Detectably and removably tagged nucleic acids

• US 6,544,738 B2
• Filed: 04/25/2000
• Issued: 04/08/2003
• 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:
  US09/558,971
• Publication Number:
  US 20020098482A1
• Time in Patent Office: 1,078 Days
• Field of Search: 536/22.1, 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. An isolated nucleic acid composition comprising:

A) a first oligonucleotide covalently linked to a removable tag, wherein the removable tag by a first photocleavable linker, is an amino acid, a peptide, a polymer, biotin, digoxygenin, a fluorescent dye or a dye impregnated bead, wherein the tag is detectable by a method selected from the group consisting of absorption spectra, antibody binding and optical sorting, and wherein at least a portion of the first oligonucleotide is complementary to a first region of a transcribed gene, and

B) a second oligonucleotide covalently linked to a selector tag by a second photocleavable linker, wherein at least a portion of the second oligonucleotide sequence is complementary to a second region of the transcribed gene, wherein the removable tag and the selector tag are distinguishable, wherein the first and the second photocleavable linker are cleavable at different wavelength and wherein the first oligonucleotide and the second oligonucleotide can be used as a primer pair to amplify a target region of the transcribed gene.

2. The composition of claim 1, wherein the selector tag is detectable by avidin/streptavidin or antibodies to digoxygenin.

3. The composition of claim 1, wherein the composition comprises 2 to 100,000 distinct oligonucleotide sequences.

4. The composition of claim 1, wherein the composition comprises 1000 to 100,000 distinct oligonucleotide sequences.

5. An isolated nucleic acid comprising a nucleic acid sequence complementary to the oligonucleotide sequence in the composition of claim 1.

13. The composition of claim 1, wherein the first oligonucleotide and the second oligonucleotide are designed to amplify the target region of the transcribed gene using the polymerase chain reaction.

6. An isolated nucleic acid composition comprising 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.

7. The composition of claim 6, wherein the first removable tag is an amino acid, a peptide, a polymer, biotin, digoxytenin, a florescent dye or a dye impregnated bead.

8. The composition of claim 6, wherein the first removable tag is detected by a method selected from the group consisting of absorption spectra, antibody binding and optical sorting.

9. The composition of claim 6, wherein the first removable tag is detectable by avidin/streptavidin or antibodies to digoxygenin.

10. The composition of claim 6, wherein the composition comprises 2 to 100,000 distinct oligonucleotide sequences.

11. The composition of claim 6, wherein the composition comprises 1000 to 100,000 distinct oligonucleotide sequences.

12. The composition of claim 6, wherein at least a portion of an oligonucleotide sequence is complementary to a region of a transcribed gene.

14. The composition of claim 6, wherein the first oligonucleotide and the second oligonucleotide can be used as a primer pair to amplify a target region of a transcribed gene.