Nucleic acid constructs and methods of use

• US 9,783,847 B2
• Filed: 05/27/2015
• Issued: 10/10/2017
• Est. Priority Date: 04/30/2009
• Status: Active Grant

• Current Assignee: Prognosys Biosciences Inc.
• Original Assignee: Prognosys Biosciences Inc.
• Inventors: Chee, Mark S.
• Primary Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US14/723,332
• Publication Number:
  US 20160024576A1
• Time in Patent Office: 867 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• 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 2539/103 Serial analysis of gene expression [SAGE]
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

The present invention provides oligonucleotide constructs, sets of such oligonucleotide constructs, and methods of using such oligonucleotide constructs to provide validated sequences or sets of validated sequences corresponding to desired ROIs. Such validated ROIs and constructs containing these have a wide variety of uses, including in synthetic biology, quantitative nucleic acid analysis, polymorphism and/or mutation screening, and the like.

Claims

1. A set of nucleic acid constructs from an individual, wherein

a) each construct of the set comprises:

a naturally occurring nucleic acid region of interest (ROI) from the individual;

at least one unique identifier attached to the ROI that distinguishes the construct from other constructs in the set with a same ROI, wherein the at least one unique identifier is not part of its attached ROI; and

at least one amplification site; and

b) the set of constructs comprises;

(i) at least two constructs with the same ROI and different unique identifiers, and (ii) two or more different ROIs.

2. The set of claim 1, wherein one or more of the constructs comprise two or more amplification sites flanking the ROI and the unique identifier.

3. The set of claim 1, wherein one or more of the constructs comprise a single amplification site adjacent to the ROI and the unique identifier.

4. The set of claim 1, wherein one or more of the constructs comprise one or more excision sites.

5. The set of claim 4, wherein one or more of the constructs comprise at least two excision sites flanking the ROI.

6. The set of claim 4, wherein one or more of the constructs comprise a single excision site adjacent to the ROI.

7. The set of claim 1, wherein one or more of the constructs are immobilized on a solid support.

8. The set of claim 7, wherein the solid support comprises a bead.

9. The set of claim 1, wherein the set comprises at least five constructs with the same ROI and different unique identifiers.

10. The set of claim 1, wherein the set comprises at least five constructs with the same ROI, different unique identifiers, and at least one excision site.

11. The set of claim 1, wherein one or more of the unique identifiers comprise a degenerate nucleic acid sequence.

12. The set of claim 1, wherein the at least one unique identifier comprises at least 10 nucleotides.

13. The set of claim 1, wherein the ROI comprises DNA.

14. The set of claim 1, wherein the ROI comprises RNA.

15. The set of claim 13, wherein the DNA is genomic DNA.

16. The set of claim 1, wherein one or more of the constructs is double-stranded.

17. The set of claim 1, wherein one or more of the constructs is single-stranded.

18. The set of claim 1, wherein one or more of the constructs comprise at least two unique identifier sequences.

19. The set of claim 2, wherein the two or more amplification sites are different from one another.

20. The set of claim 2, wherein the two or more amplification sites are the same.

21. The set of claim 2, wherein the amplification site that is opposite to the unique identifier with respect to the ROI is separate from an adjacent excision site.

22. The set of claim 2, wherein the amplification site that is opposite to the unique identifier with respect to the ROI comprises an excision site.

23. The set of claim 1, wherein the at least one amplification site comprises a universal priming site.

24. The set of claim 1, wherein the ROI comprises a rare sequence variant, a low-frequency mutation, a minor allelic variant, a polymorphism, a mutation, or a haplotype.

25. The set of claim 1, wherein the unique identifier is from a pre-defined set of sequences.

26. The set of claim 1, wherein the unique identifier is from a degenerate set of sequences.

27. The set of claim 1, wherein at least one construct of the set of constructs is enriched.

28. The set of claim 1, wherein each construct comprises two unique identifier sequences.

29. The set of claim 28, wherein the two unique identifiers are attached to one end of the ROI.

30. A set of nucleic acid constructs from an individual, wherein

a) each construct of the set comprises:

a naturally occurring nucleic acid region of interest (ROI) from the individual;

a unique identifier attached to each end of the ROI, wherein the combination of identifiers on each end of a particular ROI distinguishes the construct from other constructs in the set with a same ROI, wherein the identifiers are not part of its attached ROI; and

at least one amplification site; and

b) the set of constructs comprises;

(i) at least two constructs with the same ROI and different unique identifiers, and (ii) two or more different ROIs.