Producing, cataloging and classifying sequence tags

• US 8,114,596 B2
• Filed: 10/09/2009
• Issued: 02/14/2012
• Est. Priority Date: 06/02/2004
• Status: Active Grant

• Current Assignee: Joseph C. Kaufman
• Original Assignee: Joseph C. Kaufman
• Inventors: Kaufman, Joseph C.
• Primary Examiner(s): Kapushoc, Stephen
• Assistant Examiner(s): BHAT, NARAYAN KAMESHWAR
• Application Number:
  US12/576,287
• Publication Number:
  US 20100055745A1
• Time in Patent Office: 858 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.2, 435/91.52, 536/23.1, 536/24.2
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

The described method provides, methods, and kits to produce, identify, catalog and classify a comprehensive collection of nucleic acid targets produced from a nucleic acid sample. The method, referred to as Cataloging and Classification of Sequence Tags, involves generating a set of target nucleic acid fragments; coupling the target nucleic acid fragments to a nucleic acid bridge comprising, for example, two or more primer binding sites and two recognition sites for cleavage at a site offset from the recognition site to the fragment'"'"'s end; and cleaving the fragments to generate chimeric nucleic acids of known length. The nucleic acid bridge is thus disposed between the two nucleic acid fragments in the chimeric nucleic acid. The resulting duplex nucleic acids comprise a set of sequence tags (i.e., by amplification using universal primers), comprising an addressable portion, a target nucleic portion and a portion of the nucleic acid bridge. Single-stranded or partial duplex sequence tags may be captured by coupling to a complementary capture probe. Capture probe-sequence tag hybrids, may be detected employing a labeled detector probe. The method allows a complex sample of nucleic acids to be cataloged in a reproducible and sequence-specific manner. The method further provides methods for analysis of the above sample to classify the sequence tags; determine the presence and relative amounts of sequences of interest; derive expressed genes signatures and differential gene expression signatures; and identify putative expressed sequence tags (EST).

Claims

1. A method of forming a population of nucleic acid sequence tags,

comprising:

covalently coupling a first linear target nucleic acid having two free ends to a first end of a first nucleic acid bridge and covalently coupling a second linear target nucleic acid having two free ends to a second end of the first nucleic acid bridge to form a linear chimeric nucleic acid intermediate, wherein the first nucleic acid bridge is disposed between the first linear target nucleic acid and the second linear target nucleic acid,

cleaving the linear chimeric nucleic acid intermediate to form a shortened linear chimeric nucleic acid intermediate; and

producing from the shortened linear chimeric nucleic acid intermediate a first sequence tag and a second sequence tag, wherein the first nucleic acid bridge comprises a binding site for a first primer and a binding site for a second primer, and wherein producing comprises amplifying the shortened linear chimeric nucleic acid intermediate in the presence of the first primer, the second primer, or both, to form the first sequence tag, the second sequence tag, or both,

wherein the first sequence tag comprises at least a portion of the first target nucleic acid and at least a portion of the first nucleic acid bridge, and wherein the second sequence tag comprises at least a portion of the second target nucleic acid and at least a portion of the first nucleic acid bridge, wherein the at least a first portion of the first target nucleic acid comprises a first addressable portion and wherein the at least a first portion of the second target nucleic acid comprises a second addressable portion;

wherein the first linear target nucleic acid and the second linear target nucleic acid are not covalently linked until coupling to the nucleic acid bridge, and wherein the first target nucleic acid and the second target nucleic acid are produced from the same nucleic acid sample,

wherein the first nucleic acid bridge comprises 2 restriction endonuclease recognition sequences for endonucleases that cleave at a sequence outside of the nucleic acid bridge, wherein the cleavage sequences are outside of opposite ends of the bridge, wherein the 2 restriction endonuclease recognition sequences are separated by a plurality of nucleotides,

wherein the first sequence tag and the second sequence tag comprise at least a first bridge portion,

wherein the first sequence tag and the second sequence tag are suitable for simultaneous detection without requiring pooling, sorting, or adapter mediated indexing, and wherein the first and second addressable portions have a fixed length of 2 to 30 nucleotides.

2. The method of claim 1, further comprising producing the first target nucleic acid and the second target nucleic acid by cleaving a nucleic acid sample with a first cleaving agent.

3. The method of claim 1, wherein the first nucleic acid bridge comprises a first cleaving agent recognition site and a second cleaving agent recognition site.

4. The method of claim 3, further comprising, prior to producing,

cleaving the linear chimeric nucleic acid intermediate with a second cleaving agent to produce a shortened linear chimeric nucleic acid intermediate, wherein the second cleaving agent is one that cleaves at a site disposed greater than one nucleotide offset from its binding site.

5. The method of claim 1, wherein the first and second sequence tags are single-stranded and wherein the first single-stranded sequence tag, the second single-stranded sequence tag, or both, is isolated by coupling a nucleic acid comprising a region of complementarity with a sequence tag, and removing the coupled duplex structure formed with the first sequence tag, the second sequence tag, or both.

6. The method of claim 1, further comprising detecting, quantifying, amplifying, sequencing, cataloging, classifying, or a combination comprising one or more of the foregoing, the first sequence tag, the second sequence tag, or a combination comprising one or more of the foregoing sequence tags.

7. The method of claim 6, comprising detecting and quantifying the first sequence tag, the second sequence tag, or a combination comprising one or more of the foregoing sequence tags.

8. The method of claim 1, further comprising:

coupling a population of capture probes to an end of the population of sequence tags to form a population of capture probe-sequence probe hybrid molecules.

9. The method of claim 8, wherein the capture probe is coupled to the hydrophilic portion of a polymeric micelle.

10. The method of claim 8, further comprising incubating the population of capture probe-sequence probe hybrid molecules with a population of first labeled detector probes, wherein the population of first labeled detector probe is capable of hybridizing to the population of capture probe-sequence probe hybrid molecules.

11. The method of claim 8, further comprising, prior to coupling, contacting the sequence tag with a reagent suitable to produce a population of sequence tags having reduced reactivity.

12. The method of claim 1, wherein the population of sequence tags comprises a catalog of sequence tags for a nucleic acid sample.

13. The method of claim 1, further comprising cataloging the population of sequence tags.

14. The method of claim 1, further comprising classifying the population of sequence tags.

15. The method of claim 1, wherein the population of sequence tags comprises a direct association between a sequence tag and at least one classifier in a classifier database,

wherein a classifier database comprises biological classifiers, semantic entity classifiers, sequence tag signature classifier, differential signature classifier, semantic entities classifiers, or a combination comprising one or more of the foregoing classifiers.

16. The method of claim 1, wherein the first addressable portion consists of 2 to 30 nucleotides, and the second addressable portion consists of 2 to 30 nucleotides.