Photolabile reagents for incorporation into oligonucleotide chains

• US 5,258,506 A
• Filed: 08/25/1989
• Issued: 11/02/1993
• Est. Priority Date: 10/16/1984
• Status: Expired due to Term

• Current Assignee: Chiron Diagnostics Corp (Bayer AG)
• Original Assignee: Chiron Corporation (Novartis Ag)
• Inventors: Urdea, Michael S., Horn, Thomas
• Primary Examiner(s): Yarbrough, Amelia B.
• Application Number:
  US07/398,711
• Time in Patent Office: 1,530 Days
• Field of Search: 435/6, 435/91, 935/77, 935/78, 436/518, 436/527, 436/532, 436/501, 536/27, 536/23.1, 536/25.32
• US Class Current: 536/23.1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site

Abstract

Novel photolabile photochemical reagents are disclosed. The reagents are useful in a variety of biochemical and chemical contexts, including nucleic hybridization assays and chemical phosphorylation of hydroxyl-containing compounds. The reagents are particularly useful for introducing cleavable sites into oligonucleotide or polynucleotide chains, i.e., sites which are cleavable upon photolysis. The reagents are also useful in both 5'"'"'- and 3'"'"'-phosphorylation of oligonucleotide or polynucleotide chains.

Claims

1. A polynucleotide reagent having the structure ##STR7## wherein DNA₁ is a first segment of DNA;

DNA₂ is a second segment of DNA; and

one of x and y is zero while the other is an integer in the range of 1 to 12 inclusive.

2. The polynucleotide reagent of claim 1, wherein x is zero.

3. The polynucleotide reagent of claim 2, wherein y is an integer in the range of 1 to 4 inclusive.

4. The polynucleotide reagent of claim 3, wherein y is one.

5. The polynucleotide reagent of claim 1, wherein y is zero.

6. The polynucleotide reagent of claim 5, wherein x is an integer in the range of 1 to 4 inclusive.

7. The polynucleotide reagent of claim 6, wherein x is one.

8. A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, said method. comprising:

combining under hybridizing conditions said nucleic acid sample with the polynucleotide reagent of claim 1, wherein one of said sample or said reagent is bound to a support and hybridization of said analyte and said polynucleotide reagent results in a label being bound to said support through the cleavage site ##STR8## substantially freeing said support of label bound to said support other than through said selectable cleavage site;

cleaving said cleavage site via photolysis using light having a wavelength of at least about 350 nm; and

detecting label free of said support.

9. A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, said method comprising:

combining under hybridizing conditions in an aqueous medium, said nucleic acid sample with the polynucleotide reagent of claim 1, where one of said sample or a component of said reagent is bound to a support and hybridization of said analyte and said polynucleotide reagent results in a label being bound to said support through the cleavage site ##STR9## separating said support having bound polynucleotide reagent and nucleic acid analyte from said aqueous medium;

washing said support with a medium of different hybridizing stringency from said aqueous medium to remove label bound to said support other than through said cleavage site;

cleaving said cleavage site via photolysis using light having a wavelength of at least about 350 nm; and

detecting label free of said support.