Method for Gene Identification Signature (GIS) Analysis

• US 20070161024A1
• Filed: 12/13/2006
• Published: 07/12/2007
• Est. Priority Date: 09/17/2003
• Status: Active Grant

• Current Assignee: Agency For Science Technology and Research
• Original Assignee: Agency For Science Technology and Research
• Inventors: Ruan, Yijun, Ng, Patrick, Wei, Chialin
• Granted Patent: US 7,553,947 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

A method of identifying at least a nucleic acid molecule fragment to which a protein of interest binds, comprising: (i) preparing at least one nucleic acid molecule fragment to which a protein binds; (ii) isolating the 5′ terminus and the 3′ terminus of the nucleic acid fragment(s) and linking the 5′ terminus and 3′ terminus to create the at least one ditag; (iii) sequencing the ditag; and (iv) mapping the ditag sequence(s) to the genome.

Claims

1. A vector comprising at least one ditag, wherein the ditag comprises two joined first and second sequence tags, and wherein the first tag includes a 5′

-terminus sequence and a second tag comprises the 3′

-terminus sequence of a nucleic acid molecule fragment, and wherein the nucleic acid molecule fragment is enriched for transcription factor binding sites (TFBSs).

2. The vector of claim 1, wherein the ditag is flanked at each side by a motif comprising at least:

a first restriction site which is an asymmetric restriction site and/or at least a second restriction site, and wherein the remainder of the backbone of the vector does not comprise the asymmetric restriction site and/or the second restriction site.

3. The vector of claim 2, wherein the asymmetric restriction site is restriction endonuclease asymmetric cleavage site sequences recognizable by type II or type III restriction enzymes.

4. The vector of claim 3, wherein the type II restriction enzymes is selected from the group consisting of:

AarI, AceIII, AloI, BaeI, Bbr7I, BbvI, BbvII, BccI, Bce83I, BceAI, BcefI, BcgI, BciVI, BfiI, BinI, BplI, BsaXI, BscAI, BseMII, BseRI, BsgI, BsmI, BsmAI, BsmFI, Bsp24I, BspCNI, BspMI, BsrI, BsrDI, BstF5I, BtgZI, BtsI, CjeI, CjePI, EciI, Eco31I, Eco57I, Eco57MI, Esp3I, FalI, FauI, FokI, GsuI, HaeIV, HgaI, Hin4I, HphI, HpyAV, Ksp632I, MboII, MlyI, MmeI, MnlI, PleI, PpiI, PsrI, RleAI, SapI, SfaNI, SspD5I, Sth132I, StsI, TaqII, TspDTI, TspGWI, TspRI and Tth111II.

5. The vector of claim 3, wherein the type II restriction enzyme is MmeI.

6. The vector of claim 3, wherein the type III restriction enzyme is EcoP15I.

7. The vector of claim 2, wherein the asymmetric restriction site is a type II or III restriction site or a restriction site recognised by a homing endonuclease.

8. The vector of claim 2, wherein the asymmetric restriction site is a homing endonuclease asymmetric recognition site sequence, and wherein the enzyme recognizing the homing endonuclease asymmetric restriction site is selected from the group consisting of:

I-CeuI, PI-SceI, PI-PspI and I-SceI.

9. The vector of claim 1, wherein the vector has a sequence of SEQ ID NO:

22.

10. The vector of claim 1, wherein the ditags are joined to form a concatemer of ditags.

11. The method of claim 10, wherein the concatemer comprises 1-1000 ditags.

12. A vector having the nucleotide sequence of SEQ ID NO:

22.