CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U. S. Provisional Application No. 61/698,483, entitled "A NOVEL SHORT TANDEM REPEAT MULTIPLEX SYSTEM," filed September 7, 2012, the contents of which are incorporated by reference in their entirety.

BACKGROUND

[0002] The discovery and development of polymorphic short tandem repeats (STRs) as genetic markers has stimulated progress in the development of linkage maps, the identification and characterization of diseased genes, and the simplification and precision of DNA typing.

[0003] While the alleles at a single STR locus may be the same for two different individuals in a population, especially if the individuals are genetically related, the probability that the alleles of two individuals will be identical at several different loci becomes smaller and smaller as the number of loci which are examined increases. If a sufficient number of loci are examined, the overall allelic pattern will be unique for each individual. As a result, by determining the alleles at a sufficiently large number of loci in two different DNA samples it is possible to establish with virtual certainty whether or not the two samples originally came from the same source. Such certainty has found particular importance in biomedical research.

[0004] Cell lines are used extensively in biomedical research as in vitro models. A substantial proportion of cell lines are mislabeled or replaced by cells derived from a different individual, tissue or even species. This problem has been known of for over 50 years and has been described as the most compelling quality control issue confronting the biomedical research community. In one instance, the misidentification of esophageal cell lines brought down the findings of more than 100 scientific publications, at least three National Institutes of Health cancer research grants and 11 US patents.

[0005] Multiplex STR profiling, the simultaneous amplification and identification, of many STRs offers an unambiguous way to authenticate cell lines. SUMMARY

[0006] Accordingly, the instant disclosure provides a novel multiplex STR assay, a use for which is in cell line identification. Additionally, the instant disclosure provides ratios of relative primer concentrations in multiplex STR assay.

[0007] In some embodiments, there is disclosed a composition encompassing amplified nucleic acid from at least ten loci, wherein ten of the loci are D1S1656, D2S441, D2S1338, D6S1043, D10S1248, HUMTH01, D12S391, D19S433, D22S1045 and amelogenin.

[0008] In other embodiments, a method is provided, wherein the method encompasses selecting at least ten loci to be analyzed, wherein ten loci in the set are selected from the group of D1S1656, D2S441, D2S1338, D6S1043, D10S1248, HUMTH01, D12S391, D19S433 and D22S1045, amplifying the loci and evaluating the amplified loci to determine the alleles present in at least one DNA sample.

[0009] In some embodiments, the method encompasses co-amplifying the set of loci and evaluating the amplified alleles in the mixture to determine the alleles present in at least one DNA sample. In other embodiments, each of the amplified loci is evaluated to determine the alleles present.

[0010] In some embodiments, a method is provided, wherein the method encompasses selecting a set of at least ten loci to be analyzed, wherein ten loci are selected from the group of D1S1656, D2S441, D2S1338, D6S1043, D10S1248, HUMTH01, D12S391, D19S433,

D22S1045 and amelogenin, co-amplifying the set of loci in a multiplex amplification reaction, wherein the product of the reaction is a mixture of amplified alleles from each of the co- amplified loci in the set and evaluating the co-amplified loci present at each of the loci in the set to determine the alleles present in at least one DNA sample.

[0011] In some embodiments, a kit is provided wherein the kit encompasses primers for the amplification each of the loci D1S1656, D2S441, D2S1338, D6S1043, D10S1248, HUMTH01, D12S391, D19S433, D22S1045 and amelogenin and an allelic ladder representative of each loci. Brief Description of the Drawings

[0012] FIGURE 1 shows a schematic of the relative size ranges in base pairs of the amplicons generated by polymerase chain reaction (PCR) co-amplification of nine short tandem repeat loci (D1S1656, D2S441, D2S1338, D6S1043, D10S1248, HUMTHOl, D12S391, D19S433 and D22S1045) and amelogenin from a human DNA sample. For practical reasons, typically the length of amplicons in base pairs utilized for STR analysis is between 70 base pairs and 500 base pairs. This puts constraints on the number of loci, and in turn alleles, which can be labeled with any particular fluorescent label.

[0013] FIGURE 2 shows an electropherogram of the results of a multiplex PCR of the indicated loci and the alleles present for each locus.

[0014] FIGURE 3 shows a plot of an allelic ladder for the loci D1S1656, D2S441,

D2S1338, D6S1043, D10S1248, HUMTHOl, D12S391, D19S433 and D22S1045 and amelogenin. The allelic ladder shown depicts both physical bins, where an amplified allele product is present, and virtual bins, where no amplified allele is present, but the presence of an allele in the population is known. From top to bottom boxes representing fluorescent dye channels of an electropherogram, loci Dl OS 1248 (alleles 8-18 are physical alleles; allele 16 runs at about 110 bp) and D1S1656 (alleles 9-20.3 are physical alleles; allele 9 runs just above 150 bp, while allele 13 just below 170 bp) are labeled with the same fluorescent label; amelogenin and D2S1338 (alleles 15-20 are physical alleles; allele 22 runs just below 150 bp, while allele 23 runs just above 150 bp) are labeled with the same fluorescent label; D22S1045 (alleles 8-19 are physical alleles; allele 17 runs just below 110 bp, while allele 18 runs just above 110 bp) D19S433 (alleles 9-17.2 are physical alleles) and HUMTHOl (alleles 4-13.3 are physical alleles; allele 6 runs just below 190 bp, while allele 7 runs just above 190 bp) are labeled with the same fluorescent label; D2S441 (alleles 9-16 are physical alleles, allele 16 runs just below 110 bp), D6S1043 (alleles 9-25 are physical alleles; allele 14 runs just below 150 bp, while allele 15 runs just above 150 bp.) and D12S391 (alleles 14-27 are physical alleles; allele 14 runs at about 230 bp, while allele 24 runs about 270 bp.) are labeled with the same fluorescent label as shown in the bottom most box. DETAILED DESCRIPTION

[0015] A genetic marker is a polymorphic locus having alleles in genomic DNA with characteristics of interest for analysis. One type of genetic marker is the Short Tandem Repeat (STR). The instant disclosure provides an STR analysis kit and methods for its use which results in a unique assay layout.

[0016] A "short tandem repeat (STR)" or "STR locus" refers to a region of a genome which contains short, repetitive sequence elements of 2 to 7 nucleotides in length adjacent to one another. Each sequence element is repeated at least once within a STR and is referred to as a "repeat unit" or simply "repeat." A STR repeat unit can be a three nucleotide sequence such as ATC or a four nucleotide sequence such as GATA or a five nucleotide sequence such as ATTGC and so forth.

[0017] Because STRs are polymorphic they can be used as an identification tool. Usually, within a population about 5-20% of the individuals share the same STR allele. An "allele" refers to an alternative form of the same STR locus, such as a difference in repeat numbers. The STR locus D10S1248, for instance, contains between 8 and 19 repeats of the nucleotide sequence GGAA. The nucleotide sequence with eight repeats can be represented as [GGAA] ₈ , while the nucleotide sequence of nineteen repeats can be represented as [GGAA]i ₉ . Each of the different number of repeats represents a different Dl OS 1248 allele.

[0018] Other allelic forms of a STR locus can include "incomplete repeats," "imperfect repeats," and "variant repeats" which refer to a tandem repeat within which the repeat unit has sequence differences between one or more repeat units.

[0019] An incomplete repeat is a tandem repeat in which the number of nucleotides in a repeat unit is incomplete. Allele 9.3 of the STR locus HUMTHOl is an example of an incomplete repeat. The sequence of the repeat unit for the HUMTHOl STR locus is AATG. The 9.3 allele contains nine "AATG" repeats and one incomplete repeat, "ATG." The nucleotide sequence of the 9.3 allele can be represented as [AATG] ₆ ATG[AATG]3. Other known variant alleles of HUMTHOl include 5.3, 6.1, 6.3, 7.1, 7.3, 8.3, 9.1, 10.3 and 13.3. The HUMTHOl locus also includes non-variant alleles ranging from 3-14 repeat units. [0020] An example of imperfect repeat is the sequence ATCG ATCG AACG ATCG ATCG where the sequence of the third repeat unit is not identical to the other repeat units. A variant repeat has variation(s) within the repeat unit, for example, ATCC ATCG ATCC ATCG ATCG ATCC ATCC, where the 4-peat repeat unit has a variant base pair at the fourth position of the repeat unit, either a "C" or a "G" nucleotide.

[0021] The terms for the particular STR loci as used herein refer to the names assigned to these loci as they are known in the art. Where appropriate, the current Accession Number as of time of filing is presented, as provided by Gen Bank® (National Center for Biotechnology Information, Bethesda, Md.).

[0022] Information about specific STRs, including their sequences can be found in the STRbase on the world wide web.cstl.nist.gov/strbase (as updated on 26 August 2013). Using this resource, the STR locus D1S1656 is identified is having an allele range of 9-21, with incomplete, imperfect and variant alleles within this range. The STR locus D2S1338 is identified as having an allele range of 11-28, with incomplete, imperfect and variant alleles within this range. The STR locus D22S1045 is identified as having an allele range of 8-20. The STR locus D19S433 is identified as having an allele range of 5.2-20, with incomplete, imperfect and variant alleles within this range. The STR locus D2S441 is identified as having an allele range of 8-17, with incomplete alleles within this range. The STR locus D6S1043 is identified as having an allele range of 1 1-28, with incomplete, imperfect and variant alleles within this range. The STR locus D12S391 is identified as having an allele range of 15-26, with variant alleles within this range.

[0023] By assaying multiple, independently assorting STR loci it becomes possible to identify specific individuals within a population. This is because the product rule of probabilities can be applied to determining the likelihood of having any particular combination of STR alleles. Under the product rule the chance of having any particular combination of alleles for a first STR locus is multiplied by the chance of having another particular combination of alleles for a second STR locus and so on. Accordingly, by increasing the number of STR loci analyzed, one can increase the potential discriminatory power of an STR based assay. Commercially available STR based identification tools such as GlobalFiler interrogate 22 STR loci simultaneously, providing a discriminatory power on the order of 7.12 x 10 ^"26 . [0024] In some embodiments, a kit is provided wherein the kit encompasses primer pairs for the amplification of the STR loci D10S1248 and HUMTHOl and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391.

[0025] In some embodiments, a kit is provided wherein the kit encompasses a container having one or more locus-specific primers. A kit can also provide instructions for use. A kit can also provide other components, such as, for example, one or more of an allelic ladder directed to each of the specified loci, a sufficient quantity of enzyme for amplification, amplification buffer to facilitate the amplification, divalent cation solution to facilitate enzyme activity, dNTPs for strand extension during amplification, loading solution for preparation of the amplified material for electrophoresis, genomic DNA as a template control, a size marker to insure that materials migrate as anticipated in the separation medium, and a protocol and manual to educate the user and limit error in use. The amounts of the various reagents in the kits also can be varied depending upon a number of factors, such as the optimum sensitivity of the process. A kit may or may not include one or more of the components described in the forgoing with the exception of one or more locus-specific primers, which are necessarily included.

[0026] In some embodiments, a kit is provided wherein the kit encompasses primer pairs for the co-amplification of the STR loci D10S1248 and HUMTHOl and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus

amelogenin.

[0027] Amelogenin is not an STR locus, but it produces X and Y chromosome specific PCR products of different sizes. Amelogenin locus is identified by GenBank as HUMAMELY (when used to identify a locus on the Y chromosome as present in male DNA) or as

HUMAMELX (when used to identify a locus on the X chromosome as present in male or female DNA). Thus, amelogenin can be used in gender determination.

[0028] DNA amplification techniques, such as polymerase chain reaction (PCR), employ a "primer pair." A primer pair includes an "upstream" or "forward" primer and a "downstream" or "reverse" primer, which delineate a region of a nucleic acid template to be amplified. One primer anneals to the sense (+) strand of a denatured template DNA, and the other primer anneals to the antisense (-) strand of the denatured template DNA. The orientation of the two primer- binding sites is generally different. A first primer and a second primer may be either an upstream or a downstream primer and are used interchangeably.

[0029] "Primer" refers to a polynucleotide and analogs thereof that are capable of selectively hybridizing to a target nucleic acid. Typically a primer can be between about 10 to 100 nucleotides in length and can provide a point of initiation for template-directed synthesis of a polynucleotide complementary to the template, which can take place in the presence of appropriate enzyme(s), cofactors, and substrates such as nucleotides.

[0030] Exemplary amplification techniques include, but are not limited to, PCR or any other method employing a primer extension step. Other non-limiting examples of amplification include ligase detection reaction (LDR) and ligase chain reaction (LCR). Amplification methods can comprise thermal-cycling or can be performed isothermally.

[0031] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTH01 and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons.

[0032] "Amplicon" refers to an amplified polynucleotide sequence.

[0033] In some embodiments, an amplicon is of a length and has a sequence that is determinative of an STR allele. The amplicon may range in length from the combined length of the primer pairs plus and/or one nucleotide base pair, and/or the combined length of the primer pairs plus about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 nucleotides.

[0034] In some embodiments, a fluorophore can be used to label at least one primer of the multiplex amplification, for example, by being covalently bound to the primer, thus creating a fluorescent labeled primer.

[0035] Fluorescent labels suitable for attachment to primers are numerous and

commercially available. The following are some examples of possible fluorescent labels, also referred to as fluorophores. Exemplary fluorophores include: fluorescein (FL), which absorbs maximally at 492 nm and emits maximally at 520 nm; N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA™), which absorbs maximally at 555 nm and emits maximally at 580 nm; 5-carboxyfluorescein (5-FAM™), which absorbs maximally at 495 nm and emits maximally at 525 nm; 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE™), which absorbs maximally at 525 nm and emits maximally at 555 nm); 6-carboxy-X-rhodamine (ROX™), which absorbs maximally at 585 nm and emits maximally at 605 nm; CY3™, which absorbs maximally at 552 nm and emits maximally at 570 nm; CY5™, which absorbs maximally at 643 nm and emits maximally at 667 nm; tetrachloro-fluorescein (TET™), which absorbs maximally at 521 nm and emits maximally at 536 nm; and hexachloro-fluorescein (HEX™), which absorbs maximally at 535 nm and emits maximally at 556 nm; NED™ which absorbs maximally at 546 nm and emits maximally at 575 nm; 6-carboxyfluorescein (6-FAM™), which absorbs maximally at 495 nm and emits maximally at approximately 520 nm; VIC® which emits maximally at approximately 550 nm; PET® which emits maximally at approximately 590 nm; and LIZ™, which emits maximally at approximately 650 nm. See, S R Coticone et al., U.S. Pat. No. 6,780,588; AMPF/STR® IDENTIFILER™ PCR AMPLIFICATION KIT USERS

MANUAL, pp. 1-3, Applied Biosystems (2001). Other fluorophores include BTG, BTY and BTR. Note that the above listed emission and/or absorption wavelengths are typical and can be used for general guidance purposes only; actual peak wavelengths may vary for different applications and under different conditions. Additional fluorophores can be selected for the desired absorbance and emission spectra as well as color as is known to one of skill in the art and are provided below.

[0036] Where fluorescent labeling of primers is used in a multiplex reaction, generally at least three different labels, at least four different labels, at least five different labels or at least six different labels can be used to label the different primers. The at least four dyes may comprise any four of the above-listed dyes, or any other four dyes known in the art, or 6-FAM™, VIC®, NED™ and PET®. Other embodiments of the present teaching may comprise a single multiplex reaction comprising at least five different dyes. These at least five dyes may comprise any five of the above-listed dyes, or any other five dyes known in the art, or 6-FAM™, VIC®, NED™ PET®, and LIZ™ dyes. Other embodiments of the present teaching may comprise a single multiplex reaction comprising at least six different dyes. These at least six dyes may comprise any six of the above-listed dyes, or any other six dyes known in the art, 6-FAM™, VIC®, NED™, PET®, SID™ and LIZ™ dyes with the SID dye having a maximum emission at approximately 620 nm (LIZ™ dye was used to label the size standards). TAZ™ dye can also be used (Applied Biosystems). The dyes TMR-ET, CXR-ET and CC5 can be used (Promega, Madison, Wis.) as well as BTG, BTY and BTR (Qiagen, N.V., Venlo, Netherlands).

[0037] In some embodiments, primers for different target loci in a multiplex can be labeled with different fluorophores, each fluorophore producing a different colored product depending on the emission wavelength of the fluorophore. These variously labeled primers can be used in the same multiplex reaction, and their respective amplification products subsequently analyzed together. Either the forward or reverse primer of the pair that amplifies a specific locus can be labeled. In some embodiments, both primers of the primer pair are labeled. [0038] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTH01 and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein each of the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with the same fluorescent label. In some embodiments, a kit is provided wherein the amplicons generated by the primer pairs for amelogenin and D2S1338 are labeled with the same fluorescent label. In some embodiments, a kit is provided wherein the amplicons generated by the primer pairs for D22S1045 and D19S433 and HUMTH01 are labeled with the same fluorescent label. In some embodiments, a kit is provided wherein the amplicons generated by the primer pairs for D2S441 and D6S1043 and D12S391 are labeled with the same fluorescent label.

[0039] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTH01 and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein each of the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with the same fluorescent label, wherein the amplicons generated by the primer pairs for amelogenin and D2S1338 are labeled with the same fluorescent label, wherein the amplicons generated by the primer pairs for D22S1045 and D19S433 and HUMTH01 are labeled with the same fluorescent label and wherein the amplicons generated by the primer pairs for D2S441 and D6S1043 and D12S391 are labeled with the same fluorescent label.

[0040] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTH01 and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for amelogenin. In some embodiments, a kit is provided wherein the amplicons generated the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D2S1338. In some embodiments, a kit is provided wherein the amplicons generated the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D22S1045. In some embodiments, a kit is provided wherein the amplicons generated the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D19S433. In some embodiments, a kit is provided wherein the amplicons generated the primer pairs for Dl OS 1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for HUMTH01. In some embodiments, a kit is provided wherein the amplicons generated the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D2S441. In some embodiments, a kit is provided wherein the amplicons generated the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D6SS1043. In some embodiments, a kit is provided wherein the amplicons generated the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D12S391.

[0041] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTH01 and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for amelogenin and the primer pair for D2S1338. In some embodiments, a kit is provided wherein the amplicons generated the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D22S 1045 and the primer pair for D 19S433 and the primer pair for HUMTH01. In some embodiments, a kit is provided wherein the amplicons generated the primer pairs for Dl OS 1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D2S441 and the primer pair for D6SS1043 and the primer pair for D12S391.

[0042] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTH01 and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for amelogenin, the primer pair for D2S1338, the primer pair for D22S1045, the primer pair for D19S433, the primer pair for HUMTHOl, the primer pair for D2S441, the primer pair for D6SS1043 and the primer pair for D12S391.

[0043] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTHOl and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for amelogenin and the primer pair for D2S1338, wherein the one or more amplicons generated by the primer pair for amelogenin and the primer pair for D2S1338 are labeled with the same fluorescent label and wherein the one or more amplicons generated by the primer pair for D10S1248 and the primer pair for D1S1656 are labeled with the same fluorescent label.

[0044] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTHOl and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D22S1045 and the primer pair for D19S433 and the primer pair for HUMTHOl, wherein the one or more amplicons generated by the primer pairs for D22S1045 and D19S433 and HUMTHOl are all labeled with the same fluorescent label and wherein the one or more amplicons generated by the primer pair for D10S1248 and the primer pair for D1S1656 are labeled with the same fluorescent label.

[0045] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTHOl and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D2S441 and the primer pair for D6S1043 and the primer pair for D12S391, wherein the one or more amplicons generated by the primer pairs for D2S441 and D6S1043 and D12S391 are all labeled with the same fluorescent label and wherein the one or more amplicons generated by the primer pair for D10S1248 and the primer pair for D1S1656 are labeled with the same fluorescent label.

[0046] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTH01 and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for amelogenin and the primer pair for D2S1338, wherein the one or more amplicons generated by the primer pairs for amelogenin and D2S1338 are all labeled with the same fluorescent label, wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D22S1045 and the primer pair for D19S433 and the primer pair for HUMTH01, wherein the one or more amplicons generated by the primer pairs for D22S1045 and D19S433 and

HUMTH01 are all labeled with the same fluorescent label, wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D2S441 and the primer pair for D6S1043 and the primer pair for D12S391, wherein the one or more amplicons generated by the primer pairs for D2S441 and D6S1043 and D12S391 are all labeled with the same fluorescent label and wherein the one or more amplicons generated by the primer pair for Dl OS 1248 and the primer pair for D1S1656 are labeled with the same fluorescent label.

[0047] Besides fluorescent labels, other detection moieties and methodologies include the use of radiolabeling and non-radioactive methods of detection, such as silver staining and DNA intercalators, because fluorescent methods of detection generally reveal fewer amplification artifacts than do silver staining and DNA intercalators. This is due in part to the fact that only the amplified strands of DNA with labels attached thereto are detected in fluorescent detection, whereas both strands of every amplified product are stained and detected using the silver staining and intercalator methods of detection, which result in visualization of many non-specific amplification artifacts. Additionally, there are potential health risks associated with the use of EtBr and SYBR. EtBr is a known mutagen; SYBR, although less of a mutagen than EtBr, is generally suspended in DMSO, which can rapidly pass through skin.

[0048] The amplicons can be analyzed on a sieving or non-sieving medium. In some embodiments, the amplicons are analyzed by electrophoresis; for example, capillary

electrophoresis, or slab gel electrophoresis, or denaturing polyacrylamide gel electrophoresis. The separation of DNA fragments by electrophoresis is based primarily on differential fragment size.

[0049] When the amplicons are separated by capillary electrophoresis, typically the size of the amplicons are between 70 and 500 bp, in some instances 70 and 450 bp, in some instances 70 to 400 bp., and in some instances 70 and 350 bp and in some instances 70 and 300 bp. These size limitations put constraints on the combination of loci, and the underlying alleles, which can be labeled with the same fluorescent label. And further can put constraints on the overall assay layout.

[0050] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTH01 and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein the wherein the smallest possible amplicon generated by the primer pair for D1S 1656 is larger than the smallest possible amplicon generated by the primer pair for Dl OS 1248. In some

embodiments, the smallest possible amplicon generated by the primer pair for D2S1338 is larger than the smallest possible amplicon generated by the primer pair for amelogenin. In some embodiments, the smallest possible amplicon generated by the primer pair for HUMTH01 is larger than the smallest possible amplicon generated by the primer pair for D19S433. In some embodiments, the smallest possible amplicon generated by the primer pair for HUMTH01 is larger than the smallest possible amplicon generated by the primer pair for D22S1045. In some embodiments, the smallest possible amplicon generated by the primer pair for D19S433 is larger than the smallest possible amplicon generated by the primer pair for D22S1045. In some embodiments, the smallest possible amplicon generated by the primer pair for D12S391 is larger than the smallest possible amplicon generated by the primer pair for D6S1043. In some embodiments, the smallest possible amplicon generated by the primer pair for D12S391 is larger than the smallest possible amplicon generated by the primer pair for D2S441. In some embodiments, the smallest possible amplicon generated by the primer pair for D6S1043 is larger than the smallest possible amplicon generated by the primer pair for D2S441.

[0051] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTH01 and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein the wherein the smallest possible amplicon generated by the primer pair for D1S 1656 is larger than the smallest possible amplicon generated by the primer pair for D10S1248, wherein the smallest possible amplicon generated by the primer pair for D2S1338 is larger than the smallest possible amplicon generated by the primer pair for amelogenin, wherein the smallest possible amplicon generated by the primer pair for HUMTH01 is larger than the smallest possible amplicon generated by the primer pair for D19S433, wherein the smallest possible amplicon generated by the primer pair for HUMTH01 is larger than the smallest possible amplicon generated by the primer pair for D22S1045, wherein the smallest possible amplicon generated by the primer pair for D19S433 is larger than the smallest possible amplicon generated by the primer pair for D22S1045, wherein the smallest possible amplicon generated by the primer pair for D12S391 is larger than the smallest possible amplicon generated by the primer pair for D6S1043, wherein the smallest possible amplicon generated by the primer pair for D12S391 is larger than the smallest possible amplicon generated by the primer pair for D2S441 and wherein the smallest possible amplicon generated by the primer pair for D6S1043 is larger than the smallest possible amplicon generated by the primer pair for D2S441.

[0052] Amplicons can also be analyzed by chromatography; for example, by size exclusion chromatography (SEC). [0053] Once the amplicons are separated, each amplicon representing an amplified allele, can then be visualized and analyzed, for example by detection of a fluorescent signal. When fluorescent-labeled primers are used for detecting each locus in the multiplex reaction, amplification can be followed by detection of the labeled products employing a fiuorometric detector.

[0054] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTH01 and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein each of the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with the same fluorescent label, wherein the amplicons generated by the primer pairs for Dl OS 1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D2S1338, wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D22S1045, wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D12S391 and wherein the smallest possible amplicon generated by the primer pair for D1S1656 is larger than the smallest possible amplicon generated by the primer pair for Dl OS 1248.

[0055] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTH01 and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein each of the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with the same fluorescent label, wherein the amplicons generated by the primer pairs for Dl OS 1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for amelogenin, wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D22S1045, wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D12S391 and wherein the smallest possible amplicon generated by the primer pair for D1S1656 is larger than the smallest possible amplicon generated by the primer pair for Dl OS 1248.

[0056] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTH01 and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein each of the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with the same fluorescent label, wherein the amplicons generated by the primer pairs for Dl OS 1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for amelogenin and the one or more amplicons generated by the primer pair for D2S1338, wherein the amplicons generated by the primer pairs for amelogenin and D2S1338 are labeled with the same fluorescent label, wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D22S1045, wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D12S391 and wherein the smallest possible amplicon generated by the primer pair for D1S1656 is larger than the smallest possible amplicon generated by the primer pair for Dl OS 1248.

[0057] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTH01 and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein each of the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with the same fluorescent label, wherein the amplicons generated by the primer pairs for Dl OS 1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D2S1338, wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D19S433, wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D12S391 and wherein the smallest possible amplicon generated by the primer pair for D1S1656 is larger than the smallest possible amplicon generated by the primer pair for Dl OS 1248.

[0058] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTH01 and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein each of the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with the same fluorescent label, wherein the amplicons generated by the primer pairs for Dl OS 1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for amelogenin, wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D19S433, wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D12S391 and wherein the smallest possible amplicon generated by the primer pair for D1S1656 is larger than the smallest possible amplicon generated by the primer pair for Dl OS 1248.

[0059] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTH01 and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein each of the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with the same fluorescent label, wherein the amplicons generated by the primer pairs for Dl OS 1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for amelogenin and the one or more amplicons generated by the primer pair for D2S1338, wherein the amplicons generated by the primer pairs for amelogenin and D2S1338 are labeled with the same fluorescent label, wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D19S433, wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D12S391 and wherein the smallest possible amplicon generated by the primer pair for D1S1656 is larger than the smallest possible amplicon generated by the primer pair for Dl OS 1248.

[0060] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTHOl and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons and wherein each of the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with the same fluorescent label, wherein the amplicons generated by the primer pairs for Dl OS 1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D2S1338, wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for HUMTHOl, wherein the amplicons generated by the primer pairs for D10S1248 and D1S1656 are labeled with a fluorescent label different from the one or more amplicons generated by the primer pair for D12S391 and wherein the smallest possible amplicon generated by the primer pair for D1S1656 is larger than the smallest possible amplicon generated by the primer pair for Dl OS 1248.

[0061] In some embodiments, a kit is provided wherein the kit encompasses oligonucleotide primer pairs for the co-amplification of the STR loci D10S1248 and HUMTHOl and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin wherein the co-amplified loci generate a set of amplicons, wherein the layout of the loci in terms of fluorescent channels they occupy with other STR loci and their relative position is that as shown in Figure 1.

[0062] Visualization of can also be accomplished using any of a number of techniques known in the art, such as, for example, silver staining or by use of reporters such as radioisotopes or chemiluminescents and enzymes in combination with detectable substrates. Where fluorescent-labeled primers are used for detecting each locus in the multiplex reaction, amplification can be followed by detection of the labeled products employing a fiuorometric detector.

[0063] In some embodiments, methods of amplifying alleles of the STR loci Dl OS 1248 and HUMTHOl and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin are provided, the methods encompassing using any of the combination of primer pairs taught in the foregoing kits.

[0064] In some embodiments, compositions are provided, the compositions being the amplified alleles of the STR loci D10S1248 and HUMTHOl and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin resulting from the combination of primer pairs taught in the foregoing kits.

[0065] In some embodiments, compositions are provided, the compositions being the primers for the STR loci D10S1248 and HUMTHOl and D1S1656 and D2S1338 and D22S1045 and D19S433 and D2S441 and D6S1043 and D12S391 and the locus amelogenin, as disclosed in the foregoing kits. In some embodiments, a primer for 2, 3, 4, 5, 6, 7, 8, 9 or 10 loci are included in a single tube, the primers being those described in the foregoing kits, and a second tube including a primer for 2, 3, 4, 5, 6, 7, 8, 9 or 10 loci, the primers being those described in the foregoing kits, such that a primer pair is possible when the first and second tube are used in conjunction. In some embodiments, a primer pair for 2, 3, 4, 5, 6, 7, 8, 9 or 10 loci are included in a single tube, the primers being those described in the foregoing kits.

EXAMPLES

Example I

[0066] A DNA sample to be analyzed was combined with STR- and Amelogenin-specific primer sets in a PCR mixture to amplify the loci D1S1656, D2S441, D2S1338, D6S1043, D10S1248, HUMTHOl, D12S391, D19S433 and D22S1045) and amelogenin. Primer sets for these loci were designed according to the methodology provided herein, supra. One primer from each of the primer sets that amplify D1S1656 and D10S1248 was labeled with the 6-FAM™ fluorescent label. One primer from each of the primer sets that amplify D2S1338 and amelogenin was labeled with the VIC® fluorescent label. One primer from each of the primer sets that amplify TH01, D19S433 and D22S1045 was labeled with the NED™ fluorescent label. One primer from each of the primer sets that amplify D2S441, D6S1043 and D12S391 was labeled with the PET™ fluorescent label. A fifth fluorescent label, LIZ™ dye, was used to label a size standard. PCR Assay

[0067] The recommended PCR conditions call for 1.0 ng of genomic DNA to be amplified in a total reaction volume of 25 μί. A PCR reaction mix is prepared based on the following calculation per reaction: 10 μΐ of Master mix, 5 μΐ of 5x primer mix and 0.5 μΐ AmpliTaq Gold. These are thorough mixed followed by briefly centrifuging to remove any liquid from the cap of the vial containing the PCR reaction mix.

[0068] The PCR reaction mix is aliquoted into each reaction vial or well followed by addition of each sample to be analyzed into its own vial or well, up to 10 μί volume to have approximately 1.0 ng sample DNA/reaction. Samples of less than 10 μΐ _^ are made up to a final 10 μΐ. volume with Low-TE Buffer (consisting of 10 mM Tris-Cl pH 8.0 and 0.1 mM EDTA, was added as needed to bring the reaction volume up to 25 μί). Following sample addition the tubes or wells are covered and a brief centrifugation at 3000 rpm for about 30 seconds is performed to remove any air bubbles prior to amplification.

PCR Reaction Parameters

[0069] PCR reactions were set up in MicroAmp® 96-well reaction plates covered by MicroAmp® Clear Adhesive Film. The thermal cycling conditions are an initial incubation step at 95° C. for 1 1 min., 28 cycles of 94° C. for 1 min. denaturing, 59° C. for 1 min. annealing and 72o C. for 1 min. extension, followed by a final extension at 60° C. for 10 min. and final hold at 4° C. indefinitely.

Capillary Electrophoresis Sample Preparation and Detection

[0070] The amplified samples are analyzed by methods that resolve amplification product size and/or sequence differences as would be known to one of skill in the art. For example, capillary electrophoresis can be used following the instrument manufactures directions. Briefly, 0.5 μΕ GeneScan™-600 LIZ™ Size Standard and 8.5 μΕ of Hi-Di™ Formamide are mixed for each sample to be analyzed. 9.04 of the Formamide/GeneScan-600 LIZ solution is dispensed into each well of a MicroAmp® Optical 96-well reaction plate to which a 1.0 μί aliquot of the PCR amplified sample or allelic ladder is added and the plate is covered. The plate is briefly centrifuged to mix the contents and collect them at the bottom of the plate. The plate is heated at 95° C. for 3 minutes to heat-denature the samples and then quenched immediately by placing on ice for 3 minutes.

Capillary Electrophoresis Methods and Analysis

[0071] Capillary electrophoresis (CE) was performed on the current Applied Biosystems instruments: the Applied Biosystems 3500x1 Genetic Analyzer using the specified J6 variable binning module as described in the instrument's User's Guide. The 3500x1 Genetic Analyzer's parameters were: sample injection for 24 sec at 1.2 kV and electrophoresis at 15 kV for 1210 sec in Performance Optimized Polymer (POP-4™ polymer) with a run temperature of 60° C. as indicated in the HID36_POP4xl_G5_NT3200 protocol. Variations in instrument parameters, e.g. injection conditions, were different on other CE instruments such as the 3500, 3130x1, or 3130 Genetic Analyzers.) The data were collected using versions the Applied Biosystems Data Collection Software specific to the different instruments, such as v.3.0 for the 3130x1 and 3500 Data Collection Software vl .O that were analyzed using GeneMapper ID-X vl .2. FIG. 1 provides the spacing of an exemplary 11-plex multiplex of the present teachings.

[0072] Following instrument set-up according to the manufacturer's directions each sample is injected and analyzed by appropriate software, e.g., GeneMapper® ID Software v3.2 or GeneMapper® ID-X vl .2 software with the standard analysis settings. A peak amplitude of 50 RFU (relative fluorescence units) was used as the peak detection threshold.

DNA Samples

[0073] Anonymous whole-blood samples were purchased from Seracare Life Sciences (Oceanside, Calif.) or Interstate Blood Bank, Inc. (Memphis, Tenn.), and the control DNA 9947 A was purchased from Marligen Biosciences (Ijamsville, Md.). FTA cards, Indicating FT A cards, and EasiCollect devices were purchased from Whatman, Inc. Blood on FTA cards was prepared by spotting 75-80 uL of whole blood onto the center of the sampling spot. Buccal cells were collected using Buccal DNA Collector® (Bode Technology) EasiCollect devices or foam swabs, followed by contact transfer to the Indicating FTA cards.

[0074] Buccal or Blood were spotted on FTA paper samples. A 1.2 mm punch was removed from the center of the sample and placed into individual wells of a MicroAmp® Optical 96-well reaction plate. Manual punching was performed by placing the tip of a 1.2 mm Harris Micro- Punch on the card holding the barrel of the Harris Micro-Punch (do not touch the plunger) and gently pressing and twisting ¼-turn to cut the 1.2 mm punch which was then ejected into the appropriate well on the reaction plate. If automated punching is used refer to the User Guide of your automated or semi-automated disc punch instrument (e.g. BSD 600) for proper guidance. It is appropriate to make the punch as close as possible to the center of the sample to ensure optimum peak intensity. It is noted that increasing the size of the punch may cause inhibition during the PCR amplification phase of the assay and reduce the quality and reproducibility of the result.

[0075] As those skilled in the art will appreciate, numerous changes and modifications may be made to the various embodiments of the present teachings without departing from the spirit of these teachings. It is intended that all such variations fall within the scope of these teachings.