P450 single nucleotide polymorphism biochip analysis
First Claim
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.
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Accused Products
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.
51 Citations
48 Claims
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1. A biochip comprising a solid substrate comprising an array comprising:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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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:
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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.
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10. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:
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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.
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11. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:
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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. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 27, 28, 29)
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25. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:
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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. - View Dependent Claims (26)
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30. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:
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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. - View Dependent Claims (31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 46, 47, 48)
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44. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:
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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. - View Dependent Claims (45)
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Specification