Method of detecting a nucleic acid

• US 6,562,567 B2
• Filed: 01/27/1999
• Issued: 05/13/2003
• Est. Priority Date: 01/27/1998
• Status: Expired due to Fees

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Wold, Barbara J.
• Primary Examiner(s): Benzion, Gary
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/238,528
• Publication Number:
  US 20020098478A1
• Time in Patent Office: 1,567 Days
• Field of Search: 435/6, 435/91.2, 935/77, 935/78, 536/23.1, 536/25.32
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

Disclosed is a method of detecting specific nucleic acids using an oligonucleotide linked to a cleavable tag. The presence of a specific nucleic acid in a population of nucleic acids is determined by hybridizing an oligonucleotide containing the tag to a population of nucleic acids, separating hybridizing bound oligonucleotides, and then removing and identifying the tag. Also provided are compositions and kits comprising oligonucleotides linked to a cleavable tag.

Claims

1. A method for detecting a first target nucleic acid molecule and a second target nucleic acid molecule in a collection of at least two nucleic acid molecules having different nucleotide sequences, the method comprising:

providing a first oligonucleotide covalently linked to a first removable tag by a first photocleavable linker, and a second oligonucleotide covalently linked to a second removable tag by a second photocleavable linker, wherein the first and second photocleavable linkers are cleavable at different wavelengths;

contacting the first oligonucleotide and the second oligonucleotide with the collection of nucleic acid molecules in a single reaction under conditions which permit hybridization of the first oligonucleotide and the second oligonucleotide to complementary sequences in the collection of nucleic acid molecules, thereby forming a mixture of hybridized and non-hybridized oligonucleotides;

separating hybridized oligonucleotides from non-hybridized oligonucleotides;

removing the first tag from hybridized first oligonucleotides by irradiating the mixture of hybridized and non-hybridized oligonucleotides at a first wavelength, thereby cleavinthe first photocleavable linker;

identifying the first tag by its known chemical property, thereby detecting the first target nucleic acid molecule;

removing the second tag from hybridized second oligonucleotides by irradiating the mixture of hybridized and non-hybridized oligonucleotides at a second wavelength, thereby cleaving the second photocleavable linker; and

identifying the second tag by its known chemical property, thereby detecting the second target nucleic acid molecule in the collection of nucleic acid molecules.

2. The method of claim 1, wherein the contacting step is performed in solution.

3. The method of claim 1, wherein the contacting step is performed with the first oligonucleotide and the second oligonucleotide being attached to a solid support, or with the collection of nucleic acid molecules being attached to a solid support.

4. The method of claim 1, wherein the first oligonucleotide is about 8 to about 50 nucleotides in length.

5. The method of claim 1, wherein the first oligonucleotide is about 20 to about 40 nucleotides in length.

6. The method of claim 1, wherein the collection of nucleic acid molecules is selected from the group consisting of RNA and cDNA.

7. The method of claim 1, wherein the tag is identified from a method selected from the group consisting of mass spectra, emission spectra, absorption spectra, and antibody-binding.

8. The method of claim 1, wherein the tag is identified using a mass spectrophotometric method selected from the group consisting of time-of-flight, quadrupole, magnetic sector and ion trap mass spectrometry.

10. The method of claim 1, wherein the amount of the identified first tag determines quantitatively the presence of the first target nucleic acid molecule identified from the collection of nucleic acids.

11. The method of claim 1, wherein the first removable tag is an amino acid, a peptide, a polymer, biotin, digoxygenin, a fluorescent dye or a dye impregnated bead.

12. The method of claim 1, where the first tag is identified by a method selected from the group consisting of absorption spectra, antibody binding and optical sorting.

13. The method of claim 1, wherein the first tag and the second tag are identical.

14. The method of claim 1, wherein the first tag and the second tag are different.

15. The method of claim 1, wherein the first oligonucleotide and the second oligonucleotide are complementary to a coding region of a transcribed gene.

16. The method of claim 1, wherein the first oligonucleotide and the second oligonucleotide are complementary to a coding region of the same transcribed gene.

9. A kit useful for detection of the presence of a specific nucleic acid sequence in a collection of nucleic acid sequences, the kit comprising one or more containers comprising a first container containing a first isolated nucleic acid composition comprising a first oligonucleotide sequence covalently linked to a first removable tag by a first photocleavable linker and a second container containing a second oligonucleotide sequence covalently linked to a second removable tag by a second photocleavable linker, wherein the first and the second photocleavable linkers are cleavable at different wavelengths and a distinct correlation between the first photocleavable linker and the sequence of the first oligonucleotide is known, and a distinct correlation between the second photocleavable linker and the sequence of the second oligonucleotide is known.