Nucleic acid constructs and methods of use

• US 9,085,798 B2
• Filed: 04/29/2010
• Issued: 07/21/2015
• 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:
  US13/266,568
• Publication Number:
  US 20120065081A1
• Time in Patent Office: 1,909 Days
• Field of Search: None
• US Class Current: 1/1
• 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 method for determining the sequences of a set of nucleic acid targets, comprising:

tagging each individual nucleic acid target in a set of nucleic acid targets to create a set of tagged constructs, with;

(a) at least one unique identifier oligonucleotide that distinguishes the tagged construct from other tagged constructs in the set, wherein the at least one unique identifier oligonucleotide is not part of the nucleic acid target; and

(b) at least one amplification site;

amplifying the set of tagged constructs; and

determining the sequences of the amplified set of tagged constructs comprising the unique identifier oligonucleotides and the nucleic acid targets.

2. The method of claim 1, wherein the nucleic acid targets are isolated from a biological sample.

3. The method of claim 2, wherein the method determines one or more sequences or allelic variants of a nucleic acid target in the set of nucleic acid targets.

4. The method of claim 3, wherein the sequences or allelic variants comprise one or more haplotypes.

5. The method of claim 1, wherein in the tagging step each individual nucleic acid target is tagged with two or more amplification sites.

6. The method of claim 1, further comprising assembling the sequences of the amplified set of tagged constructs into one or more contiguous sequences.

7. The method of claim 1, wherein the set of tagged constructs comprises at least two tagged constructs with the same nucleic acid target sequence and different unique identifier oligonucleotide sequences.

8. A method for identifying a nucleic acid comprising a target ROI (region of interest), comprising:

providing a master set of nucleic acid constructs, each nucleic acid construct comprising;

(a) an ROI;

(b) at least one unique identifier oligonucleotide that distinguishes the nucleic acid construct from other nucleic acid constructs in the master set, wherein the at least one unique identifier oligonucleotide is not part of the ROI; and

(c) at least one amplification site;

amplifying all or a portion of the master set, thereby generating an amplified master set comprising at least two copies of the nucleic acid construct comprising a target ROI;

sequencing at least two nucleic acid constructs from the amplified master set; and

identifying nucleic acid constructs in the amplified master set that are amplified from the same nucleic acid construct in the master set, by identifying nucleic acid constructs in the amplified master set that comprise the same unique identifier oligonucleotide,

wherein the nucleic acid construct comprising the target ROI is identified, wherein the target ROI is distinguished from other ROIs in the amplified master set by the unique identifier oligonucleotide sequences and/or by the ROI sequences.

9. The method of claim 8, further comprising determining the sequences of a subset of nucleic acid constructs in the master set.

10. The method of claim 9, wherein the sequences of the subset of nucleic acid constructs are determined by nucleic acid sequencing.

11. The method of claim 8, wherein in the identifying step, two or more nucleic acid constructs identified in the amplified master set comprise the same unique identifier oligonucleotide and different ROIs.

12. The method of claim 8, further comprising isolating nucleic acid constructs comprising the same unique identifier oligonucleotide.

13. The method of claim 12, wherein each nucleic acid construct further comprises at least one excision site, the method further comprising excising the ROI from its nucleic acid construct at the at least one excision site following the isolating step.

14. The method of claim 13, wherein the ROI is excised from a subset of nucleic acid constructs from the amplified master set.

15. The method of claim 13, wherein the ROI is excised from the amplified master set.

16. The method of claim 8, wherein the nucleic acid constructs are immobilized on a solid support.

17. The method of claim 16, wherein the solid support is a bead.

18. The method of claim 11, wherein the different ROIs comprise an allelic variant, a polymorphism, and/or a mutation.

19. The method of claim 11, wherein at least one of the different ROIs is confirmed as an error-free ROI.

20. The method of claim 11, wherein at least one of the different ROIs results from error during nucleic acid synthesis, preparation of the nucleic acid constructs, the amplifying step, and/or the sequencing step.

21. The method of claim 8, wherein the master set of nucleic acid constructs comprises at least two constructs with the same ROI and different unique identifier oligonucleotide sequences.