WO1997039138B1 - Multiplex amplification of short tandem repeat loci - Google Patents
Multiplex amplification of short tandem repeat lociInfo
- Publication number
- WO1997039138B1 WO1997039138B1 PCT/US1997/006293 US9706293W WO9739138B1 WO 1997039138 B1 WO1997039138 B1 WO 1997039138B1 US 9706293 W US9706293 W US 9706293W WO 9739138 B1 WO9739138 B1 WO 9739138B1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- loci
- primers
- group
- alleles
- Prior art date
Links
- 230000003321 amplification Effects 0.000 title claims abstract 17
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract 17
- 229920002393 Microsatellite Polymers 0.000 title claims 10
- 229920003013 deoxyribonucleic acid Polymers 0.000 claims 22
- 229920002401 polyacrylamide Polymers 0.000 claims 9
- 239000003155 DNA primer Substances 0.000 claims 8
- 239000000203 mixture Substances 0.000 claims 8
- 210000001519 tissues Anatomy 0.000 claims 8
- 238000004458 analytical method Methods 0.000 claims 6
- 239000007850 fluorescent dye Substances 0.000 claims 6
- 101700076922 3S31 Proteins 0.000 claims 3
- 239000003550 marker Substances 0.000 claims 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 claims 3
- 238000003752 polymerase chain reaction Methods 0.000 claims 3
- BQCADISMDOOEFD-UHFFFAOYSA-N silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims 3
- 229910052709 silver Inorganic materials 0.000 claims 3
- 239000004332 silver Substances 0.000 claims 3
- GZAJOEGTZDUSKS-UHFFFAOYSA-N 6-amino-3',6'-dihydroxyspiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C21OC(=O)C1=CC(N)=CC=C21 GZAJOEGTZDUSKS-UHFFFAOYSA-N 0.000 claims 2
- 210000004381 Amniotic Fluid Anatomy 0.000 claims 2
- 210000004369 Blood Anatomy 0.000 claims 2
- 210000004209 Hair Anatomy 0.000 claims 2
- 210000003296 Saliva Anatomy 0.000 claims 2
- 210000000582 Semen Anatomy 0.000 claims 2
- 210000002700 Urine Anatomy 0.000 claims 2
- 239000008280 blood Substances 0.000 claims 2
- 210000004027 cells Anatomy 0.000 claims 2
- 230000001605 fetal Effects 0.000 claims 2
- 238000001502 gel electrophoresis Methods 0.000 claims 2
- 230000003169 placental Effects 0.000 claims 2
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims 2
- 210000000988 Bone and Bones Anatomy 0.000 claims 1
- 229920000272 Oligonucleotide Polymers 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 claims 1
- 230000002068 genetic Effects 0.000 abstract 2
Abstract
The invention is directed to the simultaneous amplification of multiple distinct genetic loci using PCR or other amplification systems to determine in one multiplex reaction the allels of each of the loci contained in the multiplex reaction. The genetic loci analyzed comprise the following: HUMvWFA31, HUMLIPOL, HUMBFXIII, HUMF13A01, HUMFESFPS, HUMTH01, HUMTPOX, HUMCSF1PO, D22S683, D20S481, D19S253, D17S1299, D17S1298, D16S753, D16S539, D16S490, D14S562, D14S548, D14S118, D13S317, D10S1239, D9S930, D7S820, D5S818, D4S2368, D3S1539.
Claims
1 . A method of simultaneously determining the alleles present in at least four 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 at least four short tandem repeat loci of the DNA sample to be analyzed which can be amplified together, wherein at least four of the loci in the set are selected from the group of loci consisting of:
D3S1539, D4S2368, D5S818, D7S820, D9S930, D10S1239, D13S317, D14S118, D14S548, D14S562, D16S490, D16S539, D16S753, D17S1298, D17S1299, D19S253, D20S481, D22S683, HUMCSFIPO, HUMTPOX, HUMTH01, HUMFESFPS, HUMF13A01, HUMBFXIII, HUMLIPOL,
HUMvWFA31; c. 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 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.
2. The method of claim 1 , wherein the set of at least four loci co-amplified therein is a set of four loci, wherein the set of four loci is selected from the group of sets of loci consisting of: D3S1539, D7S820, D13S317, D5S818; D17S1298, D7S820, D13S317, D5S818; D20S481, D7S820, D13S317, D5S818; D9S930, D7S820, D13S317, D5S818;
D10S1239, D7S820, D13S317, D5S818; - 100-
D14S118, D7S820, D13S317, D5S818; D14S562, D7S820, D13S317, D5S818; D14S548, D7S820, D13S317, D5S818; D16S490, D7S820, D13S317, D5S818; D17S1299, D7S820, D13S317, D5S818;
D16S539, D7S820, D13S317, D5S818; D22S683, D7S820, D13S317, D5S818; D16S753, D7S820, D13S317, D5S818; D3S1539, D19S253, D13S317, D20S481; D3S1539, D19S253, D4S2368, D20S481;
D10S1239, D9S930, D4S2368, D20S481; and D16S539, D7S820, D13S317, HUMvWFA31.
3. The method of claim 1 , wherein the set of at least four loci co-amplified therein is a set of six loci, wherein the set of six loci is selected from the group of sets of loci consisting of:
D16S539, D7S820, D13S317, D5S818, HUMCSFIPO, HUMTPOX; and D16S539, D7S820, D13S317, D5S818, HUMF13A01, HUMFESFPS.
4. The method of claim 1 , wherein the set of at least four loci co-amplified therein is a set of seven loci, wherein the set is selected from the group of sets of loci consisting of:
D16S539, D7S820, D13S317, D5S818, HUMCSFI PO, HUMTPOX,
HUMTH01 ; and D16S539, D7S820, D13S317, D5S818, HUMF13A01 , HUMFESFPS,
HUMBFXIII.
5. The method of claim 1 , wherein the set of at least four loci co-amplified therein is a set of at least eight loci, and wherein the set is selected from the group of sets of loci consisting of: - 101 -
D16S539, D7S820, D13S317, D5S818, HUMCSFI PO, HUMTPOX, HUMTH01 , HUMvWFA31 ; and
D16S539, D7S820, D13S317, D5S818, HUMF13A01 , HUMFESFPS, HUMBFXIII, HUMLIPOL.
6. The method of claim 1 , wherein the multiplex amplification reaction is done using at least four pair of primers flanking the at least four loci analyzed.
7. The method of claim 6, additionally comprising the step of selecting pairs of primers for the multiplex amplification reaction which produce alleles from each locus that do not overlap the alleles of the other loci in the set co-amplified therein, when the alleles are separated by gel electrophoresis.
8. The method of claim 6, wherein at least one of each of the pairs of primers used in the multiplex amplification reaction has a sequence selected from one of the groups of sequences consisting of:
SEQ ID NO:1 and SEQ ID NO:2, when one of the loci in the set is D7S820;
SEQ ID NO:3 and SEQ ID NO:4, when one of the loci in the set is D13S317;
SEQ ID NO:5 and SEQ ID NO:6, when one of the loci in the set is D5S818; SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:49, when one of the loci in the set is D3S1539;
SEQ ID NO:9, SEQ ID NO:10, when one of the loci in the set is D17S1298;
SEQ ID NO:1 1 , SEQ ID NO:12, SEQ ID NO:52, SEQ ID NO: 53, when one of the loci in the set is D20S481 ; 9138 PCΪ7US97/06293
- 102-
SEQ ID NO:1 3, SEQ ID NO:14, SEQ ID NO: 55, SEQ ID NO: 61 , when one of the loci in the set is D9S930;
SEQ ID NO: 15, SEQ ID NO:1 6, SEQ ID NO:54, when one of the loci in the set is D1 OS 1239; SEQ ID NO: 1 7, SEQ ID NO: 18, when one of the loci in the set is
D14S1 1 8;
SEQ ID NO: 1 9, SEQ ID NO:20, when one of the loci in the set is D14S562;
SEQ ID NO:21 , SEQ ID NO:22, when one of the loci in the set is D14S548;
SEQ ID NO:23, SEQ ID NO:24, when one of the loci in the set is D1 6S490;
SEQ ID NO:25, SEQ ID NO:26, when one of the loci in the set is D1 6S753; SEQ ID NO:27, SEQ ID NO:28, when one of the loci in the set is
D1 7S1 299;
SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:58, when one of the loci in the set is D1 6S539;
SEQ ID NO:31 , SEQ ID NO:32, when one of the loci in the set is D22S683;
SEQ ID NO:33, SEQ ID NO:34, when one of the loci in the set is HUMCSFI PO;
SEQ ID NO:35, SEQ ID NO:36, when one of the loci in the set is HUMTPOX; SEQ ID NO:37, SEQ ID NO:38, 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 when one of the loci in the set is HUMvWFA31 ;
SEQ ID NO:41 , SEQ ID NO:42, when one of the loci in the set is HUMF13A01 ; - 103-
SEQ ID NO:43, SEQ ID NO:44, when one of the loci in the set is HUMFESFPS;
SEQ ID NO:45, SEQ ID NO:46, when one of the loci in the set is HUMBFXIII; SEQ ID NO:47, SEQ ID NO:48, when one of the loci in the set is
HUMLIPOL;
SEQ ID NO:50, SEQ ID NO:51 , when one of the loci in the set is D1 9S253; and
SEQ ID NO:56, SEQ ID NO:57, when one of the loci in the set is D4S2368.
9. The method of claim 6, wherein the multiplex amplification reaction is a polymerase chain reaction.
10. 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.
1 1 . The method of claim 1 , wherein the amplified alleles are evaluated using polyacrylamide gel electrophoresis to separate the alleles, forming a polyacrylamide gel of separated alleles.
1 2. The method of claim 1 1 , wherein the separated alleles in the polyacrylamide gel are determined by visualizing the alleles with silver stain analysis.
1 3. The method of claim 1 1 , wherein primers capable of binding to a region flanking each of the loci in the set are used in co-amplifying the loci, wherein at least one of the primers used in co-amplifying each locus has a fluorescent label covalently attached thereto such that the amplified alleles - 104-
produced therefrom are fluorescβntly labeled, and wherein the separated alleles in the polyacrylamide gel are determined by visualizing the alleles with fluorescent analysis.
14. The method of claim 1 3, wherein the fluorescent label is selected from the group of labels consisting of fluorescein and tetramethyl rhodamine.
1 5. The method of claim 1 wherein the at least one DNA sample to be analyzed is isolated from human tissue, wherein the human tissue 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.
1 6. A method of simultaneously determining the alleles present in three 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 three short tandem repeat loci of the DNA sample to be analyzed which can be amplified together, wherein the set of three loci is selected from the group of sets of loci consisting of:
D3S1539, D19S253, D13S317; D10S1239, D9S930, D20S481; D10S1239, D4S2368, D20S481; D10S1239, D9S930, D4S2368;
D16S539, D7S820, D13S317; and D10S1239, D9S930, D13S317. c. co-amplifying the set of three 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 - 1 05-
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.
1 7. The method of claim 1 6, wherein the multiplex amplification reaction is done using three pair of primers, wherein each pair of primers flanks one of the three short tandem repeat loci in the set of loci co-amplified in the reaction.
18. The method of claim 1 7, wherein each of the three pair of primers used in the multiplex amplification reaction is designed to hybridize with an allele of a locus in the set of loci co-amplified in the reaction wherein: when D7S820 is one of the loci in the set of loci co-amplified, at least one of the primers has a sequence selected from the group consisting of SEQ
ID NO: 1 and SEQ ID NO:2; when D1 3S31 7 is one of the loci in the set of loci co-amplified, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4; when D2OS481 is one of the loci in the set of loci co-amplified, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO: 1 1 , SEQ ID NO:1 2, SEQ ID NO:52, SEQ ID NO:53; when D9S930 is one of the loci in the set of loci co-amplified, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO: 1 3, SEQ ID NO: 1 4, SEQ ID NO:55 and SEQ ID NO:61 ; when D10S1 239 is one of the loci in the set of loci co-amplified, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO: 1 5, SEQ ID NO: 1 6 and SEQ ID NO:44; when D1 6S539 is one of the loci in the set of loci co-amplified, at least one of the primers has a sequence selected from the group consisting of SEQ
ID NO:29 and SEQ ID NO:30; and - 106-
when D4S2368 is one of the loci in the set of loci co-amplified, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:56 and SEQ ID NO:57.
1 9. The method of claim 16, wherein the multiplex amplification reaction is a polymerase chain reaction.
20. The method of claim 1 6, 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.
21 . The method of claim 16, wherein the amplified alleles are evaluated using polyacrylamide gel electrophoresis to separate the alleles, forming a polyacrylamide gel of separated alleles.
22. The method of claim 21 , wherein the separated alleles in the polyacrylamide gel are determined by visualizing the alleles with silver stain analysis.
23. The method of claim 21 , wherein the separated alleles in the polyacrylamide gel are determined by visualizing the alleles with fluorescent analysis.
24. The method of claim 1 6 wherein the at least one DNA sample to be analyzed is isolated from human tissue, wherein the human tissue 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. - 107-
25. A kit for simultaneously analyzing short tandem repeat sequences in at least three loci, comprising a container which has oligonucleotide primers for co-amplifying a set of at least three short tandem repeat loci, wherein the set of loci are selected from the sets of loci consisting of:
D3S1539, D19S253, D13S317;
D10S1239, D9S930, D20S481;
D1OS1239, D4S2368, D20S481;
D10S1239, D9S930, D4S2368; D16S539, D7S820, D13S317;
D10S1239, D9S930, D13S317;
D3S1539, D7S820, D13S317, D5S818;
D17S1298, D7S820, D13S317, D5S818;
D20S481, D7S820, D13S317, D5S818; D9S930, D7S820, D13S317, D5S818;
D10S1239, D7S820, D13S317, D5S818;
D14S118, D7S820, D13S317, D5S818;
D14S562, D7S820, D13S317, D5S818;
D14S548, D7S820, D13S317, D5S818; D16S490, D7S820, D13S317, D5S818;
D17S1299, D7S820, D13S317, D5S818;
D16S539, D7S820, D13S317, D5S818;
D22S683, D7S820, D13S317, D5S818;
D16S753, D7S820, D13S317, D5S818; D3S1539, D19S253, D13S317, D20S481
D3S1539, D19S253, D4S2368, D20S481
D10S1239, D9S930, D4S2368, D20S481
D16S539, D7S820, D13S317, HUMvWFA31;
D16S539, D7S820, D13S317, D5S818, HUMCSFIPO, HUMTPOX; D16S539, D7S820, D13S317, D5S818, HUMF13A01, HUMFESFPS; D16S539, D7S820, D13S31 7, D5S818, HUMCSFI PO, HUMTPOX, HUMTH01 ;
D1 6S539, D7S820, D1 3S31 7, D5S81 8, HUMF1 3A01 , HUMFESFPS, HUMBFXIII; D1 6S539, D7S820, D1 3S31 7, D5S81 8, HUMCSF1 PO, HUMTPOX,
HUMTH01 , HUMvWFA31 ; and
D16S539, D7S820, D13S317, D5S818, HUMF13A01 , HUMFESFPS, HUMBFXIII, HUMLIPOL.
26. The kit of claim 25, wherein each of the oligonucleotide primers is designed to hybridize with an allele of one of the three loci in the set of loci selected, wherein: when D7S820 is one of the loci in the set, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:2; when D13S317 is one of the loci in the set, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4; when D5S818, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6; when D3S1 53, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:49; when D1 7S1 298, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:9 and SEQ ID NO: 10; when D20S481 , at least one of the primers has a sequence selected from the group consisting of SEQ ID NO: 1 1 , SEQ ID NO: 1 2, SEQ ID NO:52, SEQ ID NO:53; when D9S930, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO: 1 3, SEQ ID NO: 14, SEQ ID NO:55, SEQ ID NO:61 ; - 109-
when D1 OS 1239, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:1 5, SEQ ID NO:1 6, SEQ ID NO:54; when D14S1 1 8, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:1 7, SEQ ID NO:1 8; when D14S562, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:1 9, SEQ ID NO:20; when D14S548, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:21 , SEQ ID NO:22; when D1 6S490, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24; when D1 6S753, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26; when D17S1 299, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28; when D1 6S539, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:58; when D22S683, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:31 , SEQ ID NO:32; when HUMCSFI PO, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:34; when HUMTPOX, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:36; when HUMTH01 , at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:37 SEQ ID NO:38; when HUMvWFA31 , at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:60; when HUMF1 3A01 , at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:41 , SEQ ID NO:42; when HUMFESFPS, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:44; - 1 10-
when HUMBFXIII, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:45, SEQ ID NO:46; when HUMLIPOL, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:47, SEQ ID NO:48; when D19S253, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:50, SEQ ID NO:51 ; and when D4S2368, at least one of the primers has a sequence selected from the group consisting of SEQ ID NO:56, SEQ ID NO:57.
27. The kit of claim 25, further comprising a container having reagents for at least one multiplex amplification reaction.
28. The kit of claim 25, further comprising a container having an allelic ladder.
29. The kit of claim 28, wherein each rung of the allelic ladder and at least one oligonucleotide primer for each of the loci in the set each have a label covalently attached thereto.
30. The kit of claim 29, wherein the label is a fluorescent label.
31 . The kit of claim 30, wherein at least one of the oligonucleotide primers has a different fluorescent label covalently attached thereto than some of the other primer pairs in the container.
32. A pair of oligonucleotide primers flanking locus D3S1539 in human DNA, wherein one primer of the pair has a sequence of SEQ ID NO:7, and wherein the other primer of the pair has a sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:49. - 1 1 1 -
33. A pair of oligonucleotide primers flanking locus D17S1298 in human DNA, wherein one primer of the pair has a sequence of SEQ ID NO:9, and wherein the other primer of the pair has a sequence of SEQ ID NO:10.
34. A pair of oligonucleotide primers flanking locus D20S481 in human DNA, wherein one primer of the pair has a sequence selected from the group consisting of SEQ ID NO:1 1 and SEQ ID NO:52, and wherein the other primer of the pair has a sequence selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:53.
35. A pair of oligonucleotide primers flanking locus D9S930 in human DNA, wherein one primer of the pair has a sequence of SEQ ID NO:55, and wherein the other primer of the pair has a sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO:61 .
36. A pair of oligonucleotide primers flanking locus D4S2368 in human DNA, wherein one primer of the pair has a sequence of SEQ ID NO:56 and wherein the other primer of the pair has a sequence of SEQ ID NO:57.
37. A method of simultaneously determining the alleles present in at least four 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 at least four short tandem repeat loci of the
DNA sample to be analyzed which can be amplified together, wherein three of the loci in the set are D7S820, D13S317, and D5S818; c. 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 - 1 12-
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.
38. The method of claim 37, wherein the multiplex amplification reaction is done using at least four pair of primers flanking the at least four loci analyzed.
39. The method of claim 38, additionally comprising the step of selecting pairs of primers for the multiplex amplification reaction which produce alleles from each locus that do not overlap the alleles of the other loci in the set co-amplified therein, when the alleles are separated by gel electrophoresis.
40. The method of claim 37, wherein the multiplex amplification reaction is a polymerase chain reaction.
41 . The method of claim 37, 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.
42. The method of claim 37, wherein the amplified alleles are evaluated using polyacrylamide gel electrophoresis to separate the alleles, forming a polyacrylamide gel of separated alleles.
43. The method of claim 42, wherein the separated alleles in the polyacrylamide gel are determined by visualizing the alleles with silver stain analysis. - 1 13-
44. The method of claim 42, wherein primers capable of binding to a region flanking each of the loci in the set are used in co-amplifying the loci, wherein at least one of the primers used in co-amplifying each locus has a fluorescent label covalently attached thereto such that the amplified alleles produced therefrom are fluorescently labeled, and wherein the separated alleles in the polyacrylamide gel are determined by visualizing the alleles with fluorescent analysis.
45. The method of claim 44, wherein the fluorescent label is selected from the group of labels consisting of fluorescein and tetramethyl rhodamine.
- 1 14-
STATEMENT UNDER PCT ARTICLE 19
Applicant respectfully submits that none of the amendments to any of the claims presented herein adds any new matter to the subject international application. The amendments are introduced herein to clarify the subject matter of each claim, as filed, and to facilitate examination of the application by the International Search Authority.
Applicant hereby requests communication of the enclosed PCT Article amendments to the Designated Offices, in accordance with PCT Article 20(2).
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1019980708392A KR100290332B1 (en) | 1996-04-15 | 1997-04-15 | Complex Amplification Method for Short Repetition Array Loci |
| AU33675/97A AU724531B2 (en) | 1996-04-15 | 1997-04-15 | Multiplex amplification of short tandem repeat loci |
| DE69736637T DE69736637T2 (en) | 1996-04-15 | 1997-04-15 | MULTIPLEX AMPLIFICATION SHORT TANDEM REPEAT LOCI |
| BR9708567-7A BR9708567A (en) | 1996-04-15 | 1997-04-15 | Multiplex amplification of short tandem repeat loci. |
| JP53732697A JP3602142B2 (en) | 1996-04-15 | 1997-04-15 | Multiplex amplification of STR loci |
| CA2251793A CA2251793C (en) | 1996-04-15 | 1997-04-15 | Multiplex amplification of short tandem repeat loci |
| EP97929672A EP0960207B1 (en) | 1996-04-15 | 1997-04-15 | Multiplex amplification of short tandem repeat loci |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/632,575 US5843660A (en) | 1994-09-30 | 1996-04-15 | Multiplex amplification of short tandem repeat loci |
| US08/632,575 | 1996-04-15 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1997039138A1 WO1997039138A1 (en) | 1997-10-23 |
| WO1997039138B1 true WO1997039138B1 (en) | 1997-12-31 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1997/006293 WO1997039138A1 (en) | 1996-04-15 | 1997-04-15 | Multiplex amplification of short tandem repeat loci |
Country Status (11)
| Country | Link |
|---|---|
| US (1) | US5843660A (en) |
| EP (2) | EP1715058A3 (en) |
| JP (2) | JP3602142B2 (en) |
| KR (1) | KR100290332B1 (en) |
| CN (2) | CN100422339C (en) |
| AU (1) | AU724531B2 (en) |
| BR (1) | BR9708567A (en) |
| CA (1) | CA2251793C (en) |
| DE (1) | DE69736637T2 (en) |
| ES (1) | ES2273367T3 (en) |
| WO (1) | WO1997039138A1 (en) |
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1996
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1997
- 1997-04-15 EP EP06007030A patent/EP1715058A3/en not_active Withdrawn
- 1997-04-15 CN CNB971949670A patent/CN100422339C/en not_active Expired - Lifetime
- 1997-04-15 JP JP53732697A patent/JP3602142B2/en not_active Expired - Lifetime
- 1997-04-15 DE DE69736637T patent/DE69736637T2/en not_active Expired - Lifetime
- 1997-04-15 BR BR9708567-7A patent/BR9708567A/en not_active Application Discontinuation
- 1997-04-15 KR KR1019980708392A patent/KR100290332B1/en active IP Right Grant
- 1997-04-15 CN CNA2008101288222A patent/CN101323880A/en active Pending
- 1997-04-15 AU AU33675/97A patent/AU724531B2/en not_active Expired
- 1997-04-15 ES ES97929672T patent/ES2273367T3/en not_active Expired - Lifetime
- 1997-04-15 EP EP97929672A patent/EP0960207B1/en not_active Revoked
- 1997-04-15 CA CA2251793A patent/CA2251793C/en not_active Expired - Lifetime
- 1997-04-15 WO PCT/US1997/006293 patent/WO1997039138A1/en active IP Right Grant
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- 2004-06-23 JP JP2004185341A patent/JP4034293B2/en not_active Expired - Lifetime
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