Method for Archiving and Clonal Expansion
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Abstract
The present method provides methods, libraries, and kits related to the archiving and clonal expansion of sequences related to target polynucleotide sequences. The method allow for the attachment of polynucleotides with defined 3′ and or 5′ sequences to solid surfaces. The polynucleotides attached to the solid substrates can be stored or archived as libraries and can subsequently be retrieved for analysis, for example by clonal expansion. In some embodiments, nucleotides attached to solid surfaces can be used for sequencing of nucleotide sequences related to target RNA or target RNA. The methods are applicable to total RNA and/or total DNA analysis.
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Citations
382 Claims
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1-329. -329. (canceled)
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330. A method for amplifying a DNA or RNA target nucleic acid sequence or its complement on a solid support to form a plurality of amplified products comprising clonally amplifying said target sequence or its complement by:
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(i). linear amplification; (ii). amplification using a single primer; (iii). amplification from a double-stranded nucleic acid having a 3′
overhang;(iv). amplification using a DNA-RNA chimeric primer;
or(v). (i), (ii), (iii), and (iv); wherein said target nucleic acid sequence is coupled to said solid support. - View Dependent Claims (331, 332, 333, 334, 335, 336, 339)
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337. A method for clonally amplifying a target nucleic acid sequence or its complement comprising:
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delivering the target nucleic acid sequence or its complement into an emulsion; and performing linear amplification of said target nucleic acid sequence inside said emulsion. - View Dependent Claims (338)
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340. A method comprising:
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(a) extending a first primer or a set of first primers hybridized to a target nucleic acid with a DNA polymerase comprising a DNA segment and a 5′
RNA segment, wherein a 3′
portion of the primer, sequence (P) is complementary to a target nucleic acid and a portion of the 5′
RNA segment, sequence (A), is not complementary to the target nucleic acid, to form a first primer extension product or set of first primer extension products hybridized to the target nucleic acid;(b) separating or removing the first primer extension product or the set of first primer extension products from the target nucleic acid; (c) extending a second primer with a DNA polymerase comprising a 3′
DNA segment and a 5′
DNA segment, wherein a portion of the 3′
DNA segment is complementary to the first primer extension product or set of first primer extension products and a 5′
portion, sequence (B), of the of the second primer is not complementary to the first primer extension product or the set of first primer extension products, to produce a double-stranded DNA product or a set of double-stranded DNA products, each product comprising the first primer extension product hybridized to a second primer extension product, whereby the second primer extension product has a DNA sequence (A′
) that is complementary and hybridized to the RNA sequence (A) of the first primer extension product at its 3′
end, thereby forming the double stranded nucleic acid product or products with an RNA/DNA heteroduplex at one end;(d) cleaving the RNA from the RNA/DNA heteroduplex; (e) annealing a chimeric oligonucleotide comprising a 5′
end wherein the 5′
end comprises sequence (C), and the 5′
end further comprises RNA, and a 3′
end wherein the 3′
end comprises the DNA sequence (A), to the double stranded nucleic acid product or products; and(f) extending the double stranded nucleic acid product or products with a DNA polymerase to produce a sequence (C)—
(C′
) RNA-DNA heteroduplex. - View Dependent Claims (341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367)
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368. A method for generating an amplified nucleic acid of comprising:
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(a) providing a first nucleic acid or a set of first nucleic acids comprising DNA corresponding to a region of a target nucleic acid and further comprising a 5′
end and a 3′
end, wherein said 5′
end of the first nucleic or the set of first nucleic acids comprises a sequence (A) and a sequence (C), wherein said sequence (A) is 3′
of sequence (C), and wherein sequence (C) comprises RNA, and wherein said 3′
end of the first nucleic acid comprises sequence (B′
);(b) hybridizing said first nucleic acid or set of first nucleic acids to a solid support comprising an oligonucleotide comprising a sequence (B) complementary to the sequence (B′
) of the first nucleic acid;(c) extending the oligonucleotide to produce a double stranded product comprising a second nucleic acid hybridized to the first nucleic acid, wherein the second nucleic acid comprises a 3′
segment complementary to a portion of the first nucleic acid and a 5′
segment complementary to a portion of the first nucleic acid, whereby a portion of the 3′
segment comprises a sequence (A′
) and a portion of the 3′
segment comprises a sequence (C′
), and wherein the sequence (A′
) is 5′
of the sequence (C′
), and whereby a portion of the 5′
segment comprises a sequence (B). - View Dependent Claims (369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382)
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Specification