Method and devices for analyzing small RNA molecules
First Claim
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1. A method for detecting a small RNA molecule in a biological sample comprising the steps of:
- modifying a small RNA molecule contained in the biological sample with an adaptor;
attaching the modified small RNA molecule to a surface wherein individual small RNA molecules are positioned on the surface such that individual small RNA molecules are individually optically resolvable; and
analyzing the attached modified small RNA molecule, wherein at least one nucleotide is identified in at least one attached modified small RNA molecule,thereby detecting a small RNA molecule in a biological sample.
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Abstract
The instant inventon provides methods and devices for detecting, enumerating and/or identifying small RNA molecules using single molecule sequencing techniques.
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Citations
25 Claims
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1. A method for detecting a small RNA molecule in a biological sample comprising the steps of:
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modifying a small RNA molecule contained in the biological sample with an adaptor; attaching the modified small RNA molecule to a surface wherein individual small RNA molecules are positioned on the surface such that individual small RNA molecules are individually optically resolvable; and analyzing the attached modified small RNA molecule, wherein at least one nucleotide is identified in at least one attached modified small RNA molecule, thereby detecting a small RNA molecule in a biological sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method for detecting a small RNA molecule in a biological sample comprising the steps of:
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extracting RNA from a biological sample; separating the RNA by size; obtaining RNA corresponding to about 10 to about 200 nucleotides in length from the separated RNA; modifying the RNA obtained in c) with an adaptor; attaching the modified RNA to a surface wherein individual RNA molecules are positioned on the surface such that individual small RNA molecules are individually optically resolvable; and analyzing the sequence of the attached modified RNA molecules, wherein at least one nucleotide is identified in at least one attached modified RNA molecule, thereby detecting a small RNA molecule in a biological sample.
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22. An apparatus for automated detection of small RNA molecules in a biological sample comprising:
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an extractor, whereby RNA can be extracted from a biological sample; a fractionator, whereby the RNA is fractionated by size; a modification chamber, whereby the fractionated RNA can be modified with an adaptor; an attachment chamber, whereby the modified RNA can be attached to a surface wherein individual RNA molecules are positioned on the surface such that at least two of the individual RNA molecules are individually optically resolvable; and a sequencing chamber, whereby at least one nucleotide of at least one attached modified RNA can be identified, wherein the extractor, fractionator, and chambers are operably linked to allow automated detection of RNA in a biological sample.
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23. A combination article of manufacture comprising:
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an adaptor for modifying small RNA molecules contained in a biological sample; and a surface whereby small RNA molecules modified with the adaptor of a) can be attached to the surface wherein individual small RNA molecules are positioned on the surface such that at least two of the individual RNA molecules are individually optically resolvable.
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24. A method for sequencing a small RNA, the method comprising the steps of:
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obtaining a single-stranded RNA comprising between about 19 and about 27 nucleotides in length; hybridizing said single-stranded RNA to a surface-bound primer nucleic acid comprising a nucleotide sequence that is complementary to at least a portion of said single-stranded rNA, thereby forming a duplex that is individually optically resolvable; exposing said duplex to an RNA polymerase and at least one nucleotide under conditions that support template-dependent nucleotide addition to said primer; determining whether a nucleotide is added to said primer; and repeating said exposing and determining steps.
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25. A method for sequencing a small RNA molecule, the method comprising the steps of:
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obtaining a single-stranded RNA comprising between about 19 and about 27 nucleotides in length; preparing a cDNA complement of said RNA; hybridizing said cDNA to a surface-bound primer nucleic acid comprising a nucleotide sequence that is complementary to at least a portion of said cDNA, thereby forming a duplex that is individually optically resolvable; exposing said duplex to a polymerase and at least one nucleotide under conditions that support template-dependent nucleotide addition to said primer; retermining whether a nucleotide is added to said primer; and repeating said exposing and determining steps.
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Specification