Method for gene identification signature (GIS) analysis

• US 20050255501A1
• Filed: 01/31/2005
• Published: 11/17/2005
• Est. Priority Date: 09/17/2003
• Status: Abandoned Application

• Current Assignee: Agency For Science Technology and Research (Government of Singapore)
• Original Assignee: Agency For Science Technology and Research (Government of Singapore)
• Inventors: Ruan, Yijun, Ng, Patrick, Wei, Chialin
• Application Number:
  US11/045,468
• Publication Number:
  US 20050255501A1
• 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 method of identifyng 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 molecule 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.

2. The method of claim 1, wherein the nucleic acid molecule fragment to which a protein of interest binds is a nucleic acid molecule fragment enriched for transcription factor binding site(s) (TFBSs).

3. The method of claim 1, wherein before carrying out step (ii), the nucleic acid molecule fragment to which a protein binds is inserted into a vector.

4. The method of claim 3, wherein the vector comprises two motifs flanking the nucleic acid molecule fragment to be inserted into the vector, each 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.

5. The method of claim 4, wherein the asymmetric recognition sites are restriction endonuclease asymmetric cleavage site sequences recognizable by type II or type III restriction enzymes.

6. The method of claim 5, wherein the type II restriction enzyme 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, Eco571, Eco57MI, Esp3I, FalI, FauI, FokI, GsuI, HaeIV, HgaI, Hin4I, HphI, HpyAV, Ksp6321, MboII, MlyI, MmeI, MnlI, PleI, PpiI, PsrI, RleAI, SapI, SfaNI, SspD5I, Sthl32I, StsI, TaqII, TspDTI, TspGWI, TspRI and Tth111II.

7. The method of claim 5, wherein the type III restriction enzyme is EcoP15I.

8. The method of claim 4, wherein the asymmetric recognition sites are homing endonuclease asymmetric recognition site sequences, 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 method of claim 5, wherein the type II restriction site is MmeI.

10. The method of claim 3, wherein the vector has the sequence of SEQ ID NO:

22.

11. The method of claim 1, wherein the ditags are joined to form a concatemer of ditags.

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

13. The method of claim 1, wherein the nucleic acid molecule fragment of step (i) is isolated from a living cell by:

(a) cross-linking DNA binding protein in the living cell to genomic DNA of the living cell, thereby producing DNA binding protein cross-linked to genomic DNA;

(b) generating DNA fragments of the genomic DNA cross-linked to DNA binding protein in (a), thereby producing a DNA/protein complex comprising DNA fragments to which the DNA binding protein is bound;

(c) removing the DNA fragment to which the protein of interest is bound from the complex produced in (b); and

(d) isolating the DNA fragment identified in (c) from the protein of interest.

14. The method of claim 13, wherein the DNA/protein complex is isolated by antibody-mediated immunoprecipitation.

15. The method of claim 1, wherein the nucleic acid molecule fragment(s) is isolated by chromatin immunoprecipitation.

16. The method of claim 1, wherein the nucleic acid molecule fragment(s) is isolated by incorporating a photoactivable moiety into the DNA and/or the protein of interest and isolation of DNA/protein complex by antibody-mediated precipitation or by a affinity-mediated isolation method techniques.

17. The method of claim 1, comprising:

(a) cross-linking DNA binding protein(s) in living cell(s) to genomic DNA of the living cell, thereby producing DNA binding protein cross-linked to genomic DNA;

(b) generating DNA fragments of the genomic DNA cross-linked to DNA binding protein in (a), thereby producing a DNA/protein complex comprising DNA fragments to which the DNA binding protein is bound;

(c) removing the DNA fragment to which the protein of interest is bound from the complex produced in (b);

(d) isolating the DNA fragment(s) identified in (c) from the protein of interest;

(e) inserting the isolated DNA fragment(s) into a vector;

(f) isolating the 5′

terminus and the 3′

terminus of the nucleic acid fragment(s) inserted into the vector and linking the 5′

terminus and 3′

terminus to create the at least one ditag;

(g) sequencing the ditag; and

(h) mapping the ditag sequence(s) to the genome.

18. The method of claim 17, wherein in the step (a) formaldehyde is added to living cells;

and in step (b) crude extracts of the fixed cells are prepared, and sonicated to shear the chromatin.

19. The method of claim 1, wherein the protein of interest binds to the nucleic acid molecule fragment(s) at a consensus binding site, which is determined by the region of of genomic DNA encompassed by the two signatures of the ditag.

24. The method of claim 1, which is a method for the discovery of protein binding site(s).

25. The method of claim 24, wherein the protein binding site(s) is transcription factor binding site(s) (TFBSs).

20. 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).

21. The vector of claim 20, 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.

22. The vector of claim 21, wherein the asymmetric restriction site is a type II or III restriction site or a restriction site recognised by a homing endonuclease.

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

22.