Nucleic acid constructs and methods of use

• US 10,266,883 B2
• Filed: 11/11/2016
• Issued: 04/23/2019
• 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:
  US15/349,906
• Publication Number:
  US 20170058339A1
• Time in Patent Office: 893 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 to analyze nucleic acid sequences, comprising:

attaching a unique identifier from a pool of different unique identifiers to a first end of each strand of a plurality of nucleic acid target molecules to form a plurality of tagged nucleic acid target molecules,

wherein the pool of unique identifiers is in excess of the plurality of nucleic acid target molecules;

determining a sequence of uniquely identified amplicons of a tagged nucleic acid target molecule from the plurality of tagged nucleic acid target molecules, wherein sequences of amplicons having the same unique identifier are amplicons related by descent;

comparing the sequences of amplicons related by descent; and

identifying a sequence as representing a nucleic acid target molecule when multiple clonal copies of the nucleic acid target molecule result in a consensus sequence.

2. The method of claim 1, wherein prior to the step of determining a sequence, the plurality of tagged nucleic acid target molecules are amplified to form uniquely identified amplicons of the plurality of tagged nucleic acid target molecules.

3. The method of claim 1, wherein the step of attaching is performed with a primer in an amplification reaction.

4. The method of claim 1, wherein a universal priming site is attached to a second end of each strand of a plurality of tagged nucleic acid target molecules.

5. The method of claim 3, wherein the amplification reaction produces a plurality of tagged nucleic acid target molecules having a unique identifier on the first end of each strand and a universal priming site attached to a second end of each strand.

6. The method of claim 1, wherein the unique identifier is adjacent to a universal priming site.

7. The method of claim 4, wherein the unique identifier is adjacent to a universal priming site.

8. The method of claim 7, wherein prior to the step of determining a sequence, the plurality of tagged nucleic acid target molecules are amplified using universal primers complementary to the universal priming sites to form uniquely identified amplicons of the plurality of tagged nucleic acid target molecules.

9. The method of claim 6, wherein the unique identifier is attached to a 5′

end of each strand of a plurality of nucleic acid target molecules and the second universal priming site is 5′

to the unique identifier.

10. The method of claim 6, wherein the unique identifier is attached to a 3′

end of each strand of a plurality of nucleic acid target molecules and the second universal priming site is 3′

to the unique identifier.

11. The method of claim 1, wherein the plurality of nucleic acid target molecules are fragmented nucleic acid molecules.

12. The method of claim 3, wherein the primers used in the attaching step comprise a modification.

13. The method of claim 3, wherein the primers used in the attaching step comprise a phosphorothioate linkage.

14. The method of claim 3, wherein the pool of unique identifiers is in excess of the plurality of nucleic acid target molecules during the amplification reaction.

15. The method of claim 1, wherein the unique identifiers comprise about 10 to about 30 bases.

16. The method of claim 1, further comprising identifying a mutation in a sequence representing a nucleic acid target molecule when the sequence differs from a validated, error-free nucleic acid target molecule sequence.

17. The method of claim 1, further comprising identifying a determined nucleotide sequence of a target nucleic acid molecule as having a synthesis or sequencing error when it differs from a consensus sequence derived from multiple clonal copies of the target nucleic acid molecule.

18. The method of claim 16, wherein the mutation is identified when it appears in sequences of target nucleic acid molecules having the same sequence and different unique identifiers.

19. The method of claim 16, wherein the mutation is a polymorphism.

20. A method to analyze nucleic acid sequences, comprising:

attaching a unique identifier comprising a sequence of about 10 to about 30 bases from a pool of different unique identifiers to a first end of each strand of a plurality of nucleic acid target molecules using a primer in amplification reaction to form a plurality of tagged nucleic acid target molecules,

wherein the pool of unique identifiers is in excess of the plurality of nucleic acid target molecules;

determining a sequence of uniquely identified amplicons of a tagged nucleic acid target molecule from the plurality of tagged nucleic acid target molecules, wherein sequences of amplicons having the same unique identifier are amplicons related by descent;

comparing the sequences of amplicons related by descent;

identifying a sequence as representing a nucleic acid target molecule when multiple clonal copies of the nucleic acid target molecule result in a consensus sequence; and

comparing the sequence representing a nucleic acid target molecule to a validated, error-free target nucleic acid molecule and identifying a mutation in the sequence when the sequence differs from a validated, error-free nucleic acid target molecule sequence.