Nucleic acid constructs and methods of use

• US 10,119,165 B2
• Filed: 11/11/2016
• Issued: 11/06/2018
• 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 ANN
• Application Number:
  US15/349,927
• Publication Number:
  US 20170058345A1
• Time in Patent Office: 725 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 plurality of nucleic acid molecules in a set, comprising:

(a) tagging one or both ends of each nucleic acid molecule comprising a region of interest in the set of nucleic acid molecules, by ligation with a unique identifier to form a tagged construct, wherein the unique identifier distinguishes the tagged construct from other tagged constructs in the set;

(b) selectively amplifying regions of interest and unique identifiers using PCR to form an amplified set of tagged constructs;

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

(d) determining sequence of the amplified tagged constructs using the unique identifiers and sequence information from step (c) to generate a consensus sequence among molecules that are identical by descent.

2. The method of claim 1, wherein each nucleic acid molecule is tagged with two unique identifiers.

3. The method of claim 2, wherein the two unique identifiers are both on one end of the nucleic acid molecule.

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

6. The method of claim 1, wherein the determining further comprises determining allelic variants of a region of interest in the set of nucleic acid sequences.

7. The method of claim 6, wherein the allelic variants comprise one or more haplotypes.

8. The method of claim 1, wherein the set of nucleic acid sequences comprises at least two tagged constructs with substantially the same region of interest and different unique identifiers.

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

11. The method of claim 1, further comprising amplifying the tagged constructs with universal primers.

12. The method of claim 1, wherein further comprising amplifying the tagged constructs with subset-specific primers.

13. The method set of claim 1, wherein the nucleic acid comprises DNA.

14. The method of claim 1, wherein the nucleic acid comprises RNA.

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

4. A method for determining sequences of a plurality of nucleic acid molecules in a set, comprising:

(a) tagging one or both ends of each nucleic acid molecule comprising a region of interest in the set of nucleic acid molecules, by ligation with two unique identifiers to form a tagged construct, wherein the unique identifiers distinguish the tagged construct from other tagged constructs in the set;

(b) selectively amplifying regions of interest and unique identifiers using PCR to form an amplified set of tagged constructs;

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

(d) determining sequence of the amplified tagged constructs using the unique identifiers and sequence information from step (c) to generate a consensus sequence among molecules that are identical by descent,

wherein the two unique identifiers are one on each end of the nucleic acid molecule.

10. A method for determining sequences of a plurality of nucleic acid molecules in a set, comprising:

(a) tagging one or both ends of each nucleic acid molecule comprising a region of interest in the set of nucleic acid molecules, by ligation with a unique identifier to form a tagged construct, wherein the unique identifier distinguishes the tagged construct from other tagged constructs in the set;

(b) selectively amplifying regions of interest and unique identifiers using PCR to form an amplified set of tagged constructs;

(c) sequencing the amplified set of tagged constructs;

(d) determining sequence of the amplified tagged constructs using the unique identifiers and sequence information from step (c) to generate a consensus sequence among molecules that are identical by descent, and

(e) identifying a mutation in a region of interest if it appears in substantially all of the tagged constructs having an identical descent.