Method for deblocking of labeled oligonucleotides

• US 9,085,797 B2
• Filed: 08/09/2013
• Issued: 07/21/2015
• Est. Priority Date: 05/24/1999
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Gebeyehu, Gulilat, Pires, Richard
• Primary Examiner(s): Crane, Lawrence E
• Application Number:
  US13/964,036
• Publication Number:
  US 20140066612A1
• Time in Patent Office: 711 Days
• US Class Current: 1/1
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• 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 1/68 involving nucleic acids

Abstract

The invention relates to a process for deblocking substantially a blocked, detectably labeled oligonucleotide by contacting the blocked detectably labeled oligonucleotide with an effective amount of a nucleophilic amino compound under conditions that result in substantial deblocking of the oligonucleotide, thereby giving the substantially deblocked oligonucleotide.

Claims

1. A process for deblocking substantially a blocked, detectably labeled oligonucleotide comprising contacting the blocked detectably labeled oligonucleotide with an effective amount of a nucleophilic amino compound under conditions that result in the deblocking of the oligonucleotide, thereby giving the substantially deblocked oligonucleotide.

2. The process of claim 1, wherein said detectable label is a fluorescent label.

3. The process of claim 2, wherein said detectable label is hexachlorofluorescein.

4. The process of claim 2, wherein said detectable label is DABCYL.

5. The process of claim 1, wherein said nucleophilic amino compound is ammonia.

6. The process of claim 5, wherein said ammonia is present at a psi of about 20 to 200.

7. The process of claim 5, wherein said ammonia is present at a psi of about 80.

8. The process of claim 1, wherein said nucleophilic amino compound is ammonia vapors.

9. The process of claim 1, wherein said nucleophilic amino compound is a C_1-6 alkylamine.

10. The process of claim 1, further comprising dissolving the substantially deblocked oligonucleotide in a buffer.

11. The process of claim 1, wherein said conditions comprise carrying the process at about room temperature to about 150°

C.

12. The process of claim 1, wherein said conditions comprise carrying the process at about 95°

C.

13. The process of claim 1, further comprising wherein said substantially blocked, detectably labeled oligonucleotide is immobilized on a solid phase.

14. The process of claim 13, further comprising wherein said substantially deblocked oligonucleotide is released from said solid phase under said conditions.

15. A process for deblocking substantially a blocked, detectably labeled oligonucleotide comprising contacting the blocked detectably labeled oligonucleotide with an effective amount of ammonia saturated with water vapor at about 80 psi, 95°

C. for about 45 min, thereby giving the substantially deblocked oligonucleotide.

16. The process of claim 15, further comprising wherein said blocked, detectably labeled oligonucleotide is immobilized on a solid phase.

17. The process of claim 16, further comprising wherein said substantially deblocked oligonucleotide is released from said solid phase.

18. A composition comprising a blocked, detectably labeled oligonucleotide and an effective amount of a nucleophilic amino compound sufficient to deblock substantially said oligonucleotide.

19. The composition of claim 18, wherein said detectable label is a fluorescent label.

20. The composition of claim 18, wherein said nucleophilic amino compound is ammonia.