Multiplexed methylation detection methods
First Claim
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1. A method of detecting methylation of a cytosine in a target nucleic acid sequence in a sample of nucleic acids, comprising:
- a) contacting the sample of nucleic acids with bisulfite and forming treated nucleic acids, wherein non-methylated cytosines of the target nucleic acid sequences of the treated nucleic acids are converted to uracil, wherein methylated cytosines of the target nucleic acid sequences of the treated nucleic acids are not converted to uracil, and wherein the target nucleic acid sequences of the treated nucleic acids comprise locus sequences and either cytosines or uracils at the potentially methylated positions of the target nucleic acid sequences of the treated nucleic acids;
b) contacting said treated nucleic acids in a single reaction with a population of single-stranded first probes and a population of single-stranded second probes, wherein the first probes are complementary to locus sequences and uracils at the potentially methylated positions of the target nucleic acid sequences of the treated nucleic acids and each probe in said population of first probes comprises an identical first universal priming site, and wherein the second probes are complementary to locus sequences and cytosines at the potentially methylated positions of the target nucleic acid sequences of the treated nucleic acids and each probe in said population of second probes comprises an identical second universal priming site, thereby forming first double-stranded hybridization complexes comprising the first probes and second double-stranded hybridization complexes comprising the second probes, respectively;
c) contacting said first and second hybridization complexes with a polymerase or a ligase that modifies the first and second probes of the first and second hybridization complexes and forming single-stranded, first and second modified probes, respectively;
d) contacting said first and second modified probes with a composition comprising a polymerase, dNTPs, and at least first and second universal amplification primers that are complementary to said first and second universal priming sites, respectively, wherein said first and second modified probes are amplified and forming first and second amplicons, respectively; and
e) detecting said first and second amplicons, such that detection of said first amplicons indicates the presence of a non-methylated cytosine in the target nucleic acid sequence in the sample of nucleic acids, and detection of said second amplicon indicates the presence of a methylated cytosine in the target nucleic acid sequence in the sample of nucleic acids.
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Abstract
The present invention is directed to sensitive and accurate multiplexed assays for target analyte detection and detection of methylation in nucleic acid samples.
327 Citations
28 Claims
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1. A method of detecting methylation of a cytosine in a target nucleic acid sequence in a sample of nucleic acids, comprising:
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a) contacting the sample of nucleic acids with bisulfite and forming treated nucleic acids, wherein non-methylated cytosines of the target nucleic acid sequences of the treated nucleic acids are converted to uracil, wherein methylated cytosines of the target nucleic acid sequences of the treated nucleic acids are not converted to uracil, and wherein the target nucleic acid sequences of the treated nucleic acids comprise locus sequences and either cytosines or uracils at the potentially methylated positions of the target nucleic acid sequences of the treated nucleic acids; b) contacting said treated nucleic acids in a single reaction with a population of single-stranded first probes and a population of single-stranded second probes, wherein the first probes are complementary to locus sequences and uracils at the potentially methylated positions of the target nucleic acid sequences of the treated nucleic acids and each probe in said population of first probes comprises an identical first universal priming site, and wherein the second probes are complementary to locus sequences and cytosines at the potentially methylated positions of the target nucleic acid sequences of the treated nucleic acids and each probe in said population of second probes comprises an identical second universal priming site, thereby forming first double-stranded hybridization complexes comprising the first probes and second double-stranded hybridization complexes comprising the second probes, respectively; c) contacting said first and second hybridization complexes with a polymerase or a ligase that modifies the first and second probes of the first and second hybridization complexes and forming single-stranded, first and second modified probes, respectively; d) contacting said first and second modified probes with a composition comprising a polymerase, dNTPs, and at least first and second universal amplification primers that are complementary to said first and second universal priming sites, respectively, wherein said first and second modified probes are amplified and forming first and second amplicons, respectively; and e) detecting said first and second amplicons, such that detection of said first amplicons indicates the presence of a non-methylated cytosine in the target nucleic acid sequence in the sample of nucleic acids, and detection of said second amplicon indicates the presence of a methylated cytosine in the target nucleic acid sequence in the sample of nucleic acids. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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Current AssigneeIllumina Incorporated
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Original AssigneeIllumina Incorporated
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InventorsFan, Jian-Bing
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Primary Examiner(s)LU, FRANK WEI MIN
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Application NumberUS10/309,803Publication NumberTime in Patent Office2,527 DaysField of Search435/6, 435/91.1, 435/91.2, 435/183, 435/283.1, 435/287.1, 435/287.2, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32US Class Current435/91.2CPC Class CodesC12Q 1/6827 for detection of mutation o...C12Q 2521/331 Methylation site specific n...C12Q 2523/125 Bisulfite(s)C12Q 2537/143 Multiplexing, i.e. use of m...C12Q 2565/501 being an array of oligonucl...C12Q 2565/518 characterised by the immobi...
