INTEGRATED SINGLE CELL SEQUENCING
First Claim
1. A method of forming a tagged nucleic acid sequence, said method comprising:
- (i) immobilizing a target polynucleotide on a solid support;
(ii) hybridizing a recognition-oligonucleotide to said target polynucleotide before or after the immobilizing, thereby forming a polynucleotide hybrid;
(iii) cleaving said polynucleotide hybrid with a cleaving agent, thereby forming a cleaved polynucleotide hybrid comprising a cleaved target polynucleotide; and
(iv) ligating an adapter nucleic acid sequence to said cleaved target polynucleotide, thereby forming a tagged nucleic acid sequence
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Accused Products
Abstract
This disclosure provides a method of forming tagged nucleic acid sequences. A target polynucleotide is immobilized on a solid support; a recognition-oligonucleotide is hybridized thereto; the recognition-oligonucleotide-target polynucleotide hybrid is cleaved; and an adapter nucleic acid is ligated to the cleaved target polynucleotide, thereby forming a tagged nucleic acid sequence. Also provided is a method of forming a tagged single stranded cDNA; a method of forming a plurality of tagged heterogeneous nucleic acid sequences; a library of recognition-oligonucleotides; and methods for amplifying a cDNA sequence immobilized on a solid support. These methods and products can be used alone or in combination for integrated single cell sequencing, and can be adapted for use in a microfluidic apparatus or device.
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Citations
24 Claims
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1. A method of forming a tagged nucleic acid sequence, said method comprising:
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(i) immobilizing a target polynucleotide on a solid support; (ii) hybridizing a recognition-oligonucleotide to said target polynucleotide before or after the immobilizing, thereby forming a polynucleotide hybrid; (iii) cleaving said polynucleotide hybrid with a cleaving agent, thereby forming a cleaved polynucleotide hybrid comprising a cleaved target polynucleotide; and (iv) ligating an adapter nucleic acid sequence to said cleaved target polynucleotide, thereby forming a tagged nucleic acid sequence - View Dependent Claims (2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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5. The method of claim 5, wherein said first release nucleic acid sequence comprises a restriction enzyme cleavage sequence.
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16. A method of forming a tagged nucleic acid sequence, said method comprising:
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(i) immobilizing an RNA molecule on a solid support; (ii) reverse transcribing said RNA molecule, thereby forming an RNA;
cDNA hybrid;(iii) cleaving said RNA;
cDNA hybrid with a cleaving agent, thereby forming cleaved cDNA; and(iv) ligating an adapter nucleic acid sequence to said cleaved cDNA, thereby forming a tagged cDNA sequence; wherein said ribonucleic acid sequence is removed from said RNA;
cDNA hybrid before or after steps (iii) and (iv). - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24)
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Specification