In a further non-limiting embodiment, the kit and the pre-addressed sterile envelope or shipping container are labeled with a bar code corresponding to the bar code applied to identify the biological sample throughout its processing steps. In yet a further non-limiting embodiment, the kit also includes sterile and stable storage for at least a portion of the biological sample. In such non-limiting embodiments, the kit, including the biological sample and biological reference sample, may be transmitted to an independent third party for verification of the identity of the biological sample.