Nucleic acid constructs and methods of use

• US 10,000,800 B2
• Filed: 07/29/2016
• Issued: 06/19/2018
• 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): Treptow, Nancy A
• Application Number:
  US15/224,253
• Publication Number:
  US 20160333403A1
• Time in Patent Office: 690 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 method for determining sequences of a set of nucleic acid targets, comprising:

(a) tagging each end of each nucleic acid target with an identifier from a set of different identifiers to create a set of tagged constructs, wherein the combination of identifiers on each end of a particular nucleic acid target has a high probability of being unique in its association of the particular nucleic acid target from the set of nucleic acid targets;

(b) amplifying the set of tagged constructs;

(c) sequencing the amplified set of tagged constructs; and

(d) using the identifiers and sequence information from step (c) to generate a consensus sequence among molecules that are identical by descent, thereby determining sequences of the nucleic acid targets.

2. The method of claim 1, further comprising providing nucleic acid targets isolated from a biological sample.

3. The method of claim 1, further comprising attaching amplification sites to the tagged constructs, wherein the attached amplification sites flank the nucleic acid target and identifiers.

4. The method of claim 3, wherein the amplifying step (b) comprises performing a universal amplification.

5. The method of claim 1, wherein the identifiers comprise a degenerate sequence.

6. The method of claim 1, wherein the identifiers are selected from a diverse, pre-defined set of oligonucleotide sequences.

7. The method of claim 1, wherein the tagging is performed by ligation.

8. The method of claim 1, wherein the amplifying comprises PCR.

9. The method of claim 1, wherein the amplifying comprises solid-phase amplification.

10. The method of claim 1, wherein the amplifying comprises emulsion PCR.

11. The method of claim 10, wherein the emulsion PCR comprises beads.

12. The method of claim 10, wherein the emulsion PCR comprises magnetic beads.

13. The method of claim 1, wherein the sequencing comprises performing massively parallel next generation sequencing.

14. The method of claim 13, wherein the next generation sequencing comprises a one pass sequencing method.

15. The method of claim 13, wherein the next generation sequencing comprises paired-end sequencing.

16. The method of claim 13, wherein the next generation sequencing comprises a hybridization-based method.

17. The method of claim 13, wherein the next generation sequencing comprises a sequencing by synthesis method.

18. The method of claim 13, wherein the next generation sequencing comprises a ligation-based method.

19. The method of claim 1, further comprising inferring a starting number of nucleic acid targets in the set.

20. The method of claim 1, further comprising identifying a mutation in a target nucleic acid if it appears in substantially all of the tagged constructs having an identical descent.

21. The method of claim 1, further comprising determining one or more sequences or allelic variants of a nucleic acid target in the set of nucleic acid targets.