Chemical preparation of high specific activity nucleotide probes
First Claim
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1. A chemical synthesis of a labeled nucleotide probe comprising the steps of:
- a) providing a first target recognition moiety comprising a nucleotide sequence of at least about 15 nucleotide bases, said sequence containing at least one 5'"'"' end and 3'"'"' end,b) chemically altering said first target recognition moiety to contain a reactive functionality at said 5'"'"' end, said 3'"'"' end or both, andc) providing a second signal generating moiety, having at least one terminus containing a functionality that is capable of reacting with at least one reactive functionality provided on said first target recognition moiety, andd) chemically reacting said first target recognition moiety now containing reactive functionality with said at least one terminus of said second signal generating moiety, to chemically join these two moieties and produce said labeled nucleotide probe;
wherein said reactive functionality of step (b) is a carboxyl group and said terminus of said signal generating moiety is a hydroxyl group.
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Abstract
Methods of chemical synthesis of labeled nucleotide probes are described. The methods comprise providing a first target recognition moiety comprising a nucleotide sequence of at least about 15 nucleotide bases including at least one 5'"'"' end and 3'"'"' end. The first target recognition moiety is chemically altered to contain a carboxyl group. A second signal generating moiety comprising a hydroxyl group is provided. The carboxyl group of the first target recognition moiety is chemically reacted with hydroxyl group of the second signal generating moiety to chemically join the two moieties and produce said labeled nucleotide probe.
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7 Claims
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1. A chemical synthesis of a labeled nucleotide probe comprising the steps of:
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a) providing a first target recognition moiety comprising a nucleotide sequence of at least about 15 nucleotide bases, said sequence containing at least one 5'"'"' end and 3'"'"' end, b) chemically altering said first target recognition moiety to contain a reactive functionality at said 5'"'"' end, said 3'"'"' end or both, and c) providing a second signal generating moiety, having at least one terminus containing a functionality that is capable of reacting with at least one reactive functionality provided on said first target recognition moiety, and d) chemically reacting said first target recognition moiety now containing reactive functionality with said at least one terminus of said second signal generating moiety, to chemically join these two moieties and produce said labeled nucleotide probe; wherein said reactive functionality of step (b) is a carboxyl group and said terminus of said signal generating moiety is a hydroxyl group. - View Dependent Claims (2, 3)
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4. A chemical synthesis for making a labeled nucleotide probe comprising the steps of:
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a) providing a first target recognition moiety comprising a nucleotide sequence of at least about 15 nucleotide bases, said sequence containing a 5'"'"' end with a free 5'"'"' group and a 3'"'"' end with a free 3'"'"' group; b) altering said first target recognition moiety with a phosphate group at said free 5'"'"' group of said 5'"'"' end, or said free 3'"'"' group of said 3'"'"' end, or said free 5'"'"' group of said 5'"'"' end and said free 3'"'"' group of said 3'"'"' end to form a phosphorylated target recognition moiety; c) activating the phosphate group of said phosphorylated target recognition moiety of step b) with imidazole and ethyl-dimethylamino-propyl carbodiimide; d) reacting said activated phosphate group of said phosphorylated target recognition moiety of step c) with epsilon-amino caproic acid to provide a carboxyl group at said 5'"'"' end or said 3'"'"' end of said target recognition moiety, wherein said carboxyl group is formed from the carboxyl group of epsilon-amino caproic acid; e) providing a second signal generating moiety comprising a nucleic acid sequence having at least one terminus and a length of from about 50 to about 200 bases, and further having at least one detectable label attached thereto wherein said terminus contains a hydroxyl group that is capable of reacting with said carboxyl group provided on said first target recognition moiety; f) chemically reacting said carboxyl group on said first target recognition moiety with said hydroxyl group on said second signal generating moiety to covalently join said first target recognition moiety and said second signal generating moiety thereby producing said labeled nucleotide probe. - View Dependent Claims (5, 6, 7)
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Specification