Methods for identifying nucleic acid sequences
First Claim
1. A method for evaluating a nucleic acid sequence in a tissue section comprising:
- incubating an RNA containing the nucleic acid sequence in the tissue section with a reverse transcriptase and a reverse transcription primer that is complementary to the RNA to generate cDNA, wherein the reverse transcription primer is modified so as to be capable of immobilization in the cells of the tissue section;
digesting all or part of the RNA;
hybridizing one or more padlock probes to the cDNA, wherein the padlock probe(s) comprise one or two terminal regions having the nucleic acid sequence;
incubating the hybridized padlock probe(s) and cDNA with ligase under conditions to ligate the ends of the padlock probe(s);
incubating the padlock probe(s) with a polymerase to create an amplified rolling circle amplification product; and
,sequencing the amplified rolling circle amplification product.
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Accused Products
Abstract
Embodiments relate to the detection of RNA in a sample of cells. More particularly, methods concern the localized detection of RNA in situ. The method relies on the conversion of RNA to complementary DNA prior to the targeting of the cDNA with a padlock probe(s). The hybridization of the padlock probe(s) relies on the nucleotide sequence of the cDNA which is derived from the corresponding nucleotide sequence of the target RNA. Rolling circle amplification of the subsequently circularized padlock probe produces a rolling circle product which may be detected. Advantageously, this allows the RNA to be detected in situ. In additional methods, rolling circle amplification products are sequenced.
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Citations
26 Claims
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1. A method for evaluating a nucleic acid sequence in a tissue section comprising:
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incubating an RNA containing the nucleic acid sequence in the tissue section with a reverse transcriptase and a reverse transcription primer that is complementary to the RNA to generate cDNA, wherein the reverse transcription primer is modified so as to be capable of immobilization in the cells of the tissue section; digesting all or part of the RNA; hybridizing one or more padlock probes to the cDNA, wherein the padlock probe(s) comprise one or two terminal regions having the nucleic acid sequence; incubating the hybridized padlock probe(s) and cDNA with ligase under conditions to ligate the ends of the padlock probe(s); incubating the padlock probe(s) with a polymerase to create an amplified rolling circle amplification product; and
,sequencing the amplified rolling circle amplification product. - 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)
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25. A method for determining in situ a nucleic acid sequence in a formalin-fixed paraffin-embedded tissue section comprising:
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generating a cDNA complementary to an RNA containing the nucleic acid sequence in the tissue section; incubating the cDNA with a ribonuclease in the sample to digest the RNA; hybridizing one or more padlock probes to the cDNA, wherein the padlock probe(s) comprise one or two terminal regions having the nucleic acid sequence; incubating the hybridized padlock probes and cDNA with ligase under conditions to ligate the ends of the padlock probe(s); replicating the padlock probe(s) using a polymerase to create an amplified product; sequencing the complement of the nucleic acid sequence in the amplified product to determine the nucleic acid sequence and/or its complement. - View Dependent Claims (26)
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Specification