Classifications

• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics
  • G16B50/20—Heterogeneous data integration
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids

Definitions

• sample's history and/or source Most samples and their sources are given a common alphanumeric designation, and this designation is also linked to information about the source and the sample (e.g., patient name, sample type, disease condition, etc.). A loss of this designation from its association with either the source, or the sample, or the information will often result in a complete loss of utility of all three.
• the present invention relates to a method of creating a unique identifier for reliably identifying samples, their sources, and associated information.
• the use of the identification system described herein substantially decreases potential mixups and misidentification of samples, their sources, and associated information.
• the present invention provides a method for creating a unique identifier which is used to label the sample, its source, or the associated information, based on the polymo ⁇ hisms inherent in the sample and its source.
• One or more polymorphisms in the sample is detected, and the resulting polymorphism data is used to produce a unique identifier, which is then used to identify the sample.
• This unique identifier cairalso be linked with the source and/or any information that may be associated with either the sample or the source (i.e. the unique identifier can be used as a common designation for the sample, its source and/or other relevant information).
• this unique identifier is separated from the sample, then the polymorphisms within the sample simply need to be re-detected to reproduce the polymorphism data which is then used to produce the unique identifier, thereby recreating the proper unique identifier, and, ultimately, its link to its source.
• the invention features a method for producing a unique identifier for a biological sample, comprising detecting one or more polymorphisms within the biological sample, and selecting one or more polymo ⁇ hisms sufficient to form a unique identifier.
• the biological sample can be from a vertebrate, an invertebrate, a plant, or consist of microorganisms.
• the biological sample can also be from a mammal, particularly a human.
• the sample can be blood, saliva, hair, body fluid, tissues, organs, one or more cells, or a whole organism.
• the polymo ⁇ hisms can be nucleic acid polymo ⁇ hisms, protein polymo ⁇ hisms, enzyme polymo ⁇ hisms, chemical polymo ⁇ hisms, biochemical polymo ⁇ hisms, phenotypic polymo ⁇ hisms, and quantitative polymo ⁇ hisms, particularly a nucleic acid sequence polymo ⁇ hism, a nucleic acid length polymo ⁇ hism, or a short tandem repeat (STR).
• the unique identifier can also be linked to the source of the biological sample, or relevant information about the biological sample or the source of the biological sample.
• the unique identifier can be in the form of an alphanumeric string, or a bar code.
• the invention also features a method for establishing a repository containing a collection of biological samples, comprising obtaining a biological sample from a source, detecting one or more polymo ⁇ hisms in the sample, selecting one or more polymo ⁇ hisms sufficient to form a unique identifier, using the unique identifier to identify the sample, storing the sample with the unique identifier, and repeating these steps for biological samples from other sources.
• the samples are DNA-containing samples, particularly from humans, and the polymo ⁇ hisms are nucleic acid polymo ⁇ hisms, protein polymo ⁇ hisms, enzyme polymo ⁇ hisms, chemical polymo ⁇ hisms, biochemical polymo ⁇ hisms, phenotypic polymo ⁇ hisms, -4-
• the unique identifier can be in the form of an alphanumeric string, or a bar code, and can also be linked to the source of the biological sample, or relevant information about the biological sample or the source of the biological sample.
• the invention features a method of determining, by means of a u que identifier, if a source is represented by a sample within the repository, comprising obtaining a sample from the source, detecting one or more polymo ⁇ hisms in the sample selecting one or more polymo ⁇ hisms sufficient to form a unique identifier, and comparing the unique identifier so produced to the unique identifier of each sample in the repository, where shared identity between the two unique identifiers indicates that the source is already represented in the repository.
• the samples are DNA-containing samples, preferably from humans.
• the polymo ⁇ hisms are nucleic acid polymo ⁇ hisms, particularly short tandem repeats (STR), protein polymo ⁇ hisms, enzyme polymo ⁇ hisms, chemical polymo ⁇ hisms, biochemical polymo ⁇ hisms, phenotypic polymo ⁇ hisms, and quantitative polymo ⁇ hisms.
• the unique identifier can also be linked to the source of the biological sample, or relevant information about the biological sample or the source of the biological sample.
• the unique identifier can be in the form of an alphanumeric string, or a bar code.
• the invention also features a method for linking, by means of a unique identifier, a first biological object lacking a unique identifier with a second object having a unique identifier, comprising detecting one or more polymo ⁇ hisms in the first biological object, selecting one or more polymo ⁇ hisms sufficient to form a unique identifier, and comparing the unique identifier so made to the unique identifier of the second object, where shared identity between the two unique identifiers links the first biological object with the second object.
• the biological sample can be from a vertebrate, an invertebrate, a plant, or consist of microorganisms.
• the biological sample can also be from a mammal, particularly a human.
• the sample can be blood, saliva, hair, body fluid, tissues, organs, one or more cells, or a whole organism.
• the polymo ⁇ hisms can be nucleic acid polymo ⁇ hisms, particularly short tandem repeats (STR), protein polymo ⁇ hisms, enzyme polymo ⁇ hisms, chemical polymo ⁇ hisms, biochemical polymo ⁇ hisms, phenotypic polymo ⁇ hisms, and quantitative polymo ⁇ hisms, particularly a nucleic acid sequence polymo ⁇ hism, or a nucleic acid length polymo ⁇ hism.
• the unique identifier can also be linked to the source of the biological sample, or relevant information about the biological sample or the source of the biological sample.
• the unique identifier can be in the form of an alphanumeric string, or a bar code.
• the present invention provides a method for creating a unique identifier for identifying biological samples, their sources, or associated information based on the polymo ⁇ hisms inherent in the sample and source themselves.
• the nucleic acid contained within the sample itself is used to produce the unique identifier for identifying and linking the sample, its source, and associated information.
• No external material needs to be added to the sample which could dilute or alter the accuracy of other test results.
• An advantage of the invention therefore, is that it is unnecessary to add "identifying sequences" to the samples, and that without such additions, one may conduct studies of genetics, disease associations, evolutionary relationships, etc., without the results being tainted by the added identifying sequences.
• a “source” or “the source from which the sample is derived” refers to the originating material for a sample.
• a source of a biological sample for example, can be a human, any animal, plant, insect, or a population or strain of microorganisms.
• a source of a biological sample does not have to be living, and can be a deposit in a tissue repository, herbaria or museum specimens, forensic specimens, or fossils.
• a “potential source” as used herein, means a source from which the sample may possibly have been taken in the past.
• sample is meant a portion of source biological material that originated elsewhere, i.e., the sample was removed from its source.
• a sample can be any biological sample, (e.g., blood, saliva, hair, organs, biopsies, bodily fluids, one or more cells), and can be taken from any vertebrate, including mammals such as humans, or plant, insect, reptile.
• the sample can also be a strain or mixed -6-
• Samples can also be biological materials taken from defunct or extinct organisms, e.g., samples can be taken from pressed plants in herbarium collections, or from pelts, taxidermy displays, fossils, or other materials in museum collections.
• Information associated with the sample or source from which the sample is derived is meant to include, without limitation, any information that might be necessary or advantageous to be linked to the sample or the sample source, e.g. name, address, sex, medical history (in the case of human samples), species, collection data, provenance (in case of non-human samples), etc.
• a biological sample is taken from its source, the sample is tested across one or more polymo ⁇ hic loci, and the polymo ⁇ hic data produced are used to create a unique identifier, which is identifiably linked to the sample, and serves as its unique designation.
• This unique identifier can also be identifiably linked to the sample's source, and/or any information that may exist concerning the source and/or the sample.
• the umque identifier is "identifiably linked" to the sample, sample source, or related information means that it is connected in some way with any or all of these three things, e.g.
• the unique identifier may be on a label attached to a container holding the sample, the unique identifier may exist as a field in a database record containing medical data regarding the source, etc.
• the genetic code of the sample itself which is unique and forms the basis of the polymo ⁇ hisms tested, serves as the unique identifier. Because the unique identifier is based on the genetic code, which is unique between individuals, the umque identifier will also be unique between samples from different source individuals.
• a "polymo ⁇ hism" is an allelic variation between two samples.
• the term includes differences between proteins (e.g., enzymes, blood groups, blood proteins), differences in the chemicals and biochemicals (e.g., secondary metabolites) produced by the source organism(s), differences between nucleic acids involving differences in the nucleotide sequence (e.g., restriction site maps), or differences in length of a stretch of nucleic acid (e.g., RFLPs (restriction fragment length polymo ⁇ hisms), microsatellites, STRs (short tandem repeats), SSRs (simple sequence repeats), SSLPs (simple sequence length polymo ⁇ hisms), VNTRs -7-
• Allelic variation can also result in phenotypic (i.e., visually- apparent) polymo ⁇ hisms, or variations in quantitative characters (e.g. variation in height, length, yield of fruit, etc.) between the organisms that serve as the source of the samples.
• phenotypic differences may be visible in the samples themselves, e.g., kernels of different types of "Indian" corn often appear very different from each other, with red, yellow, white, blue, streaked kernels, etc. With such samples, phenotypic polymo ⁇ hisms could also be used to produce the unique identifier.
• a polymo ⁇ hism is not limited by the function or effect it may have on the organism as a whole, and can therefore include allelic differences which may also be a mutation, insertion, deletion, point mutation, or structural difference, as well as a strand break or chemical modification that results in an allelic variant.
• allelic differences may also be a mutation, insertion, deletion, point mutation, or structural difference, as well as a strand break or chemical modification that results in an allelic variant.
• a polymo ⁇ hism between two nucleic acids can occur naturally, or be caused intentionally by treatment (e.g., with chemicals or enzymes), or can be caused by circumstances normally associated with damage to nucleic acids (e.g., exposure to ultraviolet radiation, mutagens or carcinogens).
• sequence polymo ⁇ hism is a difference in the sequence of two nucleic acids or two amino acids.
• Two amino acid sequences can differ by having different residues at a particular position (i.e., and amino acid substitution), or some residues may be deleted, or new residues inserted or added to one or more ends.
• Two nucleic acids differing in sequence may have the same number of base pairs (e.g., "AT£rC” vs. "ATT_C”), but may also include some differences in overall sequence length as well (e.g., "AT£A£ ⁇ TG” vs. "ATCACACATG”).
• Types of commonly-studied polymo ⁇ hisms caused by sequence differences include restriction site polymo ⁇ hisms, isozymes, differences in protein conformation, and length polymo ⁇ hisms. If the nucleic acid is sequenced, then a sequence difference itself (as represented by the string of letters) serves as the polymo ⁇ hism.
• a "length polymo ⁇ hism” is a difference in the length of two nucleic acids. Two different nucleic acids with a length polymo ⁇ hism between them also have a sequence polymo ⁇ hism, but many methods used to detect a length polymo ⁇ hism do not reveal the exact sequence polymo ⁇ hism. Commonly-used -8-
• length polymo ⁇ hisms include RFLPs (restriction fragment length polymo ⁇ hisms), microsatellites, STRs (short tandem repeats), SSRs (simple sequence repeats), SSLPs (simple sequence length polymo ⁇ hisms), and VNTRs (variable number tandem repeats).
• RFLPs restriction endonucleases are used to cut a nucleic acid molecule into fragments, which are then separated on an agarose gel.
• the differences between two individuals are measured by the changes in size of the resultant nucleic acid fragments, and so are referred to as length polymo ⁇ hisms, yet those differences are caused by differences in the underlying sequence, which is the basis for the change in restriction sites, and therefore the changes in the sizes of the nucleic acid fragments. Because the method of detection/visualization can only differentiate on the basis of fragment length, the RFLPs are generally classed as length polymo ⁇ hisms..
• a "polymo ⁇ hic locus" is a segment of nucleic acid which may contain a polymo ⁇ hism as described above. It is not required that the precise sequence of the nucleic acid be known.
• a polymo ⁇ hic locus is not limited to those loci which are polymo ⁇ hic in all situations, e.g., a polymo ⁇ hic locus which displays an allelic variation between individuals A and B, but not between individuals A and C, remains a polymo ⁇ hic locus for purposes of comparing individuals A and B, as well as individuals B and C.
• Nucleic acid means deoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleic acids from mammals or other animals, plants, insects, bacteria, viruses, or other organisms.
• unique identifier is meant an identification tag, designation, or code to be linked to a sample, its source, or other information, such as patient case history, disease testing results, genetic testing results, geographic or temporal collection data, or any other information which may be useful when linked with the sample or source.
• the unique identifier can exist in the form of an alphanumeric string, a bar -9-
• allelic polymo ⁇ hism has been studied for decades, and there are many genetic systems which have been commonly used in assessing polymo ⁇ hism between populations or individuals. These include classical blood groups, blood proteins, isozymes, distribution of restriction endonuclease sites, restriction fragment length polymo ⁇ hisms (RFLPs), and others.
• RFLPs restriction fragment length polymo ⁇ hisms
• STRs are stretches of DNA that consist of repeated sequences repeats.
• the base sequence is usually just a few base pairs long, typically two to twelve base pairs, but longer base repeats have been seen. This base sequence is then tandemly repeated, and the number of times it is repeated can vary greatly, depending on the STR locus being studied.
• An STR can therefore be expressed as (X) n , where X is the repeated sequence, (e.g. "CA”) and n is the number of times that X is repeated.
• the base sequence may be repeated 5 times in individual A, but may be repeated 8 times in mdividual B and 20 times in individual C.
• tandem repeats are believed to be caused by "slippage" of the DNA polymerase enzyme as the DNA is replicated.
• n increases over generations, and the amount of slippage varies over time and in different lines.
• the variability of these repeated sequences is generally correlated to the length of the base repeat, with STRs composed of longer base repeats exhibiting less variability between individuals than shorter base repeats.
• a two base pair repeat may consist of a two base pair unit being repeated hundreds of time in an individual, while a 12-base pair unit may only be repeated a few times.
• Short repeats tend to exhibit higher rates of polymo ⁇ hism between individuals, while 10- or 12-base pair repeats may show little or no variability.
• STRs can be amplified and detected by known procedures. For example, they can be detected by electrophoretic separation followed by radionuclide or fluorescent labeling, or silver staining. They have many advantages over other methods of detecting polymo ⁇ hisms (e.g., RFLPs) because of their small size, the ease and speed with which they can be detected and analyzed, and the fact that the process is amenable to automation.
• RFLPs radionuclide or fluorescent labeling
• the more recent generations of large-scale genetic maps have been made using STRs (Hudson, TJ. et al., Science 270:1945- 1954 (1995); Dietrich, W.F., et al., Nature Genetics 7:220-245 (1994); Yerle, M., et al., Ma m.
• kits for amplifying STR loci are commercially available, and the rates of polymo ⁇ hism of these loci in different ethnic backgrounds are known. These include AmpFlSTRTM Profiler, AmpE/STRTM Profiler Plus, AmpE/STRTM Green I (P ⁇ Applied Biosystems, Foster City, California, USA), the GeneprintTM STR Systems (Promega Co ⁇ ., Madison, WI), including GeneprintTM PowerPlexTM 1.1, GeneprintTM PowerPlexTM 1.2, GeneprintTM PowerPlexTM 2, and GeneprintTM PowerPlexTM 16, Sex Determination Systems, and others. These STR systems were developed for use in humans, but microsatellite markers have been developed in other organisms, including horse, cattle, sheep, goat, dog, pig, mouse, rat, barley, corn, soybean, and others.
• loci can be used singly, or can be combined, depending on the power of discrimination required. As the number of organisms being studied and the number of individuals from which samples are removed and archived increases, the degree of polymo ⁇ hism required to uniquely identify each sample also increases, -11-
• Locus 2 alone could serve as the unique identifier, because by itself, it can serve to distinguish between samples from all three individuals.
• the Power of Discrimination (P D ) of a given system of loci is defined as the probability that two individuals selected at random will differ with respect to that given system of loci.
• the P D is related to the Probability of Identity (P ⁇ ) by the equation
• the allelic frequencies within different ethnic populations are known for many of the polymo ⁇ hisms of the STR loci in the commercially-available kits, so a set of STRs which will provide a unique identifier for every sample can be chosen, even if the final number of samples is not known.
• Combinations of loci can be chosen that have matching probabilities of less than 1 in several million or more (See, for example Table 1). -12-
• loci Once the polymo ⁇ hism rates are known for a series of loci, one can choose which loci and how many will go into making up the unique identifier. If it is anticipated that the final number of samples will be relatively small, than only a few loci are sufficient to form the unique identifier, and one or more of the loci may not need to have a high P D . On the other hand, if one intends to store a very large number of samples, then it would be prudent to use more loci, each with a high P D . The loci seleected will be based on considerations such as P D , anticipated size of the repository, ease of use, applicability to the organisms being sampled, cost, and availability.
• the polymo ⁇ hisms that can be used in the invention will vary depending on the types of samples being stored. STRs are well-studied in humans, and kits are commercially available for amplifying a number of STR loci. Genetic maps based on STRs have been built for other organisms (e.g., mouse, rat, pig). STRs appear to exist in most higher organisms, and are easy to isolate and characterize. Because the methods used to identify and assess STRs are virtually identical for different organisms, one skilled in the art can isolate STRs in an organism of choice, assess the polymo ⁇ hism rates, and choose those most useful in the present invention. For many organisms, STRs and their primer sequences have been published in the scientific literature.
• STRs One wishing to use previously published STRs need only order those primers (e.g., custom primers can be ordered and received in 48 hours from Research Genetics, Huntsville, Alabama, USA), and then use them to amplify the STRs in the DNA of the collected samples. -13-
• microsatellite markers are not necessary to use commercially-available primers to practice the present invention, nor is it necessary to use microsatellite markers developed by others.
• the present invention allows one to use any polymo ⁇ hic marker that is convenient, so long as it provides a power of discrimination between individuals.
• microsatellite markers have become so successful is that they are easy to develop for previously unstudied organisms.
• the method described herein will be of particular use in a pathology laboratory or testing facility, or a large-scale cryogenic repository. Maintaining the integrity of the sample labels is of paramount importance in these situations, as quality control problems often result from failure of the record-keeping system. Naturally, such a method will also be of use to blood banks, tissue banks, and veterinary hospitals and testing facilities.
• the method can also be used by large repositories to identify misplaced or misidentified samples.
• a tissue bank may take a small piece (e.g., a sample) of a stored tissue (e.g., a source) for testing (e.g., tissue typing for a potential recipient of the tissue). If the identification were disassociated from the sample (e.g., the label fell off the test tube), those test results would normally be lost.
• Using the unique identifier of the present invention one would simply test the sample for the polymo ⁇ hic loci, and recreate the unique identifier. The sample (and the tissue typing test results) could then be reassociated with the source in storage.
• the method is especially useful to maintain the long-term integrity of samples and associated information, especially in tissue repositories.
• Many biomedical studies require analysis of tissue samples from large populations of individuals with known medical, dietary, genetic, social, and cultural backgrounds. During the course of a study requiring several years to complete, it may be necessary -14-
• a blood sample repository contained samples from 100,000 individuals, and associated medical data on those same individuals in computerized form
• a medical study could be conducted by selecting individuals with desired characteristics (as listed in the computerized medical data), and then retrieving samples (or more likely, sub-samples) from those individuals, which are held in the repository.
• the method of the present invention is useful in establishing such a repository, because the method greatly reduces the likelihood of samples being misidentified and allows confident re-retrieval of samples.
• Another advantage of the invention is that if the unique identifier is de- associated from the sample within the repository (e.g., the label falls off the tube) analysis of the polymo ⁇ hisms in the sample allows re-creation of the unique identifier.
• Use of the method of the invention also provides a method for preventing repository deposit of samples from duplicate sources, because when the unique identifier is created for a sample from a new source, one need only search the repository records for that same unique identifier to see if the source is already represented by a sample in the repository.
• the invention also has uses outside of the medical field. Because of the increasing ease with which samples from various sources (e.g., plant, animal, microbial, fungal, viral) can be tested for polymo ⁇ hisms, the invention is applicable in any situation where a large number of biological samples may be stored.
• sources e.g., plant, animal, microbial, fungal, viral
• This sample identification method can be used to keep track of samples in any study or collection where there are a large number of biological samples being stored for a period of time, and where there is a chance that samples may become misplaced or mislabeled.
• a biological sample is obtained from a human, and an aliquot is taken for polymo ⁇ hism testing.
• DNA is isolated by methods well known in the art (Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York; Ausubel, F.M. et al, eds., Current Protocols in Molecular Biology). An amount of this isolated DNA is removed, GenePrintTM primers (Promega Co ⁇ ., Madison, WI) for the CSF1PO locus are added to it, and amplification is carried out, all according to the manufacturer's instructions (supplemental information on thermocycling are well known in the art, see e.g., Innis, M.S., et al.
• the unique identifier can be created for the sample.
• a convenient conversion method is to simply list each locus by letter, followed by the two alleles for that locus.
• the unique identifier would be "A35B27...HXY".
• the precise conversion method could be varied, depending on the number of repeats in the loci, e.g., a locus with 3 - 12 repeats would require 4 digits after the locus letter.
• Red blood cells lack DNA because they are enucleated, and must therefore be lysed to facilitate their separation from white blood cells, which contain genomic DNA.
• the white blood cells are then lysed with an anionic detergent in the presence of a DNA stabilizer, which limits the activity of DNase.
• Contaminating RNA is then degraded with RNase, and the RNA, proteins, and other contaminants are then removed by salt precipitation.
• the genomic DNA is recovered by alcohol preciptation, dissolved in TE buffer, and stored. Because the genomic DNA will be used in a nucleic acid amplification -17-
• Isolation of genomic DNA from whole blood can be accomplished by following any of a variety of protocols, including using the PUREGENE ® kit (Gentra Systems, Minneapolis, Minnesota, USA), and following the manufacturer's instructions. Place 30 ml of RBC Lysis Solution into a 50 ml tube, add 7 ml to 10 ml of whole blood, mix by inverting several times, and incubate for 10 minutes at room temperature. Invert the tube again at least once during the incubation.
• DNA Hydration Solution (Gentra Systems, Minneapolis, Minnesota, USA), and rehydrate the DNA by incubating at 65 °C for 1 hour, overnight at room temperature, and at 65 °C 1 hour the next day. Tap the tube periodically to help disperse the DNA.
• the DNA in solution can be stored indefinitely at 4°C.
• a blood sample is drawn from a human. Two ⁇ l of blood are placed on a piece of FTATMpaper (FITZCO, Inc., Maple Plain, Minnesota, USA), dried, and stored until ready to be processed.
• FTATMpaper FITZCO, Inc., Maple Plain, Minnesota, USA
• a 1 mm disc is punched directly into a 2 ml microcentrifuge tube, and 200 ⁇ l of FT ATM purification reagent is placed on the disc.
• the tube is capped, vortexed for 3-5 seconds, then centrifuged in a microcentrifuge at 12,000 x g for 30 seconds.
• the wash solution is then aspirated and discarded.
• the wash is then repeated with another 200 ⁇ l of purification reagent.
• the disc is washed twice with TE as follows: 200 ⁇ l of TE buffer is added, and the disc vortexed for 3-5 seconds, the tube and disc are then centrifuged at 12,000 x g for 30 seconds and the filtrate removed and discarded. After the disc has been washed twice with TE, the disc is subjected to polymo ⁇ hism analysis.
• a 1 mm punch of FT ATM paper containing a blood sample, processed as described in Example 2, supra, is placed in a 0.5 ml tube, and tested with the AmpE/STR Profiler PlusTM system (Perkin Elmer Applied Biosystems, Foster City, California, USA), according to the manufacturer's instructions.
• AmpE/STR Profiler PlusTM system Perkin Elmer Applied Biosystems, Foster City, California, USA
• to the tube is added 10.5 ⁇ l of Profiler Plus Reaction Mixture, 0.5 ⁇ l of Taq Gold, and 5.5 ⁇ l of Primer Mixture.
• the tube is sealed, and placed in a thermocycler under the following conditions: 95 °C for 11 minutes, followed by 24 cycles of: 94 °C for 1 -19-
• reaction mixture is placed at 60 °C for up to 83 minutes. After thermocycling is complete, the reaction is held at 4°C until ready for gel electrophoresis.
• Example 5 Analysis of Amplification Products by Gel Electrophoresis
• Five ⁇ l of amplification product (produced as described above in Example 3) are mixed with 5 ⁇ l of 2X loading buffer (0.25% Bromphenol Blue, 12.5% Ficoll 400, 50 mM EDTA, 5X TAN (10X TAN: 0.4 M Tris, 40 mM Na Acetate Trihydrate, 10 mM EDTA, pH to 7.9 with acetic acid)).
• the 10 ⁇ l mixture is loaded into a well in a 1% agarose gel prepared with TE buffer and containing 0.5 ⁇ g of etiiidium bromide per ml of agarose gel. Appropriate size ladder is also loaded on the gel.
• the gel is then electrophoresed in TAE buffer for 1 hour at 100 volts, and then illuminated with UV light on a fransilluminator, and photographed.
• the bands in the photograph are then compared to the literature supplied by the manufacturer to determine the precise alleles present in the sample.
• the polymo ⁇ hism data for a sample can be coded in a number of ways.
• the raw data for individual #1 for example, is as follows:
• This data can be used "raw” as the unique identifier (i.e., "as is,” as above), with no alteration. For repositories with very large numbers of samples, this may be desireable, as it is the most “foolproof method.
• the STR loci can be "coded,” i.e., each locus represented by a combination of numbers or letters, e.g., D3S1358 can be represented by "A” or "01,” vWA by "B” or "02,” etc.
• the raw data so coded would then be:

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