METHODS FOR DETECTING MODIFICATION RESISTANT NUCLEIC ACIDS
First Claim
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1. A method for detecting a plurality of RNA molecules bound to at least one ribosome, said method comprising:
- (i) contacting said plurality of RNA molecules with an enzymatic degradant or a chemical degradant thereby forming a plurality of RNA fragments, wherein each RNA fragment comprises an RNA portion protected from said enzymatic degradant or said chemical degradant by a ribosome to which the RNA portion is bound;
(ii) amplifying said RNA fragments to form a detectable number of amplified nucleic acid fragments; and
(iii) detecting said detectable number of amplified nucleic acid fragments, thereby detecting said plurality of RNA molecules bound to at least one ribosome.
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Abstract
Methods are provided for, inter alia, detecting nucleic acid molecules resistant to degradation, such as a plurality of RNA molecules bound to a ribosome, using various technologies including deep sequencing.
40 Citations
33 Claims
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1. A method for detecting a plurality of RNA molecules bound to at least one ribosome, said method comprising:
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(i) contacting said plurality of RNA molecules with an enzymatic degradant or a chemical degradant thereby forming a plurality of RNA fragments, wherein each RNA fragment comprises an RNA portion protected from said enzymatic degradant or said chemical degradant by a ribosome to which the RNA portion is bound; (ii) amplifying said RNA fragments to form a detectable number of amplified nucleic acid fragments; and (iii) detecting said detectable number of amplified nucleic acid fragments, thereby detecting said plurality of RNA molecules bound to at least one ribosome. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. A method for detecting a nucleic acid comprising a nucleic acid portion resistant to enzymatic modification or chemical modification, said method comprising:
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(i) contacting said nucleic acid with an enzymatic modifying agent or chemical modifying agent thereby forming a modified nucleic acid molecule comprising said nucleic acid portion; (ii) amplifying said modified nucleic acid molecule to form a detectable number of amplified modified nucleic acid molecules; and (iii) sequencing said detectable number of amplified modified nucleic acid molecules, thereby detecting said nucleic acid. - View Dependent Claims (18, 19, 20, 21, 22)
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23. A method for detecting an RNA molecule bound to at least one ribosome, said method comprising:
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(i) contacting said RNA molecule with an enzymatic degradant or chemical degradant thereby forming an RNA fragment, wherein the RNA fragment comprises an RNA portion protected from said enzymatic degradant or said chemical degradant by a ribosome to which the RNA portion is bound; (ii) contacting said RNA fragment with a DNA polymerase and a DNA polymerase primer thereby forming a linear DNA; (iii) contacting said linear DNA with a ligase thereby forming a circularized DNA; (iv) amplifying said circularized DNA thereby forming a detectable number of amplified DNA molecules; and (v) detecting said detectable number of amplified DNA molecules thereby detecting said RNA molecule bound to at least one ribosome.
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24. A method for determining translation activity for a plurality of RNA molecules, comprising:
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(i) treating a plurality of RNA-ribosome complexes, wherein each RNA-ribosome complex comprises an RNA molecule having an RNA portion that is bound to a ribosome, under conditions sufficient produce a plurality of RNA fragments, wherein each RNA fragment comprises an RNA portion, and wherein each RNA fragment comprises a nucleotide sequence that is longer than the nucleotide sequence of said RNA portion; (ii) sequencing the plurality of RNA fragments to determine the nucleotide sequences of the RNA portions; and (iii) comparing the nucleotide sequences of the plurality of RNA portions to the nucleotide sequences of the RNA molecules, thereby determining translation activity for the plurality of RNA molecules. - View Dependent Claims (25, 26, 27, 28, 29, 30, 31, 32, 33)
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Specification