Reagents, methods, and libraries for bead-based sequencing
First Claim
1. A method for identifying a sequence of nucleotides in a template polynucleotide, the method comprising the steps of:
- (I) extending an initializing oligonucleotide along the template polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex, wherein the oligonucleotide probe comprises a phosphorothiolate linkage and is from one of at least two distinguishably labeled probe families to which the template polynucleotide is exposed prior to the ligating, and wherein each probe family comprises a plurality of oligonucleotide probes having identical labels but different sequences that comprise;
(1) a constrained portion of least two constrained nucleosides, wherein the constrained nucleosides;
(i) are at least 2-fold degenerate among the probes in the probe family, (ii) need not be located contiguously, and (iii) are not selected independently of each other, such that(2) knowing the label of a probe eliminates one or more combinations of nucleosides as possibilities for the sequence of the constrained portion, and(3) knowing the identity of a constrained nucleoside in that probe further eliminates at least one possible identity for another constrained nucleoside in the probe;
(II) identifying one or more nucleotides of the polynucleotide; and
(III) repeating steps I and II until the sequence of nucleotides is determined;
wherein the template polynucleotide is attached to a solid support and/or immobilized in or on a semi-solid support during the extending step.
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Accused Products
Abstract
The present invention provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles comprise steps of extension, ligation, and, preferably, cleavage. In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages. The invention provides methods of determining information about a sequence using at least two distinguishably labeled probe families. In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle. In certain embodiments the sequencing reactions are performed on templates attached to immobilized beads. The invention further provides sets of labeled extension probes containing phosphorothiolate linkages In addition, the invention includes performing multiple sequencing reactions on a single template by removing initializing oligonucleotides and extended strands and performing subsequent reactions using different initializing oligonucleotides.
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Citations
31 Claims
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1. A method for identifying a sequence of nucleotides in a template polynucleotide, the method comprising the steps of:
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(I) extending an initializing oligonucleotide along the template polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex, wherein the oligonucleotide probe comprises a phosphorothiolate linkage and is from one of at least two distinguishably labeled probe families to which the template polynucleotide is exposed prior to the ligating, and wherein each probe family comprises a plurality of oligonucleotide probes having identical labels but different sequences that comprise; (1) a constrained portion of least two constrained nucleosides, wherein the constrained nucleosides;
(i) are at least 2-fold degenerate among the probes in the probe family, (ii) need not be located contiguously, and (iii) are not selected independently of each other, such that(2) knowing the label of a probe eliminates one or more combinations of nucleosides as possibilities for the sequence of the constrained portion, and (3) knowing the identity of a constrained nucleoside in that probe further eliminates at least one possible identity for another constrained nucleoside in the probe; (II) identifying one or more nucleotides of the polynucleotide; and (III) repeating steps I and II until the sequence of nucleotides is determined; wherein the template polynucleotide is attached to a solid support and/or immobilized in or on a semi-solid support during the extending step. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, 25)
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14. A method for identifying a sequence of nucleotides in a template polynucleotide attached to a support at a point of attachment, the method comprising the steps of:
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(a) extending an initializing oligonucleotide along the template polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex, wherein extension proceeds along the template towards its point of attachment to the support; (b) identifying one or more nucleotides of the polynucleotide; and (c) repeating steps (a) and (b) until the sequence of nucleotides is determined. - View Dependent Claims (15, 16, 23, 24, 26, 27, 28, 29, 30, 31)
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17. A method for identifying a sequence of nucleotides in a template polynucleotide, the method comprising the steps of:
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(a) providing a template polynucleotide attached to a microparticle that is immobilized in or on a semi-solid support; (b) extending an initializing oligonucleotide along the template polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex, wherein the oligonucleotide probe comprises a scissile linkage and is from one of at least two distinguishably labeled probe families to which the template polynucleotide is exposed prior to the ligating, and wherein each probe family comprises a plurality of oligonucleotide probes having identical labels but different sequences that comprise; (1) a constrained portion of least two constrained nucleosides, wherein the constrained nucleosides;
(i) are at least 2-fold degenerate among the probes in the probe family, (ii) need not be located contiguously, and (iii) are not selected independently of each other, such that(2) knowing the label of a probe eliminates one or more combinations of nucleotides nucleosides as possibilities for the sequence of the constrained portion, and (3) knowing the identity of a constrained residue nucleoside in that probe further eliminates at least one possible identity for another constrained residue nucleoside in the probe; (c) identifying one or more nucleotides of the polynucleotide; and (d) repeating steps (b) and (c) until the sequence of nucleotides is determined. - View Dependent Claims (18, 19, 21, 22)
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Specification