Multiplex amplification of short tandem repeat loci
DC CAFC
First Claim
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1. A method of simultaneously determining the alleles present in a set of short tandem repeat loci from one or more DNA samples, comprising:
- (a) obtaining at least one DNA sample to be analyzed, (b) selecting a set of loci of the DNA sample, comprising D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31, (c) co-amplifying the loci in the set 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
(d) evaluating the amplified alleles in the mixture to determine the alleles present at each of the loci analyzed in the set within the DNA sample.
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Abstract
Methods and materials are disclosed for use in simultaneously amplifying at least thirteen loci of genomic DNA in a single multiplex reaction, as are methods and materials for use in the analysis of the products of such reactions. Included in the present invention are materials and methods for the simultaneous amplification of at least thirteen short tandem repeat loci, including specific materials and methods for the analysis of thirteen such loci specifically selected by the United States Federal Bureau of Investigation as core loci for use in the Combined DNA Index System (CODIS) database.
134 Citations
24 Claims
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1. A method of simultaneously determining the alleles present in a set of short tandem repeat loci from one or more DNA samples, comprising:
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(a) obtaining at least one DNA sample to be analyzed, (b) selecting a set of loci of the DNA sample, comprising D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31, (c) co-amplifying the loci in the set 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
(d) evaluating the amplified alleles in the mixture to determine the alleles present at each of the loci analyzed in the set within the DNA sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
SEQ ID NO;
1, SEQ ID NO;
2, SEQ ID NO;
80 and SEQ ID NO;
81, when one of the loci in the set is D7S820;
SEQ ID NO;
3, SEQ ID NO;
4, SEQ ID NO;
82, and SEQ ID NO;
83, when one of the loci in the set is D13S317;
SEQ ID NO;
5, SEQ ID NO;
6, SEQ ID NO;
84, and SEQ ID NO;
85, when one of the loci in the set is D5S818;
SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
58, SEQ ID NO;
79, and SEQ ID NO;
97, when one of the loci in the set is D16S539;
SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
77, SEQ ID NO;
78, and SEQ ID NO;
98, when one of the loci in the set is HUMCSF1PO;
SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
72 and SEQ ID NO;
73, when one of the loci in the set is HUMTPOX;
SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
66, SEQ ID NO;
67, and SEQ ID NO;
103, when one of the loci in the set is HUMTH01;
SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
59, SEQ ID NO;
60, and SEQ ID NO;
76 when one of the loci in the set is HUMvWFA31;
SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
101, and SEQ ID NO;
102, when one of the loci in the set is D18S51;
SEQ ID NO;
64 and SEQ ID NO;
65, when one of the loci in the set is D21S11;
SEQ ID NO;
68, SEQ ID NO;
69, and SEQ ID NO;
106, when one of the loci in the set is D3S1358;
SEQ ID NO;
70, SEQ ID NO;
71, and SEQ ID NO;
107, when one of the loci in the set is HUMFIBRA; and
SEQ ID NO;
74, SEQ ID NO;
75, and SEQ ID NO;
104, when one of the loci in the set is D8S1179.
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6. The method of claim 1, wherein the amplified alleles are separated prior to evaluating in step (d), using a separation means selected from the group consisting of polyacrylamide gel electrophoresis and capillary gel electrophoresis.
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7. The method of claim 1, wherein the multiplex amplification reaction is done using pairs of oligonucleotide primers flanking the loci analyzed.
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8. The method of claim 7, wherein the set of loci is co-amplified using a polymerase chain reaction.
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9. The method of claim 7, wherein each of the loci co-amplified in the multiplex reaction in step (b) is co-amplified using a pair of primers which flank the locus, wherein at least one primer of each pair has a fluorescent label covalently attached thereto.
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10. The method of claim 9, wherein at least three of the labeled primers have different fluorescent labels covalently attached thereto.
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11. The method of claim 1 wherein the at least one DNA sample to be analyzed is prepared from human tissue, wherein the human tissue is selected from the group of human tissue consisting of blood, semen, vaginal cells, hair, saliva, urine, amniotic fluid containing placental cells or fetal cells, and mixtures of any of the tissues listed above.
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12. The method of claim 1, wherein the amplified alleles are evaluated by comparing the amplified alleles to a size standard, wherein the size standard is selected from the group of size standards consisting of a DNA marker and a locus-specific allelic ladder.
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13. A method of simultaneously identifying the alleles present in a set of loci of from one or more DNA samples, comprising:
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(a) obtaining at least one DNA sample to be analyzed;
(b) selecting a set of loci of the DNA sample, comprising short tandem repeat loci D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31;
(c) co-amplifying the loci in the set 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
(d) evaluating the amplified alleles in the mixture to determine the alleles present at each of the loci analyzed in the set within the DNA sample. - View Dependent Claims (14, 15, 16, 17)
SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
101, and SEQ ID NO;
102, when one of the loci in the set is D18S51;
SEQ ID NO;
64 and SEQ ID NO;
65, for the locus D21S11;
SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
66, SEQ ID NO;
67, and SEQ ID NO;
103, for the locus HUMTH01;
SEQ ID NO;
68, SEQ ID NO;
69, and SEQ ID NO;
106, for the locus D3S1358;
SEQ ID NO;
70, SEQ ID NO;
71, and SEQ ID NO;
107, for the locus HUMFIBRA;
SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
72, and SEQ ID NO;
73, for the locus HUMTPOX;
SEQ ID NO;
74, SEQ ID NO;
75, and SEQ ID NO;
104, for the locus D8S1179;
SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
59, SEQ ID NO;
60, and SEQ ID NO;
76, for the locus HUMvWFA31;
SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
77, SEQ ID NO;
78, and SEQ ID NO;
98, for the locus HUMCSF1PO;
SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
58, SEQ ID NO;
79, and SEQ ID NO;
97, for the locus D16S539;
SEQ ID NO;
1, SEQ ID NO;
2, SEQ ID NO;
80, and SEQ ID NO;
81, for the locus D7S820;
SEQ ID NO;
3, SEQ ID NO;
4, SEQ ID NO;
82, and SEQ ID NO;
83, for the locus D13S317; and
SEQ ID NO;
5, SEQ ID NO;
6, SEQ ID NO;
84, and SEQ ID NO;
85, for the locus D5S818.
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15. The method of claim 13, wherein the multiplex amplification reaction is a polymerase chain reaction.
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16. The method of claim 13, wherein the amplified alleles are evaluated by comparing the amplified alleles to a size standard, wherein the size standard is selected from the group of size standards consisting of a DNA marker and a locus-specific allelic ladder.
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17. The method of claim 13 wherein the at least one DNA sample to be analyzed is prepared from human tissue, wherein the human tissue is selected from the group of human tissue consisting of blood, semen, vaginal cells, hair, saliva, urine, bone, buccal sample, amniotic fluid containing placental cells or fetal cells, and mixtures of any of the tissues listed above.
- 18. A kit for simultaneously analyzing a set of loci of genomic DNA comprising oligonucleotide primers for co-amplifying a set of loci of the genomic DNA to be analyzed, wherein the set of loci comprises short tandem repeat loci which can be co-amplified, the primers are in one or more containers, the genomic DNA is human genomic DNA, and the loci comprise D3S1358, D5S818, D7S820, D8S1179, D1S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, AND HUMvWFA31.
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24. A method of simultaneously determining the alleles present in a set of loci from one or more DNA samples, comprising:
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(a) obtaining at least one DNA sample to be analyzed;
(b) selecting a set of loci of the at least one DNA sample, comprising at least thirteen short tandem repeat loci which can be co-amplified, wherein at least four of the at least thirteen short tandem repeat loci are selected from the group comprising;
D5S818, D7S820, D13S317, D16S539, D18S51, D21S11, D3S1358, D8S1179, HUMFIBRA, HUMCSF1PO, HUMTPOX, HUMTH01, and HUMvWFA31;
(c) co-amplifying the loci in the set 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
(d) evaluating the amplified alleles in the mixture to determine the alleles present at each of the loci analyzed in the set within the DNA sample.
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Specification
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Litigation Data
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Current AssigneePromega Corporation
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Original AssigneePromega Corporation
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InventorsSprecher, Cynthia J., Schumm, James W.
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Primary Examiner(s)Zitomer, Stephanie W.
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Application NumberUS09/199,542Time in Patent Office1,448 DaysField of Search435/91.2, 435/91.5, 435/91.1, 435/6, 435/5, 536/23.1, 536/24.3, 536/24.31, 536/24.33, 935/77, 935/78US Class Current435/6.11CPC Class CodesC12Q 1/6858 Allele-specific amplificationC12Q 1/6876 Nucleic acid products used ...C12Q 2525/151 repeat or repeated sequence...C12Q 2537/143 Multiplexing, i.e. use of m...C12Q 2565/125 Electrophoretic separationC12Q 2600/156 Polymorphic or mutational m...C12Q 2600/16 Primer sets for multiplex a...
