| 摘要 |
Disclosed are methods and materials for detecting RNA in a sample. In some forms, the method involves (a) bringing into contact the sample and a probe array, (b) bringing into contact the probe array and a ribonuclease specific for RNA/DNA hybrids (such as RNase H), (c) bringing into contact the probe array, labeled nucleotides, and a nucleic acid polymerase capable of extending a RNA strand using a DNA template and capable of incorporating the labeled nucleotides in the extension from the RNA strand (such as Klenow fragment DNA polymerase), and (d) detecting the labeled nucleotides in the extended nucleic acid strand. The probe array comprises one or more chimeric probes. The chimeric probes comprise a DNA region and a RNA region, where the DNA region and the RNA region are contiguous and where the DNA region is 5′ of the RNA region. The chimeric probe can also include a second DNA region. The second DNA region can also be contiguous with the RNA region and can be 3′ of the RNA region. |
| 主权项 |
1. A method of detecting RNA in a sample, the method comprising:
(a) bringing into contact the sample and a probe array, wherein the probe array comprises one or more chimeric probes, wherein the chimeric probes comprise a DNA region and a RNA region, wherein the DNA region and the RNA region are contiguous, wherein the DNA region is 5′ of the RNA region, wherein if the sample contains an RNA molecule complementary to a nucleotide sequence of at least one of the chimeric probes, the RNA molecule will hybridize with the complementary chimeric probe; (b) bringing into contact the probe array and a ribonuclease specific for RNA/DNA hybrids, wherein the portion of the RNA molecule hybridized to the DNA region of the chimeric probe is degraded; (c) bringing into contact the probe array, labeled nucleotides, and a nucleic acid polymerase capable of extending a RNA strand using a DNA template and capable of incorporating the labeled nucleotides in the extension from the RNA strand, wherein the hybridized RNA molecule is extended to form an extended nucleic acid strand, wherein at least one labeled nucleotide is incorporated into the extended nucleic acid strand, wherein each of the labeled nucleotides comprises a first label; and (d) detecting the labeled nucleotides in the extended nucleic acid strand by bringing into contact the probe array and a label conjugate, wherein the label conjugate comprises a specific binding molecule and a second label, wherein the specific binding molecule binds to the first label, wherein the labeled nucleotides in the extended nucleic acid strand are detected by detecting the second label, wherein the second label is detected by bringing into contact the probe array and a detection conjugate, wherein the detection conjugate comprises an aggregator, and wherein the aggregator mediates aggregation of detection conjugates on the label conjugate, wherein detection of the labeled nucleotides in the extended nucleic acid strand indicates the presence of the RNA molecule in the sample. |
