Nucleic acid constructs and methods of use
AssigneePrognosys Biosciences, Inc.
InventorMark S. Chee
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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.
Claims15
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.
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.
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.
Dependencies are inferred from the claim text.