Covalent attachment of nucleic acid molecules onto solid-phases via disulfide bonds
First Claim
1. A method for attaching a modified nucleic acid molecule to a coated solid-phase via a disulfide bond, said modified nucleic acid molecule being modified such that a 3'"'"' carbon atom or a 5'"'"' carbon atom of a sugar moiety of a nucleotide of said nucleic acid molecule possesses a sulfhydryl group or a disulfide group, said method comprising coupling said modified nucleic acid molecule to said coated solid-phase, wherein where said modified nucleic acid molecule is a modified nucleic acid molecule having a sulfhydryl group, said coated solid-phase is coated with a disulfidesilane, and where said modified nucleic acid molecule is a modified nucleic acid molecule having a disulfide group said coated solid-phase is coated with a mercaptosilane and wherein said disulfide bond is formed between a sulfur atom of either said sulfhydryl group or said disulfide group and a sulfur atom of either said disulfidesilane or said mercaptosilane.
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Abstract
Methods for the covalent, specific and reversible immobilization of nucleic acid molecules onto solid-phases by means of a reversible disulfide bond for nucleic acid molecule array preparation are described. These methods can be used to prepare reusable nucleic acid molecule arrays with hilgh specificity and high efficiency.
106 Citations
31 Claims
- 1. A method for attaching a modified nucleic acid molecule to a coated solid-phase via a disulfide bond, said modified nucleic acid molecule being modified such that a 3'"'"' carbon atom or a 5'"'"' carbon atom of a sugar moiety of a nucleotide of said nucleic acid molecule possesses a sulfhydryl group or a disulfide group, said method comprising coupling said modified nucleic acid molecule to said coated solid-phase, wherein where said modified nucleic acid molecule is a modified nucleic acid molecule having a sulfhydryl group, said coated solid-phase is coated with a disulfidesilane, and where said modified nucleic acid molecule is a modified nucleic acid molecule having a disulfide group said coated solid-phase is coated with a mercaptosilane and wherein said disulfide bond is formed between a sulfur atom of either said sulfhydryl group or said disulfide group and a sulfur atom of either said disulfidesilane or said mercaptosilane.
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2. A method for attaching a nucleic acid molecule to a solid-phase via a disulfide bond, said nucleic acid molecule being modified such that a 3'"'"' carbon atom or a 5'"'"' carbon atom of a sugar moiety of a nucleotide of said nucleic acid molecule possesses a sulthydryl group or a disulfide group, said method comprising the steps of:
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(A) coating the surface of the solid-phase with a mercaptosilane or a disulfidesilane, wherein said mercaptosilane comprises a sulfliydryl group and said disulfidesilane comprise a disulfide group; and (B) (i) coupling a sulfhydryl modified nucleic acid molecule to the disulfide group of said disulfidesilane by means of a covalent disulfide bond;
or(ii) coupling a disulfide modified nucleic acid molecule to the sulfhydryl groups of said mercaptosilane by means of a covalent disulfide bond; wherein said disulfide bond is formed between a sulfur atom of either said sulfhydryl group or said disulfide group and a sulfur atom of either said disulfidesilane or said mercaptosilane. - View Dependent Claims (17, 18, 19, 23)
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31. A modified nucleic acid molecule coupled via a disulfide bond to a coated solid-phase, said modified nucleic acid molecule being modified such that a 3'"'"' carbon atom or a 5'"'"' carbon atom of a sugar moiety of a nucleotide of said nucleic acid molecule possesses a sulfhydryl group or a disulfide group, wherein where said modified nucleic acid molecule is a modified nucleic acid molecule having a sulfhydryl group, said coated solid-phase is coated with a disulfidesilane, and where said modified nucleic acid molecule is a modified nucleic acid molecule having a disulfide group said coated solid-phase is coated with a mercaptosilane, and wherein said disulfide bond is formed between a sulfur atom of either said sulfhydryl group or said disulfide group and a sulfur atom of either said disulfidesilane or said mercaptosilane.
Specification