High specific activity nucleic acid probes having target recognition and signal generating moieties

• US 5,324,829 A
• Filed: 12/16/1988
• Issued: 06/28/1994
• Est. Priority Date: 12/16/1988
• Status: Expired due to Term

• Current Assignee: Ortho Diagnostic Systems, Inc. (The Carlyle Group LP (Corporate Private Equity))
• Original Assignee: Ortho Diagnostic Systems, Inc. (The Carlyle Group LP (Corporate Private Equity))
• Inventors: Mendoza, Leopoldo, Bahl, Chander
• Primary Examiner(s): Parr, Margaret
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/285,274
• Time in Patent Office: 2,020 Days
• Field of Search: 435/6, 435/91, 536/27, 536/22.1, 536/23.1, 536/25.3, 536/24.2, 536/25.4, 935/17, 935/77, 935/78
• US Class Current: 536/23.1
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

The probes of the invention are made up of a target recognition moiety and a signal generation moiety. In the first step of the preparatory method of the invention, the 5'"'"' or 3'"'"' terminus or both of the target recognition moiety is chemically altered to incorporate a reactive functionality which will allow chemical linkage of this target recognition moiety with one or both termini of the signal generation moiety of the probe. Suitable functionalities vary widely, but may, in general be described as those that allow chemical attachment of the two moieties either at the 5'"'"' or 3'"'"' end of the target recognition moiety. The two moieties should not detach from each other upon hybridization of the target moiety to the target analyte. Thus, when the probe is hybridized to target analyte, it will then carry with it, signal moiety, to label this complex, which label is then later detected.

Claims

1. A labeled nucleotide probe formed according to the chemical synthesis having the following steps:

a) providing a first target recognition moiety comprising a nucleotide sequence of at least about 15 nucleotide bases, said sequence containing a 5'"'"' end and a 3'"'"' end;

b) altering said first target recognition moiety with a phosphate group at said 5'"'"' end, said 3'"'"' end, or both 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 of 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.

2. A probe according to claim 1 wherein said signal generating moiety is labeled with a hapten.

3. A probe according to claim 2 wherein said hapten is biotin.

4. A probe according to claim 2 wherein said hapten is fluorescein.