UTR tag assay for gene function discovery

• US 7,405,282 B2
• Filed: 09/09/2005
• Issued: 07/29/2008
• Est. Priority Date: 08/29/2001
• Status: Expired due to Fees

• Current Assignee: Pioneer Hi Bred International Inc (Corteva, Inc.)
• Original Assignee: Pioneer Hi Bred International Inc (Corteva, Inc.)
• Inventors: Duvick, Jon
• Primary Examiner(s): Chunduru; Suryaprabha
• Application Number:
  US11/222,955
• Publication Number:
  US 20060008839A1
• Time in Patent Office: 1,054 Days
• Field of Search: 536/23.1, 536/22.1, 435/6, 435/320.1
• US Class Current: 536/22.1
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1044 Preparation or screening of libraries displayed on scaffold proteins
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

The methods of the invention provide a means for rapid analysis of gene function in a variety of systems. The invention allows screening of large libraries of nucleotide sequences for involvement in physiological pathways of interest. The methods of the invention also provide an efficient means of identifying and isolating nucleotide sequences that modulate a physiological pathway of interest from a population of nucleotide sequences.

Claims

1. A kit for identifying a nucleotide sequence that modulates the activity of a transcriptional regulatory region comprising a library of plasmids for incorporation into a host cell population wherein the library is a collection of plasmids each plasmid having one or more non-redundant U-tags, and wherein a plasmid in the library comprises a first DNA construct and a second DNA construct, wherein:

a) the first DNA construct comprises a multiple cloning site, a reporter sequence, and an mRNA stabilizing sequence; and

,

b) the first DNA construct further comprises one or more U-tags, wherein each U-tag is a short, random nucleotide sequence inserted into an untranslated region of the reporter sequence, and one or more intron sequences occurring within the first DNA construct; and

c) the second DNA construct comprises a promoter active in a host cell operably linked to a multiple cloning site.

2. The kit of claim 1, further comprising a complementary U-tag array.

3. A kit for identifying a transcriptional regulatory region of interest that is modulated by an agent comprising a first library of plasmids for incorporation into a host cell population wherein the library is a collection of plasmids each plasmid having one or more non-redundant U-tags, and each plasmid in the library comprises a first DNA construct, wherein

a) the first DNA construct comprises a multiple cloning site, a reporter sequence, and an mRNA stabilizing sequence;

and,

b) the first DNA construct further comprises one or more U-tags, wherein each U-tag is a short, random nucleotide sequence inserted into an untranslated region of the reporter sequence, and one or more intron sequences occurring within the first DNA construct.

4. The kit of claim 3 further comprising a complementary U-tag array.

5. A kit comprising a library of plasmids for incorporation into a host cell population wherein the library is a collection of plasmids each plasmid having one or more non-redundant U-tags, and a plasmid in the library comprises a first DNA construct and a second DNA construct, wherein:

a) the first DNA construct comprises a multiple cloning site, a reporter sequence, and an mRNA stabilizing sequence; and

b) the first DNA construct further comprises one or more U-tags, wherein each U-tag is a short, random nucleotide sequence inserted into an untranslated region of the reporter sequence, and one or more intron sequences occurring within the first DNA construct; and

,

c) the second DNA construct comprises a promoter active in a host cell of the host cell population operably linked to a multiple cloning site.

6. The kit of claim 5 further comprising a complementary U-tag array.

7. The kit of claim 5 wherein the second DNA construct fUrther comprises a nucleotide sequence of interest.

8. A kit comprising a library of plasmids for incorporation into a host cell population wherein the library is a collection of plasmids each plasmid having one or more non-redundant U-tags, and each plasmid in the library comprises a first DNA construct, wherein:

a) the first DNA construct comprises a multiple cloning site, a reporter sequence, and an mRNA stabilizing sequence; and

b) the first DNA construct further comprises one or more U-tags, wherein each U-tag is a short, random nucleotide sequence inserted into an untranslated region of the reporter sequence, and one or more intron sequences occurring within the first DNA construct.

9. The kit of claim 8 further comprising a complementary U-tag array.