CA2318880A1 - Determination of a genotype of an amplification product at multiple allelic sites - Google Patents
Determination of a genotype of an amplification product at multiple allelic sites Download PDFInfo
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Abstract
A method is provided for genotyping a target sequence at at least two allelic sites by a 5' nuclease amplification reaction. In one embodiment, the method includes performing a nucleic acid amplification on a target sequence having at least two different allelic sites using a nucleic acid polymerase having 5'~3' nuclease activity and a primer capable of hybridizing to the target sequence in the presence of two or more sets of allelic oligonucleotide probes wherein: each set of allelic oligonucleotide probes is for detecting a different allelic site of the target sequence, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes to the target sequence, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer; detecting a fluorescence spectrum of the amplification; calculating a fluorescence contribution of each fluorescer to the fluorescence spectrum; and determining a presence or absence of the different allelic variants at the two or more different allelic sites based on the fluorescence contribution of each fluorescer to the combined fluorescence spectrum.
Claims (107)
1. A method for identifying which members of two or more sets of substantially homologous sequences are present in a sample of DNA, the method comprising:
performing a nucleic acid amplification on a sample of DNA which includes a first set of substantially homologous sequences and a second, different set of substantially homologous sequences using a nucleic acid polymerase having 5'~, 3' nuclease activity and one or more sets of forward and reverse primers capable of hybridizing to the sample DNA in the presence of two or more sets of oligonucleotide probes and amplifying the sets of substantially homologous sequences wherein:
each set of substantially homologous sequences includes two or more members which each differ from each other at at least one base position, each set of oligonucleotide probes is for detecting the members of one of the sets of substantially homologous sequences, each set of oligonucleotide probes includes two or more probes which are complementary to different members of a set of substantially homologous sequences, the member being 5' relative to a sequence of the sample DNA to which the primer hybridizes, and at least all but one of the oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer;
digesting those oligonucleotide probes which hybridize to the target sequence during the amplification by the nuclease activity of the polymerase;
detecting a fluorescence spectrum of the amplification;
calculating a fluorescence contribution of each fluorescer to the fluorescence spectrum; and determining a presence or absence of the different members of substantially homologous sequences based on the fluorescence contribution of each fluorescer to the fluorescence spectrum.
performing a nucleic acid amplification on a sample of DNA which includes a first set of substantially homologous sequences and a second, different set of substantially homologous sequences using a nucleic acid polymerase having 5'~, 3' nuclease activity and one or more sets of forward and reverse primers capable of hybridizing to the sample DNA in the presence of two or more sets of oligonucleotide probes and amplifying the sets of substantially homologous sequences wherein:
each set of substantially homologous sequences includes two or more members which each differ from each other at at least one base position, each set of oligonucleotide probes is for detecting the members of one of the sets of substantially homologous sequences, each set of oligonucleotide probes includes two or more probes which are complementary to different members of a set of substantially homologous sequences, the member being 5' relative to a sequence of the sample DNA to which the primer hybridizes, and at least all but one of the oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer;
digesting those oligonucleotide probes which hybridize to the target sequence during the amplification by the nuclease activity of the polymerase;
detecting a fluorescence spectrum of the amplification;
calculating a fluorescence contribution of each fluorescer to the fluorescence spectrum; and determining a presence or absence of the different members of substantially homologous sequences based on the fluorescence contribution of each fluorescer to the fluorescence spectrum.
2. The method according to claim 1 wherein the nucleic acid amplification is performed in the presence of a passive internal standard.
3. The method according to claim 2 wherein the passive internal standard is ROX.
4. The method according to claim 1 wherein all the oligonucleotide probes include a different fluorescer.
5. The method according to claim 1 wherein the nucleic acid amplification is performed in a reaction mixture containing at between about 4 and 6 mM MgCl2.
6. The method according to claim 1 wherein the nucleic acid amplification is performed in a reaction mixture containing glycerol.
7. The method according to claim 1 wherein the nucleic acid amplification is performed in a reaction mixture containing at least one member of the group consisting of gelatin and TWEEN 20.
8. The method according to claim 1 wherein the nucleic acid amplification is performed in a reaction mixture which includes about 7-9% glycerol, 0.04-0.06%
gelatin, and 0.005-0.015% TWEEN 20.
gelatin, and 0.005-0.015% TWEEN 20.
9. The method according to claim 1 wherein the nucleic acid amplification is performed in a reaction mixture which includes about 7-9% glycerol, 0.04-0.06%
gelatin, 0.005-0.015% TWEEN 20 and 25-75 mM tris buffer.
gelatin, 0.005-0.015% TWEEN 20 and 25-75 mM tris buffer.
10. The method according to claim 1 wherein the nucleic acid amplification is performed in a reaction mixture which includes about 7-9% glycerol, 0.04-0.06%
gelatin, 0.005-0.015% TWEEN 20, 25-75 mM tris buffer, pH 8.0, 4-6 mM MgCl2, 225 uM dATP, 175-225 uM dCTP, 175-225 uM deaza dGTP, 350-450 uM dUTP, .045-.055 U/uL AMPLITAQ TM Gold, 0.5-.015 U/uL AmpErase UNG, and 57-63 nM of a Passive Reference.
gelatin, 0.005-0.015% TWEEN 20, 25-75 mM tris buffer, pH 8.0, 4-6 mM MgCl2, 225 uM dATP, 175-225 uM dCTP, 175-225 uM deaza dGTP, 350-450 uM dUTP, .045-.055 U/uL AMPLITAQ TM Gold, 0.5-.015 U/uL AmpErase UNG, and 57-63 nM of a Passive Reference.
11. The method according to claim 1 wherein the one or more sets of forward and reverse primers define amplicons between about 50 and 150 bases in length.
12. The method according to claim 1 wherein the one or more sets of forward and reverse primers define amplicons less than about 100 bases in length.
13. The method according to claim 1 wherein the %GC of all the probes are at least about 20% and less than about 80%.
14. The method according to claim 1 wherein none of the probes have four or more contiguous guanines.
15. The method according to claim 1 wherein all of the probes have a melting point temperature that is about 3-5°C greater than an annealing temperature used in the amplification and the primer melting point temperature is about 2-4°C less than the annealing temperature.
16. The method according to claim 15 wherein the annealing temperature is about 60-64°C.
17. The method according to claim 1 wherein all of the probes have a melting point temperature about 65-67°C.
18. The method according to claim 17 wherein the primer melting point temperature is about 58-60°C.
19. The method according to claim 1 wherein all of the probes have a melting point temperature about 5-10 °C greater than the melting point temperature of the primers.
20. The method according to claim 1 wherein all of the probes have a melting point temperature about 7°C greater than the melting point temperature of the primers.
21. The method according to claim 1 wherein none of the probes have a guanine at a 5' end.
22. The method according to claim 1 wherein the five nucleotides at a 3' end of the primers have two or less guanines or cytosines.
23. The method according to claim 1 wherein at least one of the probes hybridizes to itself to form a hairpin.
24. The method according to claim 1 wherein the fluorescer on at least one of the probes emits a stronger fluorescence signal when hybridized to a sequence than when not hybridized to a sequence and in a non-hairpin, single stranded form.
25. The method according to claim 1 wherein at least one of the fluorescers is an energy transfer dye.
26. A method for genotyping a sample of DNA at at least two allelic sites by a 5' nuclease amplification reaction, the method comprising:
performing a nucleic acid amplification on a sample of DNA having at least two different allelic sites using a nucleic acid polymerase having 5'~3' nuclease activity and at least one set of forward and reverse primers capable of hybridizing to the DNA sample in the presence of two or more sets of allelic oligonucleotide probes and amplifying the at least two different allelic sites wherein:
each set of allelic oligonucleotide probes is for detecting a different allelic site, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence of the sample DNA to which the primer hybridizes 5' relative to a sequence to which the primer hybridizes, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer;
digesting those allelic oligonucleotide probes which hybridize to the target sequence during the amplification by the nuclease activity of the polymerase;
detecting a fluorescence spectrum of the amplification;
calculating a fluorescence contribution of each fluorescer to the fluorescence spectrum; and determining a presence or absence of the different allelic variants at the two or more different allelic sites based on the fluorescence contribution of each fluorescer to the fluorescence spectrum.
performing a nucleic acid amplification on a sample of DNA having at least two different allelic sites using a nucleic acid polymerase having 5'~3' nuclease activity and at least one set of forward and reverse primers capable of hybridizing to the DNA sample in the presence of two or more sets of allelic oligonucleotide probes and amplifying the at least two different allelic sites wherein:
each set of allelic oligonucleotide probes is for detecting a different allelic site, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence of the sample DNA to which the primer hybridizes 5' relative to a sequence to which the primer hybridizes, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer;
digesting those allelic oligonucleotide probes which hybridize to the target sequence during the amplification by the nuclease activity of the polymerase;
detecting a fluorescence spectrum of the amplification;
calculating a fluorescence contribution of each fluorescer to the fluorescence spectrum; and determining a presence or absence of the different allelic variants at the two or more different allelic sites based on the fluorescence contribution of each fluorescer to the fluorescence spectrum.
27. The method according to claim 26 wherein the at least two different allelic sites are on a single strand of DNA and amplified by a single set of forward and reverse primers.
28. The method according to claim 26 wherein the at least two different allelic sites are on a single strand of DNA and each allelic site is amplified by a different set of forward and reverse primers.
29. The method according to claim 26 wherein the at least two different allelic sites are on a separate strands of DNA and each allelic site is amplified by a different set of forward and reverse primers.
30. The method according to claim 26 wherein the nucleic acid amplification is performed in the presence of a passive internal standard.
31. The method according to claim 30 wherein the passive internal standard is ROX.
32. The method according to claim 26 wherein all the probes include a different fluorescer.
33. The method according to claim 26 wherein the nucleic acid amplification is performed in a reaction mixture containing at between about 4 and 6 m MgCl2.
34. The method according to claim 26 wherein the nucleic acid amplification is performed in a reaction mixture containing glycerol.
35. The method according to claim 26 wherein the nucleic acid amplification is performed in a reaction mixture containing at least one member of the group consisting of gelatin and TWEEN 20.
36. The method according to claim 26 wherein the nucleic acid amplification is performed in a reaction mixture which includes about 7-9% glycerol, 0.04-0.06%
gelatin, and 0.005-0.015% TWEEN 20.
gelatin, and 0.005-0.015% TWEEN 20.
37. The method according to claim 26 wherein the nucleic acid amplification is performed in a reaction mixture which includes about 7-9% glycerol, 0.04-0.06%
gelatin, 0.005-0.015% TWEEN 20, and 25-75 mM tris buffer, pH 8Ø
gelatin, 0.005-0.015% TWEEN 20, and 25-75 mM tris buffer, pH 8Ø
38. The method according to claim 26 amplification is performed in a reaction mixture which includes about 7-9% glycerol, 0.04-0.06% gelatin, 0.005-0.015%
TWEEN 20, 25-75 mM tris buffer, pH 8.0, 4-6 mM MgCl2, 175-225 uM dATP, 175-225 uM dCTP, 175-225 uM deaza dGTP, 350-450 uM dUTP, .045-.055 U/uL AMPLITAQTM
Gold, 0.5-.015 U/uL AmpErase UNG, and 57-63 nM of a Passive Reference.
TWEEN 20, 25-75 mM tris buffer, pH 8.0, 4-6 mM MgCl2, 175-225 uM dATP, 175-225 uM dCTP, 175-225 uM deaza dGTP, 350-450 uM dUTP, .045-.055 U/uL AMPLITAQTM
Gold, 0.5-.015 U/uL AmpErase UNG, and 57-63 nM of a Passive Reference.
39. The method according to claim 26 wherein the forward and reverse primers define amplicons between about 50 and 150 bases in length.
40. The method according to claim 26 wherein the one or more sets of forward and reverse primers define amplicons less than about 100 bases in length.
41. The method according to claim 26 wherein the %GC of all the probes are at least about 20% and less than about 80%.
42. The method according to claim 26 four or more contiguous guanines.
43. The method according to claim 26 wherein all of the probes have a melting point temperature that is about 3-5°C greater than an annealing temperature used in the amplification and the primer melting point temperature is about 2-4°C less than the annealing temperature.
44. The method according to claim 43 wherein the annealing temperature is about 60-64°C.
45. The method according to claim 26 melting point temperature about 65-67°C.
46. The method according to claim 45 wherein the primer melting point temperature is about 58-60°C.
47. The method according to claim 26 melting point temperature about 5-10 °C
greater than the melting point temperature of the primers.
greater than the melting point temperature of the primers.
48. The method according to claim 26 wherein all of the probes have a melting point temperature about 7 °C greater than the melting point temperature of the primers.
49. The method according to claim 26 guanine at a 5' end.
50. The method according to claim 26 wherein the five nucleotides at a 3' end of the primers have two or less guanines or cytosines.
51. The method according to claim 26 wherein at least one of the probes hybridizes to itself to form a hairpin.
52. The method according to claim 26 wherein one of the probes emits a stronger fluorescence signal when hybridized to a sequence than when not hybridized to a sequence and in a non-hairpin, single stranded form.
53. The method according to claim 26 wherein at least one fluorescers is an energy transfer dye.
54. A method for genotyping a sample of DNA at at least two allelic sites by a 5' nuclease amplification reaction, the method comprising:
performing a nucleic acid amplification on a sample of DNA having at least two different allelic sites using a nucleic acid polymerise having 5'~ 3' nuclease activity and at least one set of forward and reverse primers capable of hybridizing to the sample of DNA in the presence of two or more sets of allelic oligonucleotide probes and amplifying the at least two different allelic sites wherein:
each set of allelic oligonucleotide probes is for detecting a different allelic site, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer;
digesting those allelic oligonucleotide probes which hybridize to the sample DNA during the amplification by the nuclease activity of the polymerase;
detecting a fluorescence spectrum of the amplification;
calculating a fluorescence contribution of each fluorescer to the fluorescence spectrum; and determining a genotype of the target sequence at the at least two different allelic sites based on the fluorescence contribution of the different fluorescers to the fluorescence spectrum.
performing a nucleic acid amplification on a sample of DNA having at least two different allelic sites using a nucleic acid polymerise having 5'~ 3' nuclease activity and at least one set of forward and reverse primers capable of hybridizing to the sample of DNA in the presence of two or more sets of allelic oligonucleotide probes and amplifying the at least two different allelic sites wherein:
each set of allelic oligonucleotide probes is for detecting a different allelic site, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer;
digesting those allelic oligonucleotide probes which hybridize to the sample DNA during the amplification by the nuclease activity of the polymerase;
detecting a fluorescence spectrum of the amplification;
calculating a fluorescence contribution of each fluorescer to the fluorescence spectrum; and determining a genotype of the target sequence at the at least two different allelic sites based on the fluorescence contribution of the different fluorescers to the fluorescence spectrum.
55. A fluorescence spectrum for genotyping a sample of DNA at at least two allelic sites comprising:
a fluorescence spectrum derived from having performed a nucleic acid amplification on a sample of DNA having at least two different allelic sites using a nucleic acid polymerase having 5'~ 3' nuclease activity and a primer capable of hybridizing to the DNA sample in the presence of two or more sets of allelic oligonucleotide probes wherein:
each set of allelic oligonucleotide probes is for detecting a different allelic site, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer.
a fluorescence spectrum derived from having performed a nucleic acid amplification on a sample of DNA having at least two different allelic sites using a nucleic acid polymerase having 5'~ 3' nuclease activity and a primer capable of hybridizing to the DNA sample in the presence of two or more sets of allelic oligonucleotide probes wherein:
each set of allelic oligonucleotide probes is for detecting a different allelic site, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer.
56. The spectrum according to claim 55 wherein the at least two different allelic sites are on a single strand of DNA and amplified by a single set of forward and reverse primers.
57. The spectrum according to claim 55 wherein the at least two different allelic sites are on a single strand of DNA and each allelic site is amplified by a different set of forward and reverse primers.
58. The spectrum according to claim 55 wherein the at least two different allelic sites are on a separate strands of DNA and each allelic site is amplified by a different set of forward and reverse primers.
59. The spectrum according to claim 55 wherein the nucleic acid amplification is performed in the presence of a passive internal standard.
60. The spectrum according to claim 59 wherein the passive internal standard is ROX.
61. The spectrum according to claim 55 wherein all the oligonucleotide probes include a different fluorescer.
62. The spectrum according to claim 55 wherein the forward and reverse primers define amplicons between about 50 and 150 bases in length.
63. The spectrum according to claim 55 wherein the one or more sets of forward and reverse primers define amplicons less than about 100 bases in length.
64. The spectrum according to claim 55 wherein at least one of the fluorescers is an energy transfer dye.
65. A fluorescence signature for genotyping a sample of DNA at at least two allelic sites, the signature comprising:
fluorescence signal contributions of at least three fluorescers to a fluorescence spectrum derived from having performed a nucleic acid amplification on a sample of DNA having at least two different allelic sites using a nucleic acid polymerase having 5'~ 3' nuclease activity and forward and reverse primers capable of hybridizing to the DNA sample in the presence of two or more sets of allelic oligonucleotide probes and amplifying the at least two different allelic sites wherein:
each set of allelic oligonucleotide probes is for detecting a different allelic site, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer.
fluorescence signal contributions of at least three fluorescers to a fluorescence spectrum derived from having performed a nucleic acid amplification on a sample of DNA having at least two different allelic sites using a nucleic acid polymerase having 5'~ 3' nuclease activity and forward and reverse primers capable of hybridizing to the DNA sample in the presence of two or more sets of allelic oligonucleotide probes and amplifying the at least two different allelic sites wherein:
each set of allelic oligonucleotide probes is for detecting a different allelic site, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer.
66. The fluorescence signature according to claim 65 wherein the at least two different allelic sites are on a single strand of DNA and amplified by a single set of forward and reverse primers.
67. The fluorescence signature according to claim 65 wherein the at least two different allelic sites are on a single strand of DNA and each allelic site is amplified by a different set of forward and reverse primers.
68. The fluorescence signature according to claim 65 wherein the at least two different allelic sites are on a separate strands of DNA and each allelic site is amplified by a different set of forward and reverse primers.
69. The fluorescence signature according to claim 65 wherein the nucleic acid amplification is performed in the presence of a passive internal standard.
70. The fluorescence signature according to claim 69 wherein the passive internal standard is ROX.
71. The fluorescence signature to claim 65 wherein all the oligonucleotide probes include a different fluorescer.
72. The fluorescence signature according to claim 65 wherein the forward and reverse primers define amplicons between about 50 and 150 bases in length.
73. The fluorescence signature according to claim 65 wherein the one or more sets of forward and reverse primers define amplicons less than about 100 bases in length.
74. The fluorescence signature according to claim 65 wherein at least one of the fluorescers is an energy transfer dye.
75. A library of fluorescence signatures for genotyping a sample of DNA at at least two allelic sites, the library comprising:
fluorescence signal contributions of at least three fluorescers to a series of fluorescence spectra derived from having performed nucleic acid amplifications on a series of control sequences having known allelic variants at at least two different allelic sites using a nucleic acid polymerise having 5'~ 3' nuclease activity and forward and reverse primers capable of hybridizing to the DNA sample in the presence of two or more sets of allelic oligonucleotide probes and amplifying the at least two different allelic sites wherein:
each set of allelic oligonucleotide probes is for detecting a different allelic site, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer.
fluorescence signal contributions of at least three fluorescers to a series of fluorescence spectra derived from having performed nucleic acid amplifications on a series of control sequences having known allelic variants at at least two different allelic sites using a nucleic acid polymerise having 5'~ 3' nuclease activity and forward and reverse primers capable of hybridizing to the DNA sample in the presence of two or more sets of allelic oligonucleotide probes and amplifying the at least two different allelic sites wherein:
each set of allelic oligonucleotide probes is for detecting a different allelic site, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer.
76. A method for determining a fluorescence signature of a sample of DNA
comprising:
calculating fluorescence contributions of at least three fluorescers to a fluorescence spectrum taken of a nucleic acid amplification performed on a sample of DNA having at least two different allelic sites using a nucleic acid polymerise having 5'~ 3' nuclease activity and a primer capable of hybridizing to the DNA
sample in the presence of two or more sets of allelic oligonucleotide probes and an internal standard and amplifying the at least two different allelic sites wherein:
each set of allelic oligonucleotide probes is for detecting a different allelic site, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer;
and normalizing the fluorescence contributions of each fluorescer relative to an internal standard, the normalized fluorescence contributions corresponding to a fluorescence signature for the DNA sample for the at least two different allelic sites.
comprising:
calculating fluorescence contributions of at least three fluorescers to a fluorescence spectrum taken of a nucleic acid amplification performed on a sample of DNA having at least two different allelic sites using a nucleic acid polymerise having 5'~ 3' nuclease activity and a primer capable of hybridizing to the DNA
sample in the presence of two or more sets of allelic oligonucleotide probes and an internal standard and amplifying the at least two different allelic sites wherein:
each set of allelic oligonucleotide probes is for detecting a different allelic site, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer;
and normalizing the fluorescence contributions of each fluorescer relative to an internal standard, the normalized fluorescence contributions corresponding to a fluorescence signature for the DNA sample for the at least two different allelic sites.
77. A method for genotyping a sample of DNA at two or more different allelic sites comprising:
calculating fluorescence contributions of at least three fluorescers to a fluorescence spectrum taken of a nucleic acid amplification performed on a target sequence having at least two different allelic sites using a nucleic acid polymerase having 5'~ 3' nuclease activity and a primer capable of hybridizing to the sample of DNA in the presence of two or more sets of allelic oligonucleotide probes and an internal standard and amplifying the at least two different allelic sites wherein:
each set of allelic oligonucleotide probes is for detecting a different allelic site, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer;
normalizing the fluorescence contributions of each fluorescer relative to an internal standard, the normalized fluorescence contributions corresponding to a fluorescence signature for the DNA sample for the at least two different allelic sites; and determining the genotype of the DNA sample at the at least two allelic sites by comparing the normalized fluorescence contributions of the DNA sample to normalized fluorescence contributions of control sequences having a known genotype at the at least two allelic sites.
calculating fluorescence contributions of at least three fluorescers to a fluorescence spectrum taken of a nucleic acid amplification performed on a target sequence having at least two different allelic sites using a nucleic acid polymerase having 5'~ 3' nuclease activity and a primer capable of hybridizing to the sample of DNA in the presence of two or more sets of allelic oligonucleotide probes and an internal standard and amplifying the at least two different allelic sites wherein:
each set of allelic oligonucleotide probes is for detecting a different allelic site, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer;
normalizing the fluorescence contributions of each fluorescer relative to an internal standard, the normalized fluorescence contributions corresponding to a fluorescence signature for the DNA sample for the at least two different allelic sites; and determining the genotype of the DNA sample at the at least two allelic sites by comparing the normalized fluorescence contributions of the DNA sample to normalized fluorescence contributions of control sequences having a known genotype at the at least two allelic sites.
78. A processor for genotyping a sample of DNA at at least two allelic sites by a 5' nuclease assay, the processor comprising:
logic for taking fluorescence spectra of control samples and at least one unknown sample which have undergone a 5' nuclease assay in the presence of allelic probes for the at least two allelic sites and fluorescence spectra of at least three fluorescers used in the 5' nuclease assay and using the spectra to calculate normalized fluorescence contributions of the at least three fluorescers to the unknown and control fluorescence spectra; and logic for determining a genotype of the at least one unknown sample at two or more different allelic sites based on a comparison of the normalized fluorescence contributions of the at least three fluorescers to the spectrum of the unknown sample and to the spectra of the control samples.
logic for taking fluorescence spectra of control samples and at least one unknown sample which have undergone a 5' nuclease assay in the presence of allelic probes for the at least two allelic sites and fluorescence spectra of at least three fluorescers used in the 5' nuclease assay and using the spectra to calculate normalized fluorescence contributions of the at least three fluorescers to the unknown and control fluorescence spectra; and logic for determining a genotype of the at least one unknown sample at two or more different allelic sites based on a comparison of the normalized fluorescence contributions of the at least three fluorescers to the spectrum of the unknown sample and to the spectra of the control samples.
79. A kit for identifying which members of two or more sets of substantially homologous sequences are present in a sample of DNA, the kit comprising:
two or more sets of oligonucleotide probes wherein:
each set of oligonucleotide probes is for detecting members of one of the sets of substantially homologous sequences, each set of oligonucleotide probes includes two or more probes which are complementary to different members of a set of substantially homologous sequences, each member differing from another member in the set at at least one base position, and at least all but one of the oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer.
two or more sets of oligonucleotide probes wherein:
each set of oligonucleotide probes is for detecting members of one of the sets of substantially homologous sequences, each set of oligonucleotide probes includes two or more probes which are complementary to different members of a set of substantially homologous sequences, each member differing from another member in the set at at least one base position, and at least all but one of the oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer.
80. The kit according to claim 79, further including one or more sets of forward and reverse primers.
81. The kit according to claim 80, wherein each set of forward and reverse primers define an amplicon between about 50 and 150 bases in length.
82. The kit according to claim 80, wherein each set of forward and reverse primers define an amplicon less than about 100 bases in length.
83. The kit according to claim 80 wherein all of the probes have a melting point temperature that is about 3-5°C greater than an annealing temperature used in the amplification and the primer melting point temperature is about 2-4°C
less than the annealing temperature.
less than the annealing temperature.
84. The kit according to claim 80 wherein the primer has a melting point temperature of about 58-60°C.
85. The kit according to claim 80 wherein all of the probes have a melting point temperature about 5-10 °C greater than the melting point temperature of the primers.
86. The kit according to claim 80 wherein all of the probes have a melting point temperature about 7 °C greater than the melting point temperature of the primers.
87. The kit according to claim 79 wherein the %GC of all the probes are at least about 20% and less than about 80%.
88. The kit according to claim 79 wherein none of the probes have four or more contiguous guanines.
89. The kit according to claim 79 wherein all of the probes have a melting point temperature about 65-67°C.
90. The kit according to claim 79 wherein none of the probes have a guanine at a 5' end.
91. The kit according to claim 79 wherein at least one of the fluorescers is an energy transfer dye.
92. A kit for genotyping a sample of DNA at at least two allelic sites comprising:
two or more sets of allelic oligonucleotide probes wherein:
each set of allelic oligonucleotide probes is for detecting a different allelic site, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer.
two or more sets of allelic oligonucleotide probes wherein:
each set of allelic oligonucleotide probes is for detecting a different allelic site, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer.
93. The kit according to claim 92, further including one or more sets of forward and reverse primers.
94. The kit according to claim 93, wherein each set of forward and reverse primers define an amplicon between about 50 and 150 bases in length.
95. The kit according to claim 93, wherein each set of forward and reverse primers define an amplicon less than about 100 bases in length.
96. The kit according to claim 93 wherein all of the probes have a melting point temperature that is about 3-5°C greater than an annealing temperature used in the amplification and the primer melting point temperature is about 2-4°C
less than the annealing temperature.
less than the annealing temperature.
97. The kit according to claim 93 wherein the primer has a melting point temperature of about 58-60°C.
98. The kit according to claim 93 wherein all of the probes have a melting point temperature about 5-10 °C greater than the melting point temperature of the primers.
99. The kit according to claim 93 wherein all of the probes have a melting point temperature about 7 °C greater than the melting point temperature of the primers.
100. The kit according to claim 92 wherein the %GC of all the probes are at least about 20% and less than about 80%.
101. The kit according to claim 92 wherein none of the probes have four or more contiguous guanines.
102. The kit according to claim 92 wherein all of the probes have a melting point temperature about 65-67°C.
103. The kit according to claim 92 wherein none of the probes have a guanine at a 5' end.
104. The kit according to claim 92 wherein at least one of the fluorescers is an energy transfer dye.
105. A kit for performing a 5' nuclease assay comprising:
a reaction mixture including 14-18% glycerol, 0.04-0.06% gelatin, and 0.01-0.03% TWEEN 20.
a reaction mixture including 14-18% glycerol, 0.04-0.06% gelatin, and 0.01-0.03% TWEEN 20.
106. The kit according to claim 105, the kit further including 25-75 mM tris buffer.
107. The kit according to claim 106, the kit further including 4-6 mM MgCl2, uM dATP, 175-225 uM dCTP, 175-225 uM deaza dGTP, 350-450 uM dUTP, .045-.055 U/uL AMPLITAQ TM Gold, 0.5-.015 U/uL AmpErase UNG, and 57-63 nM of a Passive Reference.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
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| US1859598A | 1998-02-04 | 1998-02-04 | |
| US09/018,595 | 1998-02-04 | ||
| PCT/US1999/000499 WO1999040226A2 (en) | 1998-02-04 | 1999-01-08 | Determination of a genotype of an amplification product at multiple allelic sites |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2318880A1 true CA2318880A1 (en) | 1999-08-12 |
| CA2318880C CA2318880C (en) | 2010-07-27 |
Family
ID=21788757
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2318880A Expired - Fee Related CA2318880C (en) | 1998-02-04 | 1999-01-08 | Determination of a genotype of an amplification product at multiple allelic sites |
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| Country | Link |
|---|---|
| US (5) | US6154707A (en) |
| EP (1) | EP1053348B1 (en) |
| JP (1) | JP4388694B2 (en) |
| AT (1) | ATE469982T1 (en) |
| AU (1) | AU758463B2 (en) |
| CA (1) | CA2318880C (en) |
| DE (1) | DE69942444D1 (en) |
| WO (1) | WO1999040226A2 (en) |
Families Citing this family (81)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2255599C (en) | 1996-04-25 | 2006-09-05 | Bioarray Solutions, Llc | Light-controlled electrokinetic assembly of particles near surfaces |
| CA2308762A1 (en) * | 1997-11-04 | 1999-05-14 | Roche Diagnostics Gmbh | Specific and sensitive nucleic acid detection method |
| ATE469982T1 (en) * | 1998-02-04 | 2010-06-15 | Life Technologies Corp | DETERMINATION OF THE GENOTYPE OF AN AMPLIFICATION PRODUCT AT MULTIPLE ALLELE SITES |
| EP1230387B1 (en) * | 1999-10-22 | 2006-08-16 | The Public Health Research Institute of the City of New York, Inc. | Assays for short sequence variants |
| US7585632B2 (en) * | 1999-10-29 | 2009-09-08 | Hologic, Inc. | Compositions and methods for the detection of a nucleic acid using a cleavage reaction |
| US6660845B1 (en) | 1999-11-23 | 2003-12-09 | Epoch Biosciences, Inc. | Non-aggregating, non-quenching oligomers comprising nucleotide analogues; methods of synthesis and use thereof |
| US20040081959A9 (en) | 1999-12-08 | 2004-04-29 | Epoch Biosciences, Inc. | Fluorescent quenching detection reagents and methods |
| US6727356B1 (en) | 1999-12-08 | 2004-04-27 | Epoch Pharmaceuticals, Inc. | Fluorescent quenching detection reagents and methods |
| JP2001299392A (en) * | 2000-04-25 | 2001-10-30 | Otsuka Pharmaceut Co Ltd | Method of detecting catch22 syndromes |
| JP2004511210A (en) * | 2000-05-23 | 2004-04-15 | バリアジェニックス インコーポレーテッド | Method for genetic analysis of DNA to detect sequence variation |
| US7435541B2 (en) | 2000-05-23 | 2008-10-14 | Sequenom, Inc. | Restriction enzyme genotyping |
| US7892854B2 (en) | 2000-06-21 | 2011-02-22 | Bioarray Solutions, Ltd. | Multianalyte molecular analysis using application-specific random particle arrays |
| US9709559B2 (en) | 2000-06-21 | 2017-07-18 | Bioarray Solutions, Ltd. | Multianalyte molecular analysis using application-specific random particle arrays |
| GB0016516D0 (en) * | 2000-07-06 | 2000-08-23 | Owen Mumford Ltd | Improvements relating to needle protection devices |
| US7267945B2 (en) * | 2001-03-26 | 2007-09-11 | Applera Corporation | Methods of determining the presence of polynucleotides employing amplification |
| US20030143554A1 (en) * | 2001-03-31 | 2003-07-31 | Berres Mark E. | Method of genotyping by determination of allele copy number |
| US7262063B2 (en) | 2001-06-21 | 2007-08-28 | Bio Array Solutions, Ltd. | Directed assembly of functional heterostructures |
| BRPI0211191B8 (en) * | 2001-06-28 | 2021-07-27 | Chiron Corp | nucleic acid-based assays to accurately diagnose parvovirus b19 infection and initiators and markers for use in these assays |
| KR20040068122A (en) | 2001-10-15 | 2004-07-30 | 바이오어레이 솔루션스 리미티드 | Multiplexed analysis of polymorphic loci by concurrent interrogation and enzyme-mediated detection |
| EP1439222A1 (en) * | 2001-10-26 | 2004-07-21 | Matsushita Electric Industrial Co., Ltd. | Method of detecting target nucleic acid and nucleic acid probe |
| US7732138B2 (en) * | 2001-11-07 | 2010-06-08 | Diagcor Bioscience Incorporation Limited | Rapid genotyping analysis and the device thereof |
| US20110111389A1 (en) | 2001-11-07 | 2011-05-12 | Diagcor Bioscience Incorporation Limited | Rapid genotyping analysis for human papillomavirus and the device thereof |
| US20050079493A1 (en) * | 2002-11-09 | 2005-04-14 | Tam Joseph Wing On | DNA fingerprinting using allelic specific oligonucleotide reversed dot blot (ASO-RDB) flow through hybridization process and device |
| DK1487261T3 (en) * | 2002-03-05 | 2008-11-24 | Mcw Res Found Inc | Methods and compositions for pharmacological and toxicological evaluation of test preparations |
| US7176002B2 (en) * | 2002-05-16 | 2007-02-13 | Applera Corporation | Universal-tagged oligonucleotide primers and methods of use |
| JP4457001B2 (en) * | 2002-05-31 | 2010-04-28 | セクレタリー・デパートメント・オブ・アトミック・エナジー | MET / FRET based method for target nucleic acid detection in which donor / acceptor moieties are on complementary strands |
| EP1514089B1 (en) * | 2002-06-20 | 2014-05-14 | Leica Biosystems Melbourne Pty Ltd | Biological reaction apparatus with draining mechanism |
| AU2003901871A0 (en) * | 2003-03-31 | 2003-05-08 | Vision Biosystems Limited | A method and apparatus for fluid dispensation, preparation and dilation |
| US7252940B2 (en) * | 2002-08-21 | 2007-08-07 | Epoch Biosciences, Inc. | Abasic site endonuclease assay |
| US7526114B2 (en) | 2002-11-15 | 2009-04-28 | Bioarray Solutions Ltd. | Analysis, secure access to, and transmission of array images |
| WO2004055709A2 (en) * | 2002-12-13 | 2004-07-01 | Applera Corporation | Methods for identifying, viewing, and analyzing syntenic and orthologous genomic regions between two or more species |
| US7851150B2 (en) | 2002-12-18 | 2010-12-14 | Third Wave Technologies, Inc. | Detection of small nucleic acids |
| US7468248B2 (en) | 2002-12-31 | 2008-12-23 | Cargill, Incorporated | Methods and systems for inferring bovine traits |
| US7858301B2 (en) * | 2003-05-07 | 2010-12-28 | Bioarray Solutions, Ltd. | Method of probe design for nucleic acid analysis by multiplexed hybridization |
| CN1833034B (en) | 2003-06-20 | 2014-04-16 | 埃克斯魁恩公司 | Probes, libraries and kits for analysis of mixtures of nucleic acids and methods for constructing the same |
| US20050038776A1 (en) * | 2003-08-15 | 2005-02-17 | Ramin Cyrus | Information system for biological and life sciences research |
| WO2005029705A2 (en) | 2003-09-18 | 2005-03-31 | Bioarray Solutions, Ltd. | Number coding for identification of subtypes of coded types of solid phase carriers |
| US7417726B2 (en) * | 2003-09-19 | 2008-08-26 | Applied Biosystems Inc. | Normalization of data using controls |
| US20050221357A1 (en) * | 2003-09-19 | 2005-10-06 | Mark Shannon | Normalization of gene expression data |
| CA2539824C (en) | 2003-09-22 | 2015-02-03 | Xinwen Wang | Surface immobilized polyelectrolyte with multiple functional groups capable of covalently bonding to biomolecules |
| US7332280B2 (en) * | 2003-10-14 | 2008-02-19 | Ronald Levy | Classification of patients having diffuse large B-cell lymphoma based upon gene expression |
| AU2004286252A1 (en) | 2003-10-28 | 2005-05-12 | Bioarray Solutions Ltd. | Optimization of gene expression analysis using immobilized capture probes |
| WO2005045060A2 (en) | 2003-10-29 | 2005-05-19 | Bioarray Solutions, Ltd. | Multiplexed nucleic acid analysis by fragmentation of double-stranded dna |
| WO2005049849A2 (en) | 2003-11-14 | 2005-06-02 | Integrated Dna Technologies, Inc. | Fluorescence quenching azo dyes, their methods of preparation and use |
| EP1561823A1 (en) * | 2004-02-04 | 2005-08-10 | Biotez Berlin-Buch GmbH | Method for the detection of single nucleotid polymorphisms (SNP) of genes of drug metabolism and test system for performing such a method |
| US20050255485A1 (en) * | 2004-05-14 | 2005-11-17 | Livak Kenneth J | Detection of gene duplications |
| US7575863B2 (en) | 2004-05-28 | 2009-08-18 | Applied Biosystems, Llc | Methods, compositions, and kits comprising linker probes for quantifying polynucleotides |
| US7848889B2 (en) | 2004-08-02 | 2010-12-07 | Bioarray Solutions, Ltd. | Automated analysis of multiplexed probe-target interaction patterns: pattern matching and allele identification |
| EP1791982B1 (en) * | 2004-09-21 | 2011-01-05 | Life Technologies Corporation | TWO-COLOR REAL-TIME/END-POINT QUANTITATION OF MICRORNAS (MIRNAs) |
| US20060111915A1 (en) * | 2004-11-23 | 2006-05-25 | Applera Corporation | Hypothesis generation |
| US8084260B2 (en) * | 2004-11-24 | 2011-12-27 | Applied Biosystems, Llc | Spectral calibration method and system for multiple instruments |
| KR20070090233A (en) * | 2004-12-08 | 2007-09-05 | 타케시 야마모토 | Method of examining gene sequence |
| US20060166238A1 (en) * | 2004-12-22 | 2006-07-27 | Ramsing Niels B | Probes, libraries and kits for analysis of mixtures of nucleic acids and methods for constructing the same |
| WO2006127507A2 (en) * | 2005-05-20 | 2006-11-30 | Integrated Dna Technologies, Inc. | Compounds and methods for labeling oligonucleotides |
| US8486629B2 (en) | 2005-06-01 | 2013-07-16 | Bioarray Solutions, Ltd. | Creation of functionalized microparticle libraries by oligonucleotide ligation or elongation |
| WO2007044091A2 (en) | 2005-06-02 | 2007-04-19 | Fluidigm Corporation | Analysis using microfluidic partitioning devices |
| EP2660482B1 (en) * | 2005-08-22 | 2019-08-07 | Life Technologies Corporation | Vorrichtung, System und Verfahren unter Verwendung von nichtmischbaren Flüssigkeiten mit unterschiedlichen Volumen |
| US8137912B2 (en) | 2006-06-14 | 2012-03-20 | The General Hospital Corporation | Methods for the diagnosis of fetal abnormalities |
| EP2589668A1 (en) | 2006-06-14 | 2013-05-08 | Verinata Health, Inc | Rare cell analysis using sample splitting and DNA tags |
| US20080070792A1 (en) | 2006-06-14 | 2008-03-20 | Roland Stoughton | Use of highly parallel snp genotyping for fetal diagnosis |
| US20080050739A1 (en) | 2006-06-14 | 2008-02-28 | Roland Stoughton | Diagnosis of fetal abnormalities using polymorphisms including short tandem repeats |
| CA2655272C (en) * | 2006-06-14 | 2017-04-18 | Living Microsystems, Inc. | Rare cell analysis using sample splitting and dna tags |
| WO2008111990A1 (en) * | 2006-06-14 | 2008-09-18 | Cellpoint Diagnostics, Inc. | Rare cell analysis using sample splitting and dna tags |
| EP1911852B1 (en) * | 2006-10-12 | 2009-07-29 | Bio-Rad Pasteur | Double-stranded probes for the fluorescent detection of nucleic acids |
| DE102007013099A1 (en) * | 2007-03-14 | 2008-09-18 | Aj Innuscreen Gmbh | Method and test kit for the rapid detection of specific nucleic acid sequences, in particular for the detection of mutations or SNPs |
| EP2116614A1 (en) * | 2008-05-06 | 2009-11-11 | Qiagen GmbH | Simultaneous detection of multiple nucleic acid sequences in a reaction |
| US20090312196A1 (en) * | 2008-06-13 | 2009-12-17 | Codexis, Inc. | Method of synthesizing polynucleotide variants |
| CA2726850C (en) * | 2008-06-13 | 2015-06-02 | Codexis, Inc. | Method of synthesizing polynucleotide variants |
| US8383346B2 (en) * | 2008-06-13 | 2013-02-26 | Codexis, Inc. | Combined automated parallel synthesis of polynucleotide variants |
| SI2334812T1 (en) | 2008-09-20 | 2017-05-31 | The Board of Trustees of the Leland Stanford Junior University Office of the General Counsel Building 170 | Noninvasive diagnosis of fetal aneuploidy by sequencing |
| JP5709840B2 (en) * | 2009-04-13 | 2015-04-30 | キヤノン ユー．エス． ライフ サイエンシズ， インコーポレイテッドＣａｎｏｎ Ｕ．Ｓ． Ｌｉｆｅ Ｓｃｉｅｎｃｅｓ， Ｉｎｃ． | Rapid method of pattern recognition, machine learning, and automatic genotyping with dynamic signal correlation analysis |
| US20110312503A1 (en) | 2010-01-23 | 2011-12-22 | Artemis Health, Inc. | Methods of fetal abnormality detection |
| US9506057B2 (en) | 2010-03-26 | 2016-11-29 | Integrated Dna Technologies, Inc. | Modifications for antisense compounds |
| AU2011230496B2 (en) | 2010-03-26 | 2015-09-17 | Integrated Dna Technologies, Inc. | Methods for enhancing nucleic acid hybridization |
| WO2012033848A1 (en) | 2010-09-07 | 2012-03-15 | Integrated Dna Technologies, Inc. | Modifications for antisense compounds |
| US20120083035A1 (en) | 2010-09-30 | 2012-04-05 | Dharmacon, Inc. | Modified Cell Lines for Increasing Lentiviral Titers |
| US9617588B2 (en) | 2010-10-05 | 2017-04-11 | Thermo Fisher Scientific Baltics Uab | Enzyme mixture |
| EP2852682B1 (en) | 2012-05-21 | 2017-10-04 | Fluidigm Corporation | Single-particle analysis of particle populations |
| AU2017248219B2 (en) * | 2016-04-06 | 2023-09-07 | Life Technologies Corporation | Compositions, methods, and kits for synthesis and detection of nucleic acids |
| JP6846636B2 (en) * | 2017-02-28 | 2021-03-24 | パナソニックＩｐマネジメント株式会社 | Oligonucleotide for nucleic acid detection |
| EP4278009A1 (en) * | 2021-01-15 | 2023-11-22 | Nuprobe USA, Inc. | Cycle multiplexing for highly multiplexed quantitative pcr |
Family Cites Families (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5075216A (en) | 1988-09-23 | 1991-12-24 | Cetus Corporation | Methods for dna sequencing with thermus aquaticus dna polymerase |
| ATE176002T1 (en) | 1990-07-24 | 1999-02-15 | Hoffmann La Roche | REDUCING NON-SPECIFIC AMPLIFICATION DURING (IN VITRO) NUCLEIC ACID AMPLIFICATION USING MODIFIED NUCLEIC ACID BASES |
| US5210015A (en) * | 1990-08-06 | 1993-05-11 | Hoffman-La Roche Inc. | Homogeneous assay system using the nuclease activity of a nucleic acid polymerase |
| US5403707A (en) * | 1993-05-14 | 1995-04-04 | Eastman Kodak Company | Diagnostic compositions, elements, methods and test kits for amplification and detection of retroviral DNA using primers having matched melting temperatures |
| US5538848A (en) | 1994-11-16 | 1996-07-23 | Applied Biosystems Division, Perkin-Elmer Corp. | Method for detecting nucleic acid amplification using self-quenching fluorescence probe |
| US5834181A (en) | 1994-07-28 | 1998-11-10 | Genzyme Corporation | High throughput screening method for sequences or genetic alterations in nucleic acids |
| WO1996021144A1 (en) * | 1994-12-30 | 1996-07-11 | Georgetown University | Fluorometric assay for detecting nucleic acid cleavage |
| DE19524579C2 (en) * | 1995-07-06 | 1997-12-18 | Daimler Benz Ag | Transformer as a current limiter |
| US5792610A (en) * | 1996-05-01 | 1998-08-11 | Biorad Laboratories, Inc. | Method for conducting multiparametric fluorescence in situ hybridization |
| US5759781A (en) * | 1995-12-22 | 1998-06-02 | Yale University | Multiparametric fluorescence in situ hybridization |
| EP0892808B1 (en) * | 1996-04-12 | 2008-05-14 | PHRI Properties, Inc. | Detection probes, kits and assays |
| US6015667A (en) * | 1996-06-03 | 2000-01-18 | The Perkin-Emer Corporation | Multicomponent analysis method including the determination of a statistical confidence interval |
| US5736333A (en) * | 1996-06-04 | 1998-04-07 | The Perkin-Elmer Corporation | Passive internal references for the detection of nucleic acid amplification products |
| ATE469982T1 (en) | 1998-02-04 | 2010-06-15 | Life Technologies Corp | DETERMINATION OF THE GENOTYPE OF AN AMPLIFICATION PRODUCT AT MULTIPLE ALLELE SITES |
| US5952202A (en) * | 1998-03-26 | 1999-09-14 | The Perkin Elmer Corporation | Methods using exogenous, internal controls and analogue blocks during nucleic acid amplification |
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1999
- 1999-01-08 AT AT99903026T patent/ATE469982T1/en not_active IP Right Cessation
- 1999-01-08 CA CA2318880A patent/CA2318880C/en not_active Expired - Fee Related
- 1999-01-08 AU AU23144/99A patent/AU758463B2/en not_active Ceased
- 1999-01-08 WO PCT/US1999/000499 patent/WO1999040226A2/en active IP Right Grant
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- 1999-01-08 DE DE69942444T patent/DE69942444D1/en not_active Expired - Lifetime
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2002
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2006
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2009
- 2009-11-13 US US12/618,686 patent/US20120053069A1/en not_active Abandoned
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| US20020164630A1 (en) | 2002-11-07 |
| DE69942444D1 (en) | 2010-07-15 |
| EP1053348A2 (en) | 2000-11-22 |
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| US20080187913A1 (en) | 2008-08-07 |
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| EP1053348B1 (en) | 2010-06-02 |
| US20040053302A1 (en) | 2004-03-18 |
| JP2002502615A (en) | 2002-01-29 |
| JP4388694B2 (en) | 2009-12-24 |
| US6154707A (en) | 2000-11-28 |
| AU2314499A (en) | 1999-08-23 |
| WO1999040226A3 (en) | 2000-08-17 |
| WO1999040226A2 (en) | 1999-08-12 |
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