Method for detecting protein that interacts with target substance

Patent metadata

Field	Value
Publication number	US-9513295-B2
Application number	US-201214347707-A
Country	US
Kind code	B2
Filing date	Sep 28, 2012
Priority date	Sep 28, 2011
Publication date	Dec 6, 2016
Grant date	Dec 6, 2016

Abstract

The present invention provides a method for detecting an interaction, which method can solve not only the problem of false negatives but also the problem of false positives. This method is a method for detecting a protein(s) that interact(s) with a target substance(s), the method comprising repeating a (1) transcription step, (2) assignment step, (3) selection step, and (4) amplification step, wherein: (a) in each of a plurality of times of preparation of a cDNA library among the initial preparation of a cDNA library and the round(s) of preparation of a cDNA library in the later amplification step(s), the cDNA library is prepared using a primer(s) having a sequence(s) specific to the time of preparation; (b) the cDNA libraries prepared using the primers having sequences specific to the times of preparation are mixed, and sequences in the cDNA library mixture are determined; (c) the determined sequences are subjected to measurement of the number(s) of molecules encoding the same candidate protein(s) for each time of preparation based on the sequence(s) specific to the time of preparation; and (d) a candidate protein(s) encoded by a molecule(s) that significantly increase(s) as the preparation rounds proceed is/are detected as the protein(s) that interact(s) with the target substance(s).

First claim

The invention claimed is:

1. A method for detecting a protein(s) that interact(s) with a target substance(s), said method comprising:
(1) a transcription step of transcribing a cDNA library encoding a candidate protein(s) to prepare an mRNA library;
(2) an assignment step of preparing an mRNA-protein assignment molecular library from said mRNA library prepared in the transcription step;
(3) a selection step of selecting an mRNA-protein assignment molecule(s) that interact(s) with a target substance(s) from the mRNA-protein assignment molecular library prepared in the assignment step;
(4) an amplification step of preparing a cDNA library encoding the candidate protein(s) by nucleic acid amplification based on the mRNA portion(s) of said mRNA-protein assignment molecule(s) selected in the selection step; and
(5) repeating Steps (1) to (4) using said cDNA library prepared in the amplification step; wherein said method comprises:
(a) preparing, in each of a plurality of times of preparation of a cDNA library among the initial preparation of a cDNA library and the round(s) of preparation of a cDNA library in the later amplification step(s), the cDNA library using a primer(s) having a sequence(s) specific to the time of preparation;
(b) mixing the cDNA libraries prepared using said primers having sequences specific to the times of preparation, and determining sequences in the cDNA library mixture;
(c) subjecting the determined sequences to measurement of the number(s) of molecules encoding the same candidate protein(s) for each time of preparation based on the sequence(s) specific to the time of preparation; and
(d) detecting, as the protein(s) that interact(s) with said target substance(s), a candidate protein(s) encoded by a molecule(s) that significantly increase(s) as the preparation rounds proceed.

2. The method according to claim 1 , wherein, in Step (b), the mixing ratio of the cDNA library obtained by an earlier time of preparation is not less than the mixing ratio of the cDNA library obtained by the subsequent time of preparation.

3. The method according to claim 1 , wherein said sequence specific to each time of preparation has a length of 4 to 10 bases.

4. The method according to claim 1 , wherein said primer has said sequence specific to the time of preparation at the 5′-end.

5. The method according to claim 1 , wherein the number of cDNA libraries mixed in Step (b) is not less than 3.

6. The method according to claim 1 , wherein the cDNA libraries mixed in Step (b) comprise the initial cDNA library.

7. The method according to claim 1 , comprising evaluating the increase in the molecules in Step (d) by a statistical method.

8. The method according to claim 1 , wherein said target substance is a protein.

Assignees

• Univ Tokyo Science Found

Inventors

• Miyamoto Etsuko
• Fujimori Shigeo

Classifications

• C12N15/1034 Primary

  Isolating an individual clone by screening libraries · CPC title

• C40B30/04

  by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding · CPC title

• C12N15/1062

  mRNA-Display, e.g. polypeptide and encoding template are connected covalently · CPC title

• G01N33/6818 Primary

  Sequencing of polypeptides · CPC title