Methods and compositions for generating mixtures of nucleic acid molecules
First Claim
1. A method for synthesizing nucleic acid molecules, said method comprising the steps of:
- a) synthesizing an array of proximal nucleic acid molecules on a substrate,b) incorporating a cleavable linker by contacting the array of proximal nucleic acid molecules with a cleavable phosphoramidite building block comprising;
R-Lc-Prwherein Pr is a hydroxyl protecting group,Lc is a cleavable linker, andR is a phosphoramidite group,c) extending the building block to form distal nucleic acid molecules, andd) cleaving the cleavable linker to release the distal nucleic acid molecules under conditions which do not release the proximal nucleic acid molecules.
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Abstract
In some embodiments, the present disclosure provides methods of making a mixture of nucleic acid molecules, the methods comprising the steps of: synthesizing on a substrate a population of nucleic acid molecules wherein each synthesized nucleic acid molecule comprises a substrate-attached proximal nucleic acid molecule, a distal nucleic acid molecule, and a cleavable linker linking the proximal nucleic acid molecule to the distal nucleic acid molecule, and harvesting distal nucleic acid molecules from the substrate by cleaving the cleavable linker under conditions that do not release the proximal nucleic acid molecule. Related compositions and kits are also provided.
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Citations
35 Claims
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1. A method for synthesizing nucleic acid molecules, said method comprising the steps of:
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a) synthesizing an array of proximal nucleic acid molecules on a substrate, b) incorporating a cleavable linker by contacting the array of proximal nucleic acid molecules with a cleavable phosphoramidite building block comprising;
R-Lc-Prwherein Pr is a hydroxyl protecting group, Lc is a cleavable linker, and R is a phosphoramidite group, c) extending the building block to form distal nucleic acid molecules, and d) cleaving the cleavable linker to release the distal nucleic acid molecules under conditions which do not release the proximal nucleic acid molecules. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. A method for preparing a nucleic acid, the method comprising the steps of:
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a) providing a solid support comprising;
sm-PN1-Lc-PN2,wherein sm is a substrate medium, wherein PN1 is non-cleavably attached to the support, wherein Lc comprises a cleavable linker, and b) selectively releasing said PN2.
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24. A method for the synthesis of a plurality of oligonucleotides comprising the steps of
(i) forming an array of first oligonucleotides on a non-cleavable link attached to a solid support; -
(ii) attaching to the first oligonucleotides a cleavable linker moiety; (iii) forming second oligonucleotides on the cleavable linker moiety; and (iv) cleaving the cleavable linker moiety to give a plurality of oligonucleotides, wherein the first oligonucleotides are bound to the surface by an attachment linkage, and wherein the attachment linkage is devoid of a cleavable moiety. - View Dependent Claims (25)
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26. A composition comprising:
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a modified substrate medium according to the following formula;
sm-PN1-Lc-PN2wherein sm is a substrate medium, wherein Lc comprises a cleavable linker, wherein PN1 is a polynucleotide from 2-100 residues in length, wherein PN2 is a polynucleotide from 5 to 1000 residues in length, wherein PN1 is attached to the substrate medium by a non-cleavable attachment. - View Dependent Claims (27, 28, 29, 30, 31)
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32. A composition comprising:
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a modified substrate medium according to the following formula;
sm-PN1-Lc-Prwherein sm is a substrate medium, wherein Pr is a hydroxyl protecting group, wherein Lc comprises a cleavable linker, wherein PN1 is a polynucleotide from 2-100 residues in length, wherein said PN1 is bound to the surface by an attachment linkage means, and wherein the attachment means is devoid of cleavable moieties.
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33. A compound of formula I:
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sm-PN1-Lc-PN2
Iwherein sm is a substrate medium, wherein Lc comprises a cleavable linker, wherein PN1 is a polynucleotide from 2-100 residues in length, wherein PN2 is a polynucleotide from 5 to 1000 residues in length, wherein said sm is coupled to said PN1 by a non-cleavable linker, and said PN1 is coupled to said PN2 by a cleavable linker.
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34. A kit for preparing a mixture of nucleic acids, comprising:
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a) a modified substrate medium according to the following formula;
sm-PN1-Lc-PN2wherein sm is a substrate medium, wherein Lc comprises a cleavable linker, wherein PN1 is a polynucleotide from 2-100 residues in length, wherein PN2 is a polynucleotide from 5 to 1000 residues in length, wherein PN1 is attached to the substrate medium by a non-cleavable linkage, and b) a cleavage agent capable of cleaving said cleavable linker.
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35. A kit for preparing a mixture of nucleic acids, comprising:
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a) a cleavable phosphoramidite building block comprising a cleavable linker, b) a cleavage agent for cleaving said cleavable linker, c) a modified substrate medium according to the following formula;
sm-PN1-Prwherein sm is a substrate medium, wherein Pr is a hydroxyl protecting group, wherein PN1 is bound to the surface by an attachment linkage means, and wherein the attachment linkage is non-cleavable.
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Specification