Nucleic acid constructs and methods of use

Patent metadata

Field	Value
Publication number	US-2017088881-A1
Application number	US-201615372159-A
Country	US
Kind code	A1
Filing date	Dec 7, 2016
Priority date	Apr 30, 2009
Publication date	Mar 30, 2017
Grant date	—

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.

First claim

1 . (canceled)

2 . A method for determining sequences of a set of nucleic acid targets, comprising:
(a) tagging an end of each nucleic acid target with two identifiers from a set of different identifiers to create a set of tagged constructs, wherein the combination of identifiers on 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 to form a set of tagged amplicons; (c) sequencing all or a portion each tagged amplicon of the set of tagged amplicons; and (d) using the identifiers and sequence information from step (c), determining sequence of the nucleic acid targets by generating a consensus sequence among molecules that are identical by descent.

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

4 . The method of claim 2 , further comprising attaching amplification sites to the tagged constructs, wherein the attached amplification sites flank the nucleic acid target and identifiers.

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

6 . The method of claim 2 , wherein the identifiers comprise a degenerate sequence.

7 . The method of claim 2 , wherein the identifiers are selected from a diverse, pre-defined set of oligonucleotide sequences.

8 . The method of claim 2 , wherein the tagging is performed by ligation.

9 . The method of claim 2 , wherein the amplifying comprises PCR.

10 . The method of claim 2 , wherein the amplifying comprises solid-phase amplification.

11 . The method of claim 2 , wherein the amplifying comprises emulsion PCR.

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

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

14 . The method of claim 2 , wherein the sequencing comprises performing massively parallel next generation sequencing.

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

16 . The method of claim 14 , wherein the next generation sequencing comprises paired-end sequencing.

17 . The method of claim 14 , wherein the next generation sequencing comprises a hybridization-based method.

18 . The method of claim 14 , wherein the next generation sequencing comprises a sequencing by synthesis method.

19 . The method of claim 14 , wherein the next generation sequencing comprises a ligation-based method.

20 . The method of claim 2 , further comprising inferring a starting number of nucleic acid targets in the set.

21 . The method of claim 2 , further comprising identifying a mutation in a target nucleic acid if it appears in substantially all of the tagged constructs having an identical descent.

22 . The method of claim 2 , further comprising determining one or more sequences or allelic variants of a nucleic acid target in the set of nucleic acid targets.

Assignees

• Prognosys Biosciences Inc

Inventors

• Chee Mark S

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title