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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/16—Primer sets for multiplex assays

Definitions

• STRs polymorphic short tandem repeats
• 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.
• 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.
• compositions 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.
• 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.
• 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.
• a 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.
• kits 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.
• 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.
• PCR polymerase chain reaction
• 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.
• FIGURE 2 shows an electropherogram of the results of a multiplex PCR of the indicated loci and the alleles present for each locus.
• FIGURE 3 shows a plot of an allelic ladder for the loci D1S1656, D2S441,
• 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.
• loci Dl OS 1248 alleles 8-18 are physical alleles; allele 16 runs at about 110 bp
• 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
• HUMTHOl alleles 4-13.3 are physical alleles; allele 6 runs just below 190 bp, while allele 7 runs just above 190 bp
• 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.
• 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.
• 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
• 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.
• 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.
• 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.
• 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 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.
• STR loci 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.).
• 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.
• STR loci 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 .
• 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 .
• kits 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.
• kits 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.
• kits 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.
• kits 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 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.
• amelogenin can be used in gender determination.
• 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.
• Primer refers to a polynucleotide and analogs thereof that are capable of selectively hybridizing to a target nucleic acid.
• 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.
• 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.
• kits 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.
• Amplicon refers to an amplified polynucleotide sequence.
• 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,
• 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.
• Fluorescent labels suitable for attachment to primers are numerous and
• fluorophores include: fluorescein (FL), which absorbs maximally at 492 nm and emits maximally at 520 nm; N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRATM), which absorbs maximally at 555 nm and emits maximally at 580 nm; 5-carboxyfluorescein (5-FAMTM), which absorbs maximally at 495 nm and emits maximally at 525 nm; 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOETM), which absorbs maximally at 525 nm and emits maximally at 555 nm); 6-carboxy-X-rhodamine (ROXTM), which absorbs maximally at 585 nm and emits maximally at 605 nm; CY3TM, which absorb
• 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.
• fluorescent labeling of primers is used in a multiplex reaction
• 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-FAMTM, VIC®, NEDTM 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-FAMTM, VIC®, NEDTM PET®, and LIZTM dyes.
• inventions 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-FAMTM, VIC®, NEDTM, PET®, SIDTM and LIZTM dyes with the SID dye having a maximum emission at approximately 620 nm (LIZTM dye was used to label the size standards).
• TAZTM 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).
• 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.
• both primers of the primer pair are labeled.
• kits 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.
• 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.
• kits 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 D12
• kits 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.
• a kit 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.
• a kit 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.
• kits 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.
• a kit 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.
• 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.
• kits 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.
• kits 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 D1S16
• kits 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
• kits 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 D
• kits 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
• 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.
• 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.
• the amplicons can be analyzed on a sieving or non-sieving medium.
• 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.
• 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.
• kits 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.
• 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
• 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.
• 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.
• a 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
• Amplicons can also be analyzed by chromatography; for example, by size exclusion chromatography (SEC).
• SEC size exclusion chromatography
• each amplicon representing an amplified allele can then be visualized and analyzed, for example by detection of a fluorescent signal.
• 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.
• kits 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
• kits 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 amelogenin, wherein the amplicons generated
• kits 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 labele
• kits 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
• kits 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 amelogenin, wherein the amplicons generated
• kits 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 labele
• kits 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 D2S1338, wherein the amplicons generated by
• kits 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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-FAMTM 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 NEDTM fluorescent label.
• One primer from each of the primer sets that amplify D2S441, D6S1043 and D12S391 was labeled with the PETTM fluorescent label.
• a fifth fluorescent label, LIZTM dye, was used to label a size standard.
• the recommended PCR conditions call for 1.0 ng of genomic DNA to be amplified in a total reaction volume of 25 ⁇ .
• a PCR reaction mix is prepared based on the following calculation per reaction: 10 ⁇ of Master mix, 5 ⁇ of 5x primer mix and 0.5 ⁇ 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.
• 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 ⁇ volume to have approximately 1.0 ng sample DNA/reaction. Samples of less than 10 ⁇ ⁇ are made up to a final 10 ⁇ . 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 ⁇ ). 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.
• Low-TE Buffer consisting of 10 mM Tris-Cl pH 8.0 and 0.1 mM EDTA
• 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.
• 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.
• capillary electrophoresis can be used following the instrument manufactures directions. Briefly, 0.5 ⁇ GeneScanTM-600 LIZTM Size Standard and 8.5 ⁇ of Hi-DiTM 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 ⁇ 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 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-4TM polymer) with a run temperature of 60° C. as indicated in the HID36_POP4xl_G5_NT3200 protocol. Variations in instrument parameters, e.g.
• FIG. 1 provides the spacing of an exemplary 11-plex multiplex of the present teachings.
• 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.
• 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.

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