Multiplexed genetic reporter assays and compositions
AssigneeThe Broad Institute, Inc.
InventorsTarjei Mikkelsen, Andreas Gnirke, Alexandre Melnikov, Eric S. Lander, Li Wang, Xiaolan Zhang
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Abstract
The invention provides methods for determining the activity of a plurality of nucleic acid regulatory elements. These methods may facilitate, e.g., the systematic reverse engineering, and optimization of mammalian cis-regulatory elements at high resolution and at a large scale. The method may include integration of multiplexed DNA synthesis and sequencing technologies to generate and quantify the transcriptional regulatory activity of e.g., thousands of arbitrary DNA sequences in parallel in cell-based as says (e.g., mammalian cell based assays).
Claims20
A plurality of expression vectors,
- wherein each of said expression vectors comprises a nucleic acid regulatory element, an open reading frame, a pair of universal primer sites, and one or more identifying nucleic acid tags;
- wherein the nucleic acid regulatory element, the open reading frame, and the one or more identifying nucleic acid tags are between the pair of universal primer sites;
- wherein the open reading frame is located 3′ to the nucleic acid regulatory element and the one or more identifying nucleic acid tags are located 3′ to the open reading frame;
- wherein the open reading frame of each of said plurality of expression vectors is identical, and optionally encodes a fluorescent protein or a luciferase;
- wherein the plurality of expression vectors comprises a plurality of distinct nucleic acid regulatory elements, wherein the plurality of distinct regulatory elements comprises synthetic variants of a single regulatory element;
- wherein each nucleic acid regulatory element is paired with the one or more identifying nucleic acid tags;
- and
- wherein the pair of universal primer sites comprise a first primer site comprising the sequence of SEQ ID NO:
- 5 and a second primer site comprising the sequence of SEQ ID NO:
- 7.
A plurality of nucleic acid constructs comprised within expression vectors,
- wherein the plurality of nucleic acid constructs comprises a plurality of distinct nucleic acid regulatory elements, wherein the distinct regulatory elements comprise synthetic variants of a single regulatory element;
- wherein each of said constructs comprises one or more identifying nucleic acid tags, two or more restriction enzyme sites, a pair of universal primer sites, and a particular nucleic acid regulatory element paired to said one or more nucleic acid tags,
- wherein the nucleic acid regulatory element, two or more restriction enzyme sites, and one or more identifying nucleic acid tags are between the pair of universal primer sites;
- wherein the two or more restriction enzyme sites are located 3′ to the nucleic acid regulatory element and the one or more identifying nucleic acid tags are located 3′ to the two or more restriction enzyme sites;
- and
- wherein the pair of universal primer sites comprise a first primer site comprising the sequence of SEQ ID NO:
- 5 and a second primer site comprising the sequence of SEQ ID NO:
- 7.
Dependencies are inferred from the claim text.