P450 single nucleotide polymorphism biochip analysis

• US 20040229222A1
• Filed: 04/01/2002
• Published: 11/18/2004
• Est. Priority Date: 03/30/2001
• Status: Active Grant

• Current Assignee: Amersham Biosciences (General Electric Company)
• Original Assignee: Amersham Biosciences (General Electric Company)
• Inventors: Luehrsen, Kenneth R., Li, Changming, Gupta, Vineet, Elghanian, Robert, Mazumder, Abhijit, Ramakrishnan, Ramesh, Yamashiro, Carl, Liu, Chang-Gong, Jayaraman, Krishnamurthy, Wang, David G., Yowanto, Handy, Kiser, Gretchen, Chui, Buena, Gu, Zhijie John, Tuggle, Todd, Fermin, David R., Silbergleyt, Arkadiy, Pestova, Ekaterina
• Granted Patent: US 6,986,992 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/172 Haplotypes

Abstract

This invention relates to methods and compositions for determining single nucleotide polymorphisms (SNPs) in P450 genes. In preferred embodiments, self extension of interrogation probes is prevented by using novel non self-extension probes and/or methods, thereby improving the specificity and efficiency of P450 SNP detection in target samples with minimal false positive results. The invention thus describes a variety of methods to decrease self-extension of interrogation probes. In addition, this invention provides a unique collection of P450 SNP probes on one assay, primer sequences for specific amplification of each of the seven P450 genes and amplicon control probes to evaluate whether the intended p450 gene targets were amplified successfully. The invention also describes a variety of array platforms for performing the assays of the invention; for example: CodeLink™, eSensor™, multiplex arrays with cartridges etc., all described herein.

Claims

1. A biochip comprising a solid substrate comprising an array comprising:

a) at least one capture probe substantially homologous to a portion of the sense strand of a nucleic acid encoding CPY1A1;

b) at least one capture probe substantially homologous to a first portion of the sense strand of a nucleic acid encoding CPY1A2;

c) at least one capture probe substantially homologous to a first portion of the sense strand of a nucleic acid encoding CPY1B 1;

d) at least one capture probe substantially homologous to a first portion of the sense strand of a nucleic acid encoding CPY2C 19;

e) at least one capture probe substantially homologous to a first portion of the sense strand of a nucleic acid encoding CPY2D6;

f) at least one capture probe substantially homologous to a first portion of the sense strand of a nucleic acid encoding CPY2E1; and

g) at least one capture probe substantially homologous to a first portion of the sense strand of a nucleic acid encoding CPY3A4.

2. A biochip according to claim 1 wherein said first portion of said sense strand of said nucleic acid encoding CPY2D6 is adjacent to a single nucleotide polymorphism (SNP) position of interest.

3. A biochip according to claim 1 wherein said first portion of said sense strand of said nucleic acid encoding CPY2D6 includes at a terminus a single nucleotide polymorphism (SNP) position of interest.

4. A biochip according to claim 1 wherein said array further comprises at least one capture probe substantially homologous to a portion of the antisense strand of a nucleic acid encoding a protein selected from the group consisting of CYP1A1;

CYP1A2;

CYP1B1;

CYP2C19;

CYP2D6;

CYP2E1 and CYP3A4.

5. A biochip according to claim 1 wherein said array further comprises at least one capture probe substantially homologous to a portion of a CYP pseudogene.

6. A biochip according to claim 1 wherein said solid support is selected from the group consisting of glass, plastic, ceramic, and PC board.

7. A biochip according to claim 1 wherein said array comprises an array of electrodes.

8. A biochip according to claim 1 wherein said array comprises an array of polymer gel pads.

9. A method of determining the identification of a nucleotide at a detection position in at least one target sequence selected from the group consisting of CYP1A1, CYP1A2, CYP1B1, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, said method comprising:

a) providing an array comprising;

i) at least one first capture probe substantially homologous to a first portion of a nucleic acid encoding CPY1A1, wherein said first capture probe is directly adjacent to or includes at its terminus a detection position;

ii) at least one second capture probe substantially homologous to a first portion of the sense strand of a nucleic acid encoding CPY1A2, wherein said second capture probe is directly adjacent to or includes at its terminus a detection position;

iii) at least one third capture probe substantially homologous to a first portion of the sense strand of a nucleic acid encoding CPY1 B1, wherein said third capture probe is directly adjacent to or includes at its terminus a detection position;

iv) at least one fourth capture probe substantially homologous to a first portion of the sense strand of a nucleic acid encoding CPY2C19, wherein said fourth capture probe is directly adjacent to or includes at its terminus a detection position;

v) at least one fifth capture probe substantially homologous to a first portion of the sense strand of a nucleic acid encoding CPY2D6, wherein said fifth capture probe is directly adjacent to or includes at its terminus a detection position;

vi) at least one sixth capture probe substantially homologous to a first portion of the sense strand of a nucleic acid encoding CPY2E1, wherein said sixth capture probe is directly adjacent to or includes at its terminus a detection position; and

vii) at least one seventh capture probe substantially homologous to a first portion of the sense strand of a nucleic acid encoding CPY3A4, wherein said seventh capture probe is directly adjacent to or includes at its terminus a detection position;

b) hybridizing at least one target sequence to its corresponding capture probe to form a hybridization complex;

c) adding a polymerase and at least one dNTP comprising a label, under conditions whereby if said dNTP is perfectly complementary to a detection position, said dNTP is added to a capture probe to form an extended probe;

d) determining the nucleotide at the interrogation position of said extended probe.

10. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:

a) providing an array comprising;

i) a solid support with a first surface comprising a hydrogel layer comprising an array of capture probes;

b) hybridizing said target sequence to at least one of said capture probes to form a hybridization complex; and

c) determining the nucleotide at said detection position.

11. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:

a) providing a solid support with a first surface comprising at least one non self-extension probe wherein self-extension of said non self-extension probe does not occur in the absence of said target and wherein, said non self-extension probe includes an interrogation nucleotide;

b) hybridizing said target sequence to said non self-extension probe to form a hybridization complex;

c) contacting said surface with;

i) an extension enzyme; and

ii) at least one chain terminating nucleotide comprising a hapten;

under conditions whereby if said chain terminating nucleotide is perfectly complementary to the base of the target sequence immediately adjacent to the 3′

end of said non self-extension probe in the hybridization complex, said chain terminating nucleotide is added to said non self-extension probe to form a modified extension probe;

d) contacting said modified extension probe with the binding partner of said hapten, wherein said hapten is labeled; and

e) detecting the presence of said label to determine the nucleotide at said detection position.

12. A method according to claim 11 wherein said interrogation nucleotide in said non self-extension probe is within two bases of its 3′

terminal end and wherein, said 3′

terminal end nucleotide is non-complementary to a corresponding base when a self-hybridizing structure of said non self-extension probe is formed.

13. A method according to claim 12 wherein said interrogation nucleotide is the 3′

terminal nucleotide.

14. A method according to claim 12 wherein said interrogation nucleotide is the penultimate 3′

terminal nucleotide.

15. A method according to claim 11 wherein said non self-extension probe comprises at least two modified nucleotides.

16. A method according to claim 15 wherein said modified nucleotides are exo-cyclic amine modified bases.

17. A method according to claim 15 wherein said modified nucleotides are terminator bases.

18. A method according to claim 16 wherein said exo-cyclic amine modified bases are selected from a group consisting of 2-thio thymine, 2-amino adenine, amine modified cytosine and amine modified guanine.

19. A method according to claim 17 wherein said terminator base is 4-methylindole.

20. A method according to claim 15 wherein said modified nucleotides alter protein binding and are present in the stem region of said non self-extension probe.

21. A method according to claim 20 wherein said modified nucleotide comprises a sugar modification.

22. A method according to claim 20 wherein said modified nucleotide comprises a phosphate modification.

23. A method according to claim 22 wherein said phosphate modifications are selected from a group consisting of phosphorothioates, phosphoramidates, methyl phosphonates, methyl phosphates, H-phosphonates.

24. A method according to claim 11 wherein self-extension of said non self-extension probe is inhibited by short complementary oligonucleotides.

27. A method according to claim 11 or 25 wherein said hapten is biotin.

28. A method according to claim 11 or 25 wherein said binding partner is streptavidin.

29. A method according to claim 11 or 25 wherein said binding partner is Alexa dye labeled streptavidin.

25. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:

a) amplifying the target DNA using random primers to generate DNA amplicons;

b) transcribing said DNA amplicons to generate RNA target sequences (in vitro transcription);

c) providing a solid support with a first surface comprising at least one extension probe wherein said extension probe includes an interrogation nucleotide;

b) hybridizing said RNA target sequence to said extension probe to form a hybridization complex;

c) contacting said surface with;

i) a modified reverse transcriptase; and

ii) at least one chain terminating nucleotide comprising a hapten;

under conditions whereby if said chain terminating nucleotide is perfectly complementary to the base of the target sequence immediately adjacent to the 3′

end of said non self-extension probe in the hybridization complex, said chain terminating nucleotide is added to said non self-extension probe to form a modified extension probe;

d) contacting said modified extension probe with the binding partner of said hapten, wherein said binding partner is labeled; and

e) detecting the presence of said label to determine the nucleotide at said detection position.

26. A method according to claim 25 wherein said modified reverse transcriptase only extends extension probes bound to RNA.

30. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:

a) providing a solid support with a first surface comprising a solid support with a first surface comprising a hydrogel layer comprising at least one non self-extension probe, wherein self-extension said non self-extension probe does not occur in the absence of said target and wherein, said non self-extension probe includes an interrogation nucleotide;

b) hybridizing said target sequence to said non self-extension probe to form a hybridization complex;

c) contacting said surface with;

i) an extension enzyme; and

ii) at least one chain terminating nucleotide comprising a hapten;

under conditions whereby if said chain terminating nucleotide is perfectly complementary to the base of the target sequence immediately adjacent to the 3′

end of said non self-extension probe in the hybridization complex, said chain terminating nucleotide is added to said non self-extension probe to form a modified extension probe;

d) contacting said modified extension probe with the binding partner of said hapten, wherein said binding partner is labeled; and

e) detecting the presence of said label to determine the nucleotide at said detection position.

31. A method according to claim 30 wherein said interrogation nucleotide in said non self-extension probe is within two bases of its 3′

terminal end and wherein, said 3′

terminal end nucleotide is non-complementary to a corresponding base when a self-hybridizing structure of said non self-extension probe is formed.

32. A method according to claim 31 wherein said interrogation nucleotide is the 3′

terminal nucleotide.

33. A method according to claim 31 wherein said interrogation nucleotide is the penultimate 3′

terminal nucleotide.

34. A method according to claim 30 wherein said non self-extension probe comprises at least two modified nucleotides.

35. A method according to claim 34 wherein said modified nucleotides are exo-cyclic amine modified bases.

36. A method according to claim 34 wherein said modified nucleotides are terminator bases.

37. A method according to claim 35 wherein said exo-cyclic amine modified bases are selected from a group consisting of 2-thio thymine, 2-amino adenine, amine modified cytosine and amine modified guanine.

38. A method according to claim 36 wherein said terminator base is 4-methylindole.

39. A method according to claim 34 wherein said modified nucleotides alter protein binding and are present in the stem region of said non self-extension probe.

40. A method according to claim 39 wherein said modified nucleotide comprises a sugar modification.

41. A method according to claim 39 wherein said modified nucleotide comprises a phosphate modification.

42. A method according to claim 41 wherein said phosphate modifications are selected from a group consisting of phosphorothioates, phosphoramidates, methyl phosphonates, methyl phosphates, H-phosphonates.

43. A method according to claim 30 wherein self-extension of said non self-extension probe is inhibited by short complementary oligonucleotides.

46. A method according to claim 30 or 44 wherein said hapten is biotin.

47. A method according to claim 30 or 44 wherein said binding partner is streptavidin.

48. A method according to claim 30 or 44 wherein said binding partner is Alexa dye labeled streptavidin.

44. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:

a) amplifying the target DNA using random primers to generate DNA amplicons;

b) transcribing said DNA amplicons to generate RNA target sequences (in vitro transcription);

c) providing a solid support with a first surface comprising at least one extension probe wherein said extension probe includes an interrogation nucleotide;

b) hybridizing said RNA target sequence to said extension probe to form a hybridization complex;

c) contacting said surface with;

i) a modified reverse transcriptase; and

ii) at least one chain terminating nucleotide comprising a hapten;

under conditions whereby if said chain terminating nucleotide is perfectly complementary to the base of the target sequence immediately adjacent to the 3′

end of said non self-extension probe in the hybridization complex, said chain terminating nucleotide is added to said non self-extension probe to form a modified extension probe;

d) contacting said modified extension probe with the binding partner of said hapten, wherein said binding partner is labeled; and

e) detecting the presence of said label to determine the nucleotide at said detection position.

45. A method according to claim 44 wherein said modified reverse transcriptase only extends extension probes bound to RNA.