Polynucleotide reagents containing selectable cleavage sites

• US 5,380,833 A
• Filed: 12/13/1991
• Issued: 01/10/1995
• Est. Priority Date: 10/16/1984
• Status: Expired due to Fees

• Current Assignee: Chiron Corporation (Bayer AG)
• Original Assignee: Chiron Corporation (Bayer AG)
• Inventors: Urdea, Michael S.
• Primary Examiner(s): Parr, Margaret
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/806,642
• Time in Patent Office: 1,124 Days
• Field of Search: 435/6, 435/810, 435/91, 435/91.1, 435/91.2, 436/501, 536/25-27, 536/22.1, 536/25.3, 536/23.1, 536/25.4, 536/24.1, 536/24.2, 536/24.3-24.33, 935/7, 935/9, 935/88, 935/78
• US Class Current: 536/22.1
• 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: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Novel methods for assaying a nucleic acid analyte are provided, which employ polynucleotides having oligonucleotide sequences substantially homologous to a sequence of interest in the analyte, where the presence or absence of hybridization at a predetermined stringency provides for the release of a label from a support. Particularly, various techniques are employed for binding a label to a support, whereupon cleavage of either a single or double strand, a label may be released from a support, where the release of the label can be detected as indicative of the presence of a particular oligonucleotide sequence in a sample. The method finds use in diagnosis of disease, genetic monitoring, and analysis of nucleic acid mixtures.

Claims

1. A DNA probe useful for detecting the presence of an oligonucleotide containing a sequence of interest, comprising a polydeoxyribonucleotide reagent bound proximal at one end to a support and at its opposite end having a sequence complementary to said sequence of interest, wherein a selectable cleavage site X is present within the polynucleotide reagent, and further wherein the selectable cleavage site X (a) is chemically cleavable;

(b) is other than a restriction enzyme cleavable site;

(c) is other than a phosphodiester linkage; and

(d) provides for a complete break between adjacent nucleotides in the polynucleotide reagent upon cleavage.

2. A polynucleotide reagent having the structure ##STR9## wherein DNA₁ is a first strand of DNA, DNA₂ is a second strand of DNA, and X comprises a selectable cleavage site which:

(a) is chemically cleavable;

(b) is other than a restriction enzyme clearable site;

(c) is other than a phosphodiester linkage; and

(d) provides for a complete break between adjacent nucleotides in the reagent upon cleavage.

3. The polynucleotide reagent of claim 2, wherein X comprises a linkage cleavable by a reagent selected from the group consisting of OH^-, hydroxylamine, SH^-, or periodate.

4. The polynucleotide reagent of claim 2, wherein X comprises a linkage selected from the group consisting of ##STR10##

5. The polynucleotide reagent of claim 2, wherein X contains the moiety ##STR11##

6. The polynucleotide reagent of claim 2, wherein X is ##STR12##

9. The polynucleotide reagent of claim 3, wherein X is cleavable by OH^-.

10. The polynucleotide reagent of claim 3, wherein X is cleavable by hydroxylamine.

11. The polynucleotide reagent of claim 3, wherein X is cleavable by SH^-.

12. The polynucleotide reagent of claim 3, wherein X is clearable by periodate.

13. The polynucleotide reagent of claim 9, wherein X comprises ##STR15##

14. The polynucleotide reagent of claim 9, wherein X comprises ##STR16##

15. The polynucleotide reagent of claim 10, wherein X comprises ##STR17##

16. The polynucleotide reagent of claim 11, wherein X comprises

17. The polynucleotide reagent of claim 12, wherein X comprises ##STR18##

7. A reagent useful in polynucleotide synthesis, having the structure ##STR13## wherein R¹ is an acid-sensitive, base-stable protecting group, and R² is selected from the group consisting of H, phosphoramidite, phosphotriester, phosphodiester, phosphite, H-phosphonate, and phosphonothioate.

8. The reagent of claim 7, wherein R¹ is DMT and R² is ##STR14## where iPr is isopropyl.