Linkers and co-coupling agents for optimization of oligonucleotide synthesis and purification on solid supports
First Claim
1. A method of providing complementary nucleic acid comprising:
- a) providing oligonucleotides, wherein said oligonucleotides are generated according to the method of;
A) providing;
i) a substrate comprising an array of oligonucleotides attached to said substrate via anchor moieties attached to non-cleavable linkers attached to said substrate, wherein said anchor moieties comprise the structure —
C(X)—
C(Y), wherein X comprises —
OPO2O—
, Y is a nucleophile and wherein said structure is part of a ring moiety;
ii) a deprotecting solution; and
iii) and a wash solution;
B) deprotecting said oligonucleotides with said deprotecting solution;
C) washing said oligonucleotides with said wash solution; and
D) cleaving said oligonucleotides at said anchor moieties to provide purified oligonucleotides;
b) mixing said oligonucleotides with a polymerase and template strands having a complementary sequence to the oligonucleotide in a polymerization buffer; and
c) recovering copies of complementary nucleic acid strands in the duplex with the template strand.
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Accused Products
Abstract
A method of modulation of synthesis capacity on and cleavage properties of synthetic oligomers from solid support is described. The method utilizes linker molecules attached to a solid surface and co-coupling agents that have similar reactivities to the coupling compounds with the surface functional groups. The preferred linker molecules provide an increased density of polymers and more resistance to cleavage from the support surface. The method is particularly useful for synthesis of oligonucleotides, oligonucleotides microarrays, peptides, and peptide microarrays. The stable linkers are also coupled to anchor molecules for synthesis of DNA oligonucleotides using on support purification, eliminating time-consuming chromatography and metal cation presence. Oligonucleotides thus obtained can be directly used for mass analysis, DNA amplification and ligation, hybridization, and many other applications.
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Citations
3 Claims
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1. A method of providing complementary nucleic acid comprising:
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a) providing oligonucleotides, wherein said oligonucleotides are generated according to the method of; A) providing; i) a substrate comprising an array of oligonucleotides attached to said substrate via anchor moieties attached to non-cleavable linkers attached to said substrate, wherein said anchor moieties comprise the structure —
C(X)—
C(Y), wherein X comprises —
OPO2O—
, Y is a nucleophile and wherein said structure is part of a ring moiety;ii) a deprotecting solution; and iii) and a wash solution; B) deprotecting said oligonucleotides with said deprotecting solution; C) washing said oligonucleotides with said wash solution; and D) cleaving said oligonucleotides at said anchor moieties to provide purified oligonucleotides; b) mixing said oligonucleotides with a polymerase and template strands having a complementary sequence to the oligonucleotide in a polymerization buffer; and c) recovering copies of complementary nucleic acid strands in the duplex with the template strand.
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2. A method of providing amplified copies of complementary deoxyribonucleic acid (DNA) comprising:
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a) providing oligonucleotides, wherein said oligonucleotides are generated according to the method of; A) providing; i) a substrate comprising an array of oligonucleotides attached to said substrate via anchor moieties attached to non-cleavable linkers attached to said substrate, wherein said anchor moieties comprise the structure —
C(X)—
C(Y), wherein X comprises —
OPO2O—
, Y is a nucleophile and wherein said structure is part of a ring moiety;ii) a deprotecting solution; and iii) and a wash solution; B) deprotecting said oligonucleotides with said deprotecting solution; C) washing said oligonucleotides with said wash solution; and D) cleaving said oligonucleotides at said anchor moieties to provide purified oligonucleotides; b) mixing said oligonucleotides with a DNA polymerase and DNA template strands having a complementary sequence to the oligonucleotide at the 3′
-end in a PCR buffer; andc) recovering amplified copies of complementary DNA strands in the duplex with the template strand.
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3. A method of providing ligated DNA comprising
a) providing: -
i) oligonucleotides obtained according to the method of; A) providing; i) a substrate comprising an array of oligonucleotides attached to said substrate via anchor moieties attached to non-cleavable linkers attached to said substrate, wherein said anchor moieties comprise the structure —
C(X)—
C(Y), wherein X comprises —
OPO2O—
, Y is a nucleophile and wherein said structure is part of a ring moiety;ii) a deprotecting solution; and iii) and a wash solution; B) deprotecting said oligonucleotides with said deprotecting solution; C) washing said oligonucleotides with said wash solution; and D) cleaving said oligonucleotides at said anchor moieties to provide purified oligonucleotides; and ii) oligonucleotides which in part form duplexes with the oligonucleotides of step(i); b) mixing said oligonucleotides of (i) and (ii) with a DNA ligase in a ligation buffer; and c) recovering ligated DNA.
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Specification