Method for detecting oligonucleotides using UV light source

• US 6,218,124 B1
• Filed: 08/27/1999
• Issued: 04/17/2001
• Est. Priority Date: 08/27/1999
• Status: Expired due to Term

• Current Assignee: Perkinelmer Incorporated, Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: PE Corp.
• Inventors: Lee, Linda G.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/385,230
• Time in Patent Office: 599 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24.3
• US Class Current: 435/6.12
• 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 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: G01N 21/33 using ultraviolet light G01N21/39 takes precedence

Abstract

A method for detecting oligonucleotides is provided and comprises forming a series of different sized oligonucleotides labeled with an energy transfer dye; separating the series of labeled oligonucleotides based on size; and detecting the separated labeled oligonucleotide by exposing the oligonucleotides to light having a wavelength between about 250 and 450 nm and measuring light emitted by the energy transfer dye at a wavelength greater than about 500 nm.

Claims

1. A method for detecting an oligonucleotide comprising:

forming a series of different sized oligonucleotides labeled with an energy transfer dye;

separating the series of labeled oligonucleotides based on size; and

detecting the separated labeled oligonucleotide by exciting a donor dye of the energy transfer dye using light having a wavelength between 250 and 450 nm and measuring light emitted by an acceptor dye of the energy transfer dye at a wavelength greater than 500 nm.

2. The method of claim 1, wherein exciting the donor dye of the energy transfer dye is performed using light having a wavelength between 300 and 450 nm.

3. The method of claim 1, wherein exciting the donor dye of the energy transfer dye is performed using light having a wavelength between 350 and 400 nm.

4. The method of claim 1, wherein the light emitted by the acceptor dye of the energy transfer dye has a wavelength greater than 550 nm.

5. The method of claim 1, wherein the light emitted by the acceptor dye of the energy transfer dye has a wavelength between 500 and 700 nm.

6. The method of claim 1, wherein the light emitted by the acceptor dye of the energy transfer dye has a wavelength at least 150 nm greater than the wavelength of the light used to excite the donor dye.

7. The method of claim 1, wherein the acceptor dye is a member of a class of dyes selected from the group consisting of fluorescein, rhodamine, asymmetric benzoxanthene, xanthene, cyanine, phthalocyanine and squaraine dyes.

8. The method of claim 1, wherein the donor dye is a member of a class of dyes having a coumarin or pyrene ring structure.

9. The method of claim 1, wherein the energy transfer dye includes a linker attaching the donor dye to the acceptor dye.

10. A method for sequencing a nucleic acid sequence comprising:

forming a mixture of extended primers by hybridizing a nucleic acid sequence with an oligonucleotide primer in the presence of deoxynucleoside triphosphates, at least one dideoxynucleoside triphosphate labeled with an energy transfer dye, and a DNA polymerase, the DNA polymerase extending the primer with the deoxynucleoside triphosphates until a fluorescently labeled dideoxynucleoside triphosphate is incorporated onto the extended primer which terminates extension of the primer;

separating the mixture of extended primers; and

determining the sequence of the nucleic acid sequence by detecting the extended primer by exciting a donor dye of the energy transfer dye using light having a wavelength between 250 and 450 nm and measuring light emitted by an acceptor dye of the energy transfer dye at a wavelength greater than 500 nm.

11. The method of claim 10, wherein exciting the donor dye of the energy transfer dye is performed using light having a wavelength between 300 and 450 nm.

12. The method of claim 10, wherein exciting the energy transfer dye is performed using light having a wavelength between 350 and 400 nm.

13. The method of claim 10, wherein the light emitted by the acceptor dye of the energy transfer dye has a wavelength greater than 550 nm.

14. The method of claim 10, wherein the light emitted by the acceptor dye of the energy transfer dye has a wavelength between 500 and 700 nm.

15. The method of claim 10, wherein the light emitted by the acceptor dye of the energy transfer dye has a wavelength at least 150 nm greater than the wavelength of the light used to excite the donor dye.

16. The method of claim 10, wherein the acceptor dye is a member of a class of dyes selected from the group consisting of fluorescein, rhodamine, asymmetric benzoxanthene, xanthene, cyanine, phthalocyanine and squaraine dyes.

17. The method of claim 10, wherein the donor dye is a member of a class of dyes having a coumarin or pyrene ring structure.

18. The method of claim 10, wherein the energy transfer dye includes a linker attaching the donor dye to the acceptor dye.

19. A method for sequencing a nucleic acid sequence comprising:

forming a mixture of extended labeled primers by hybridizing a nucleic acid sequence with an oligonucleotide primer labeled with an energy transfer dye, in the presence of deoxynucleoside triphosphates, at least one dideoxynucleoside triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxynucleoside triphosphates until a dideoxynucleoside triphosphate is incorporated which terminates extension of the primer;

separating the mixture of extended primers; and

determining the sequence of the nucleic acid sequence by detecting the extended primer by exciting a donor dye of the energy transfer dye using light having a wavelength between 250 and 450 nm and measuring light emitted by an acceptor dye of the energy transfer dye at a wavelength greater than 500 nm.

20. The method of claim 19, wherein exciting the donor dye of the energy transfer dye is performed using light having a wavelength between 300 and 450 nm.

21. The method of claim 19, wherein exciting the donor dye of the energy transfer dye is performed using light having a wavelength between 350 and 400 nm.

22. The method of claim 19, wherein the light emitted by the acceptor dye of the energy transfer dye has a wavelength greater than 550 nm.

23. The method of claim 19, wherein the light emitted by the acceptor dye of the energy transfer dye has a wavelength between 500 and 700 nm.

24. The method of claim 19, wherein the light emitted by the acceptor dye of the energy transfer dye has a wavelength at least 150 nm greater than the wavelength of the light used to excite the donor dye.

25. The method of claim 19, wherein the acceptor dye is a member of a class of dyes selected from the group consisting of fluorescein, rhodamine, asymmetric benzoxanthene, xanthene, cyanine, phthalocyanine and squaraine dyes.

26. The method of claim 19, wherein the donor dye is a member of a class of dyes having a coumarin or pyrene ring structure.

27. The method of claim 19, wherein the energy transfer dye includes a linker attaching the donor dye to the acceptor dye.