Methods of Nucleic Acid Sequencing
First Claim
1. A method of detecting one or more target nucleic acids comprising the steps of:
- (a) capturing one or more target nucleic acids on a support;
(b) using the one or more captured target nucleic acids as a template strand to produce one or more nucleic acid duplexes immobilized on the support;
(c) contacting a transposase and transposon with the one or more nucleic acid duplexes under conditions wherein the one or more nucleic acid duplexes and transposon composition undergo a transposition reaction to produce one or more tagged nucleic acid duplexes, wherein the transposon composition comprises a double stranded nucleic acid molecule comprising a transferred strand and a non-transferred strand;
(d) contacting the one or more tagged nucleic acid duplexes with a nucleic acid modifying enzyme under conditions to extend the 3′
end of the immobilized strand to the 5′
end of the template strand to produce one or more end-extended tagged nucleic acid duplexes;
(e) amplifying the one or more end-extended tagged nucleic acid duplexes to produce a plurality of tagged nucleic acid strands; and
(f) detecting the plurality of tagged nucleic acid strands.
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Accused Products
Abstract
Provided herein is a method of using transposition to improve methods of sequencing RNA molecules. Provided herein is a method of tagging nucleic acid duplexes, such as DNA:RNA duplexes or DNA:DNA duplexes. The method includes the steps of providing a transposase and a transposon composition, providing one or more nucleic acid duplexes immobilized on a support, and contacting the transposase and transposon composition with the one or more nucleic acid duplexes under conditions wherein the one or more nucleic acid duplexes and transposon composition undergo a transposition reaction to produce one or more tagged nucleic acid duplexes, wherein the transposon composition comprises a double stranded nucleic acid molecule comprising a transferred strand and a non-transferred strand.
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Citations
20 Claims
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1. A method of detecting one or more target nucleic acids comprising the steps of:
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(a) capturing one or more target nucleic acids on a support; (b) using the one or more captured target nucleic acids as a template strand to produce one or more nucleic acid duplexes immobilized on the support; (c) contacting a transposase and transposon with the one or more nucleic acid duplexes under conditions wherein the one or more nucleic acid duplexes and transposon composition undergo a transposition reaction to produce one or more tagged nucleic acid duplexes, wherein the transposon composition comprises a double stranded nucleic acid molecule comprising a transferred strand and a non-transferred strand; (d) contacting the one or more tagged nucleic acid duplexes with a nucleic acid modifying enzyme under conditions to extend the 3′
end of the immobilized strand to the 5′
end of the template strand to produce one or more end-extended tagged nucleic acid duplexes;(e) amplifying the one or more end-extended tagged nucleic acid duplexes to produce a plurality of tagged nucleic acid strands; and (f) detecting the plurality of tagged nucleic acid strands. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method of detecting one or more target nucleic acids comprising the steps of:
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(a) capturing the one or more target nucleic acids on a support by hybridizing a 3′
adaptor of the one or more target nucleic acids to a plurality of primers or a subset thereof, wherein the primers are immobilized on a support;(b) using the one or more captured target nucleic acids as a template strand to produce one or more nucleic acid duplexes immobilized on the support; (c) contacting a transposase and transposon with the one or more nucleic acid duplexes under conditions wherein the one or more nucleic acid duplexes and transposon composition undergo a transposition reaction to produce one or more tagged nucleic acid duplexes, wherein the transposon composition comprises a double stranded nucleic acid molecule comprising a transferred strand and a non-transferred strand; (d) contacting the one or more tagged nucleic acid duplexes with a nucleic acid modifying enzyme under conditions to extend the 3′
end of the immobilized strand to the 5′
end of the template strand to produce one or more end-extended tagged nucleic acid duplexes;(e) amplifying the one or more end-extended tagged nucleic acid duplexes to produce a plurality of tagged nucleic acid strands; and (f) detecting the plurality of tagged nucleic acid strands. - View Dependent Claims (14, 15, 16)
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17. A method of detecting one or more target nucleic acids comprising the steps of:
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(a) providing one or more target nucleic acids; (b) capturing the one or more target nucleic acids on a support, the support comprising a plurality of immobilized nucleic acids each having a sequence complementary to at least a portion of the one or more target nucleic acids; (b) using the one or more captured target nucleic acids as a template strand to produce one or more nucleic acid duplexes immobilized on the support; (c) tagging the one or more nucleic acid duplexes via a transposition reaction to produce tagged nucleic acid duplexes; (d) amplifying the one or more tagged nucleic acid duplexes to produce a plurality of tagged nucleic acid strands; and (e) detecting the plurality of tagged nucleic acid strands. - View Dependent Claims (18, 19, 20)
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Specification