Determination of a genotype of an amplification product at multiple allelic sites
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'.fwdarw.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, andat 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; anddetermining 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.
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Claims
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'.fwdarw.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 MgCl.sub.2.
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.
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.
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 MgCl.sub.2, 175-225 uM dATP, 175-225 uM dCTP, 175-225 uM deaza dGTP, 350-450 uM dUTP,.045-0.055 U/uL AMPLITAQ.TM. Gold, 0.5-0.015 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.degree. C. greater than an annealing temperature used in the amplification and the primer melting point temperature is about 2-4.degree. C. less than the annealing temperature.
16. The method according to claim 15 wherein the annealing temperature is about 60-64.degree. C.
17. The method according to claim 1 wherein all of the probes have a melting point temperature about 65-67.degree. C.
18. The method according to claim 17 wherein the primer melting point temperature is about 58-60.degree. C.
19. The method according to claim 1 wherein all of the probes have a melting point temperature about 5-10.degree. 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.degree. 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'.fwdarw.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 oligonucleotide 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 MgCl.sub.2.
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.
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 8.0.
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 MgCl.sub.2, 175-225 uM dATP, 175-225 uM dCTP, 175-225 uM deaza dGTP, 350-450 uM dUTP, 0.045-055 U/uL AMPLITAQ.TM. Gold, 0.5-0.015 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.degree. C. greater than an annealing temperature used in the amplification and the primer melting point temperature is about 2-4.degree. C. less than the annealing temperature.
44. The method according to claim 43 wherein the annealing temperature is about 60-64.degree. C.
45. The method according to claim 26 melting point temperature about 65-67.degree. C.
46. The method according to claim 45 wherein the primer melting point temperature is about 58-60.degree. C.
47. The method according to claim 26 melting point temperature about 5-10.degree. C. 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.degree. 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 polymerase having 5'.fwdarw.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 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 polymerase having 5'.fwdarw.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.
56. 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'.fwdarw.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.
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Type: Grant
Filed: Feb 4, 1998
Date of Patent: Oct 5, 1999
Assignee: The Perkin-Elmer Corporation (Foster City, CA)
Inventors: Kenneth J. Livak (San Jose, CA), Federico Goodsaid (San Jose, CA)
Primary Examiner: Jeffrey Fredman
Attorney: David J. Wilson Sonsini Goodrich & Rosati Weitz
Application Number: 9/18,595
International Classification: C12Q 168; C12P 1934; C07H 2104;
