Polynucleotide hybridization probes

• US 4,840,892 A
• Filed: 04/15/1985
• Issued: 06/20/1989
• Est. Priority Date: 05/15/1984
• Status: Expired due to Fees

• Current Assignee: Smithkline Beecham Corporation (Smithkline Beecham PLC)
• Original Assignee: Smithkline Beecham Corporation (Smithkline Beecham PLC)
• Inventors: Adams, Craig W., Leary, Jeffry J., Rosenberg, Martin
• Primary Examiner(s): Kepplinger, Esther M.
• Application Number:
  US06/723,388
• Time in Patent Office: 1,527 Days
• Field of Search: 435/6, 435/8, 435/9, 435/172.3, 435/810, 435/91, 435/5, 436/501, 436/518, 935/1, 935/78, 935/82, 935/22, 935/3, 935/6, 935/9, 935/78, 536/27
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983

Abstract

Polynucleotide hybridization probes are labeled in non-probe regions but not in probe regions by selectively protecting probe regions prior to modification of the polynucleotide.

Claims

1. A method for preparing a polynucleotide hybridization probe which comprises selectively protecting a probe region within a single stranded polynucleotide having a probe region and a non-probe region both 5'"'"' and 3'"'"' to the probe region by hybridizing the probe region to a complementary sequence and then chemically modifying the non-probe region of the remaining single-stranded portion of the polynucleotide.

2. The method of claim 1 in which the single stranded polynucleotide is bound, directly or indirectly, to a solid support prior to selectively protecting the probe region.

3. The method of claim 1 which comprises modifying the probe by transamination after selectively protecting the probe region.

4. The method of claim 1 which comprises modifying the probe by biotinylation after selectively protecting the probe region.

5. The method of claim 4 in which the modified residues are labelled with a label comprising avidin conjugated to a detectable moiety.

6. The method of claim 1 in which the probe region is complementary to a human DNA or RNA sequence or to a characteristic nucleic acid sequence of a human pathogen or of a class of human pathogens.

7. The method of claim 1 in which the probe region is complementary to a non-human DNA or RNA sequence or to a characteristic nucleic acid sequence of a non-human pathogen or of a class of non-human pathogens.

8. The method of claim 1 in which the probe region is complementary to a DNA or RNA sequence characteristic of a host cell or of a vector used in the manufacture of a recombinant DNA product.

9. The method of claim 1 in which the probe is single stranded phage DNA or RNA having a probe region therein.

10. The method of claim 9 in which the single stranded phage DNA or RNA is M13 DNA.

11. The method of claim 10 in which the single stranded polynucleotide is bound, directly or indirectly, to a solid support prior to selectively protecting the probe region.

12. The method of claim 10 which comprises modifying the probe by transamination after selectively protecting the probe region.

13. The method of claim 10 which comprises modifying the probe by biotinylatio after selectively protecting the probe region.

14. The method of claim 13 in which the modified residues are labelled with a label comprising avidin conjugated to a detectable moiety.

15. The method of claim 10 in which the probe region is complementary to a human DNA or RNA sequence or to a characteristic nucleic acid sequence of a human pathogen or of a class of human pathogens.

16. The method of claim 10 in which the probe region is complementary to a non-human DNA or RNA sequence or to a characteristic nucleic acid sequence of a non-human pathogen or of a class of non-human pathogens.

17. The method of claim 10 in which the probe region is complementary to a DNA or RNA sequence characteristic of a host cell or of a vector used in the manufacture of a recombinant DNA product.