Detection of gene duplications

• US 20050255485A1
• Filed: 06/30/2004
• Published: 11/17/2005
• Est. Priority Date: 05/14/2004
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Livak, Kenneth J., Stevens, Junko, Lazaruk, Katherine D., Wong, Lily Y., Ziegle, Janet S.
• Application Number:
  US10/881,368
• Publication Number:
  US 20050255485A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/10 Ploidy or copy number detection
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection

Abstract

Methods of detecting a candidate genetic anomaly such as a candidate duplication in a genome are disclosed. The methods comprise quantifying fluorogenic assays for alleles of a genetic locus from a plurality of individual genomes, identifying ranges of fluorescent intensities indicative of individual genomes homozygous for a first allele, homozygous for a second allele, or heterozygous for both alleles, and identifying individual genomes in which the fluorescence intensities are outside the range of intensities indicative of homozygosity or heterozygosity for the genetic locus.

Claims

1. A method of identifying a candidate genetic anomaly in one or more individuals of a population, the method comprising:

determining, in a fluorogenic assay for alleles of a genetic locus comprised by genomes of a plurality of individuals of the population, a range of fluorescence intensities of a first fluorophore indicative of a genome homozygous for a first allele, a range of fluorescence intensities of a second fluorophore indicative of a genome homozygous for a second allele, and a range of fluorescence intensities of the first and second fluorophores indicative of a genome heterozygous for the first and second alleles; and

determining if the fluorescence intensities of the first and the second fluorophores of the fluorogenic assay for the alleles of the genetic locus of a genome of one or more individuals of the population are outside the ranges of fluorescence intensities indicative of a genome that is homozygous for the first allele, homozygous for the second SNP allele, or heterozygous for the first and second alleles.

2. A method in accordance with claim 1, wherein a fluorogenic assay comprises:

forming a mixture comprising a genome of an individual, a first nucleobase primer comprising a sequence which maps upstream from the genetic locus, a second primer complementary to a sequence which maps downstream from the genetic locus, a first probe comprising the first fluorophore and a nucleic acid sequence complementary to a genomic sequence comprising the first allele, and a second probe comprising the second fluorophore, and a nucleic acid sequence complementary to a genomic sequence comprising the second allele, and a thermostable DNA polymerase;

subjecting the mixture to thermal cycling; and

detecting fluorescence intensities of each of the first fluorophore and the second fluorophore.

3. A method in accordance with claim 2, wherein the first probe and the second probe each further comprise a fluorescence quencher.

4. A method in accordance with claim 2, wherein the first probe and the second probe each comprise from about 10 nucleotides up to about 50 nucleotides.

5. A method in accordance with claim 2, wherein the first probe and the second probe each comprise from about 12 nucleotides up to about 25 nucleotides.

6. A method in accordance with claim 2, wherein the first probe and the second probe each comprise from about 13 nucleotides up to about 18 nucleotides.

7. A method in accordance with claim 2, wherein the upstream primer and the downstream primer each comprise from about 10 nucleotides up to about 50 nucleotides.

8. A method in accordance with claim 2, wherein the upstream primer and the downstream primer each comprise from about 12 nucleotides up to about 25 nucleotides.

9. A method in accordance with claim 2, wherein the upstream primer and the downstream primer each comprise from about 13 nucleotides up to about 18 nucleotides.

10. A method in accordance with claim 2, wherein the detecting fluorescence intensity comprises end point detection of fluorescence intensity.

11. A method in accordance with claim 2, wherein the mixture further comprises a control fluorophore, and the detecting fluorescence intensity comprises detecting fluorescence intensity normalized to a control fluorophore fluorescence intensity.

12. A method in accordance with claim 11, further comprising generating a likelihood model that predicts the probability that the intensity of a selected sample will reside within a range indicative of a genome homozygous for the first allele, a genome homozygous for the second allele, and a range of fluorescence intensities of the first and second fluorophores indicative of a genome heterozygous for the first and second alleles.

13. A method in accordance with claim 11, wherein each range of fluorescence intensities comprises a range of fluorescence intensities normalized to the control fluorophore fluorescence intensity, wherein each of the normalized fluorescence intensities within a range differ from at least one other normalized fluorescence intensity within the range by no more than about 20%.

14. A method in accordance with claim 11, wherein each range of fluorescence intensities comprises a range of fluorescence intensities normalized to the control fluorophore fluorescence intensity, wherein each of the normalized fluorescence intensities within a range differ from at least one other normalized fluorescence intensity within the range by no more than about 15%.

15. A method in accordance with claim 11, wherein each range of fluorescence intensities comprises a range of fluorescence intensities normalized to the control fluorophore fluorescence intensity, wherein each of the normalized fluorescence intensities within a range differ from at least one other normalized fluorescence intensity within the range by no more than about 10%.

16. A method in accordance with claim 11, wherein each range of fluorescence intensities comprises a range of fluorescence intensities normalized to the control fluorophore fluorescence intensity, wherein each of the normalized fluorescence intensities within a range differ from at least one other normalized fluorescence intensity within the range by no more than about 5%.

17. A method in accordance with claim 11, wherein each range of fluorescence intensities comprises a range of fluorescence intensities normalized to the control fluorophore fluorescence intensity, wherein each of the normalized fluorescence intensities within a range differ from at least one other normalized fluorescence intensity within the range by no more than about 2%.

18. A method in accordance with claim 1, wherein the candidate genetic anomaly comprises a candidate genetic duplication.

19. A method in accordance with claim 1, wherein the candidate genetic anomaly comprises a genetic locus mapping in the genome to a sequence comprised by the first primer, its complement, the second primer or its complement.

20. A method in accordance with claim 1, wherein the first fluorophore and the second fluorophore are different and are each selected from the group consisting of FAM, VIC, Sybra Green, TET, HEX, JOE, NED, LIZ, TAMRA, ROX, ALEXA, Texas Red, Cy3, Cy5, Cy7, Cy9, and dR6G.

21. A method in accordance with claim 20, wherein the first fluorophore and the second fluorophore are selected from FAM and VIC.

22. A method in accordance with claim 11, wherein the control fluorophore is different from the first fluorophore and the second fluorophore, and is selected from the group consisting of FAM, VIC, Sybra Green, TET, HEX, JOE, NED, LIZ, TAMRA, ROX, ALEXA, Texas Red, Cy3, Cy5, Cy7, Cy9, and dR6G.

23. A method in accordance with claim 1, wherein the genetic anomaly is a genetic anomaly of a gene selected from the group consisting of a cytochrome 450 gene, a CYP1A1 gene, a CYP1A2 gene, a CYP2A1 gene, a CYP2A6 gene, a CYP2A7 gene, a CYP2B6 gene, a CYP2C8 gene, a CYP2C9 gene, a CYP2C19 gene, a CYP2D6 gene, a CYP2E1 gene, a CYP3A4 gene, a CYP3A5 gene, a CYP3A7 gene, a CYP4B1 gene, a CYP5A1 gene, a CYP8A1 gene, a CYP21 gene, a NAT1 gene, a NAT2 gene, a COMT gene, a TMPT gene, a TYMS gene, a constitutive androstane receptor gene, a pregnane X receptor gene, an alcohol dehydrogenase gene, a flavin monooxygenase gene, a glutathione S-transferase gene, a transporter gene, an oATP-C gene, an epoxide hydrolase gene, a carboxylesterase gene, a monoamine oxidase gene, a paraoxonase gene, sulfotransferase gene, a UDP-glucuronosyl-transferase gene, an ADH1A gene, an ADH1B gene, an ADH1C gene, an ADH4 gene, an ADH5 gene, an ADH6 gene, an ADH7 gene, an FM01 gene, an FM03 gene, an FM04 gene, an FM05 gene, a GSTM1 gene, a GSTT1 gene, an MDR1 gene, an MRP1 gene, an MRP2 gene, and an MXR gene.

24. A method in accordance with claim 1, wherein the alleles of the genetic locus are SNP alleles.

25. A system for identifying a candidate genetic anomaly in a population, the system comprising a graphical interface which exhibits a plurality of data points, wherein each data point occupies a position representing fluorescence intensities of a first fluorophore and a second fluorophore from an individual genomic sample subjected to a fluorogenic assay for alleles of a genetic locus, wherein fluorescence intensity of a first fluorophore is indicative of the presence of a first allele and fluorescence intensity of a second fluorophore is indicative of the presence of a second allele, and wherein a cluster of data points is indicative of a genome homozygous for the first allele, a genome homozygous for the second allele, or a genome heterozygous for the first and second alleles, and wherein a data point outside any cluster represents an individual genome comprising a candidate genetic anomaly.

26. A system in accordance with claim 25, wherein a fluorogenic assay comprises:

forming a mixture comprising a genome of an individual, a first nucleobase primer comprising a sequence which maps upstream from the genetic locus, a second primer complementary to a sequence which maps downstream from the genetic locus, a first probe comprising the first fluorophore and a nucleic acid sequence complementary to a genomic sequence comprising the first allele, and a second probe comprising the second fluorophore and a nucleic acid sequence complementary to a genomic sequence comprising the second allele, and a thermostable DNA polymerase having 5′

exonuclease activity;

subjecting the mixture to thermal cycling; and

detecting fluorescence intensities of each of the first fluorophore and the second fluorophore.

27. A system in accordance with claim 26, wherein the detecting fluorescence intensity comprises end point detection of fluorescence intensity.

28. A system in accordance with claim 26, wherein the mixture further comprises a control fluorophore, and the detecting fluorescence intensity comprises detecting fluorescence intensity normalized to a control fluorophore fluorescence intensity.

29. A system in accordance with claim 28, wherein each cluster of data points comprises a cluster of fluorescence intensities normalized to the control fluorophore fluorescence intensity, and wherein data points outside of any cluster are data points indicating the presence of a gene duplication.

30. A system in accordance with claim 29, wherein each cluster of data points comprises a cluster of fluorescence intensities normalized to the control fluorophore fluorescence intensity, and wherein data points outside of any cluster are data points indicating the presence of a candidate gene duplication.

31. A system in accordance with claim 28, wherein each cluster of data points comprises a cluster of fluorescence intensities normalized to the control fluorophore fluorescence intensity, and wherein data points outside of any cluster are data points indicating the presence of a candidate third allele.

32. A system in accordance with claim 28, wherein each cluster of data points comprises a cluster of fluorescence intensities normalized to the control fluorophore fluorescence intensity, and wherein data points outside of any cluster are data points indicating the presence of a candidate non-specific primer.

33. A system in accordance with claim 28, wherein each cluster of data points comprises a cluster of fluorescence intensities normalized to the control fluorophore fluorescence intensity, and wherein data points outside of any cluster are data points indicating the presence of a candidate allele under a primer.

34. A system in accordance with claim 28, wherein each cluster of data points comprises a cluster of fluorescence intensities normalized to the control fluorophore fluorescence intensity, and wherein data points outside of any cluster are data points indicating the presence of a candidate allele under a probe.

35. A system in accordance with claim 25, wherein the first fluorophore and the second fluorophore are different and are each selected from the group consisting of FAM, VIC, Sybra Green, TET, HEX, JOE, NED, LIZ, TAMRA, ROX, ALEXA, Texas Red, Cy3, Cy5, Cy7, Cy9, and dR6G.

36. A system in accordance with claim 25, wherein the first fluorophore and the second fluorophore are selected from FAM and VIC.

37. A system in accordance with claim 28, wherein the control fluorophore is different from the first fluorophore and the second fluorophore, and is selected from the group consisting of FAM, VIC, Sybra Green, TET, HEX, JOE, NED, LIZ, TAMRA, ROX, ALEXA, Texas Red, Cy3, Cy5, Cy7, Cy9, and dR6G.

38. A system in accordance with claim 25, wherein a cluster comprises at least two data points.

39. A system in accordance with claim 25, wherein the graphical interface further comprises a scatterplot displayed on coordinate axes.

40. A system in accordance with claim 39, wherein coordinate axes are orthogonal coordinate axes.

41. A system in accordance with claim 25, wherein the graphical interface is comprised by a digital computer monitor.

42. A system in accordance with claim 25, wherein the alleles of the genetic locus are SNP alleles.

43. A method of identifying a candidate genetic anomaly in a genome of a test individual, the method comprising:

exhibiting in a graphical interface a plurality of data points, wherein each data point occupies a position representing fluorescence intensities of a first fluorophore and a second fluorophore from a genomic sample of a reference population subjected to a fluorogenic assay for alleles of a genetic locus, wherein fluorescence of a first fluorophore is indicative of the presence of a first allele and fluorescence of a second fluorophore is indicative of the presence of a second allele, and wherein a cluster of data points is indicative of a genome homozygous for the first allele, a genome homozygous for the second allele, or a genome heterozygous for the first and second alleles;

exhibiting in the graphical interface a data point occupying a position representing fluorescence intensities of the first fluorophore and the second fluorophore from the fluorogenic assay for alleles of the genetic locus in the test individual;

generating a likelihood model that predicts the probability that an individual data point will reside within a particular cluster of data points; and

determining if a data point from the test individual falls outside any cluster.

44. A method in accordance with claim 43, wherein a fluorogenic assay comprises:

forming a mixture comprising a genome of an individual, a first nucleobase primer comprising a sequence which maps upstream from the genetic locus, a second primer complementary to a sequence which maps downstream from the genetic locus, a first probe comprising the first fluorophore and a nucleic acid sequence complementary to a genomic sequence comprising the first allele, and a second probe comprising the second fluorophore and a nucleic acid sequence complementary to a genomic sequence comprising the second allele, and a thermostable DNA polymerase having 5′

exonuclease activity;

subjecting the mixture to thermal cycling; and

detecting fluorescence intensities of each of the first fluorophore and the second fluorophore.

45. A method in accordance with claim 44, wherein the first probe and the second probe each comprise from about 10 nucleotides up to about 50 nucleotides.

46. A method in accordance with claim 44, wherein the upstream primer and the downstream primer each comprise from about 10 nucleotides up to about 50 nucleotides.

47. A method in accordance with claim 44, wherein the detecting fluorescence intensity comprises end point detection of fluorescence intensity.

48. A method in accordance with claim 44, wherein the mixture further comprises a control fluorophore, and the detecting fluorescence intensity comprises detecting fluorescence intensity normalized to a control fluorophore fluorescence intensity.

49. A method in accordance with claim 48, wherein a data point falling outside of any cluster indicates the presence of a candidate extra copy of a gene.

50. A method in accordance with claim 48 wherein a data point falling outside of any cluster indicates the presence of a candidate third allele.

51. A method in accordance with claim 48, wherein a data point falling outside of any cluster indicates the presence of a candidate non-specific primer.

52. A method in accordance with claim 48, wherein a data point falling outside of any cluster indicates the presence of a candidate allele under a primer.

53. A method in accordance with claim 48, wherein a data point falling outside of any cluster indicates the presence of a candidate allele under a probe.

54. A method in accordance with claim 43, wherein the first fluorophore and the second fluorophore are different and are each selected from the group consisting of FAM, VIC, Sybra Green, TET, HEX, JOE, NED, LIZ, TAMRA, ROX, ALEXA, Texas Red, Cy3, Cy5, Cy7, Cy9, and dR6G.

55. A method in accordance with claim 54, wherein the first fluorophore and the second fluorophore are selected from FAM and VIC.

56. A method in accordance with claim 54, wherein the control fluorophore is different from the first fluorophore and the second fluorophore, and is selected from the group consisting of FAM, VIC, Sybra Green, TET, HEX, JOE, NED, LIZ, TAMRA, ROX, ALEXA, Texas Red, Cy3, Cy5, Cy7, Cy9, and dR6G.

57. A method in accordance with claim 43, wherein a cluster comprises at least two data points.

58. A method in accordance with claim 43, wherein the graphical interface further comprises a scatterplot displayed on coordinate axes.

59. A method in accordance with claim 58, wherein coordinate axes are orthogonal coordinate axes.

60. A method in accordance with claim 43, wherein the graphical interface is comprised by a digital computer monitor.

61. A method in accordance with claim 43, wherein the alleles of the genetic locus are SNP alleles.

62. A method of determining a copy number of a target sequence in a sample genome, the method comprising:

1) forming a reaction mixture comprising;

a) a sample comprising the sample genome;

b) a target sequence primer pair;

c) a target sequence detection probe comprising a first fluorophore;

d) an endogenous reference sequence primer pair;

e) an endogenous reference sequence detection probe comprising a second fluorophore;

f) a DNA polymerase;

2) amplifying the target sequence and the reference sequence in the sample and in a calibrator;

3) determining threshold cycle values for the target and the reference; and

4) determining the amount of target sequence, normalized to the reference sequence and relative to the calibrator.

63. A method in accordance with claim 62, wherein the target sequence detection probe and the endogenous reference sequence detection probe each comprise a fluorescence quencher.

64. A method in accordance with claim 62, wherein the target sequence detection probe and the endogenous reference sequence detection probe each comprise from about 10 to about 50 nucleotides.

65. A method in accordance with claim 62, wherein the target sequence detection probe and the endogenous reference sequence detection probe each comprise from about 12 to about 25 nucleotides.

66. A method in accordance with claim 62, wherein the target sequence detection probe and the endogenous reference sequence detection probe each comprise from about 13 to about 18 nucleotides.

67. A method in accordance with claim 62, wherein the calibrator is a genome sample comprising a known copy number of the target sequence.

68. A method in accordance with claim 67, wherein the determining the amount of target sequence, normalized to the reference sequence and relative to a calibrator, comprises:

determining −

Δ

Δ

C_T, wherein C_T is the threshold number of cycles for detection of a fluorophore in a real time PCR assay;

C_T,q is the threshold number of cycles for detection of a fluorophore for the target sample in the real time PCR assay, C_T,cb is the threshold number of cycles for detection of a fluorophore for a calibrator sample in the real time PCR assay, Δ

C_T,q is a difference in threshold cycles for the target and the endogenous reference, Δ

C_T,cb is a difference in threshold cycles for the calibrator sample and the endogenous reference, −

Δ

Δ

C_T=Δ

C_T,q−

Δ

C_T,cb;

determining relative quantity of the target sequence, wherein the relative quantity is equal to 2<sup>−

Δ

Δ

CT</sup>; and

multiplying the relative quantity by the number of copies of the endogenous reference sequence.

69. A method in accordance with claim 62, wherein the reference sequence is comprised by an RNase P gene.

70. A method in accordance with claim 62, wherein the first fluorophore and the second fluorophore are different and each is selected from the group consisting of FAM, VIC, Sybra Green, TET, HEX, JOE, NED, LIZ, TAMRA, ROX, ALEXA, Texas Red, Cy3, Cy5, Cy7, Cy9, and dR6G.

71. A method in accordance with claim 70, wherein the first fluorophore and the second fluorophore are selected from FAM and VIC.

72. A method in accordance with claim 62, wherein the target sequence is comprised by a gene selected from the group consisting of a cytochrome 450 gene, a CYP1A1 gene, a CYP1A2 gene, a CYP2A1 gene, a CYP2A6 gene, a CYP2A7 gene, a CYP2B6 gene, a CYP2C8 gene, a CYP2C9 gene, a CYP2C19 gene, a CYP2D6 gene, a CYP2E1 gene, a CYP3A4 gene, a CYP3A5 gene, a CYP3A7 gene, a CYP4B1 gene, a CYP5A1 gene, a CYP8A1 gene, a CYP21 gene, .a NAT1 gene, a NAT2 gene, a COMT gene, a TMPT gene, a TYMS gene, a constitutive androstane receptor gene, a pregnane X receptor gene, an alcohol dehydrogenase gene, a flavin monooxygenase gene, a glutathione S-transferase gene, a transporter gene, an oATP-C gene, an epoxide hydrolase gene, a carboxylesterase gene, a monoamine oxidase gene, a paraoxonase gene, sulfotransferase gene, a UDP-glucuronosyl-transferase gene, an ADH1A gene, an ADH1B gene, an ADH1C gene, an ADH4 gene, an ADH5 gene, an ADH6 gene, an ADH7 gene, an FM01 gene, an FM03 gene, an FM04 gene, an FM05 gene, a GSTM1 gene, a GSTT1 gene, an MDR1 gene, an MRP1 gene, an MRP2 gene, and an MXR gene.