Method and kit for concentrating target double-stranded nucleic acid molecules using a pyrrole-imidazole-containing polyamide

Patent metadata

Field	Value
Publication number	US-10858646-B2
Application number	US-201815948124-A
Country	US
Kind code	B2
Filing date	Apr 9, 2018
Priority date	Oct 8, 2015
Publication date	Dec 8, 2020
Grant date	Dec 8, 2020

Abstract

A method of separating a target double-stranded nucleic acid molecule from a sample including the target double-stranded nucleic acid molecule and a non-target double-stranded nucleic acid molecule, including (1) mixing the sample, a pyrrole-imidazole-containing polyamide (first PI polyamide) modified with a first linker molecule and capable of specifically binding to a sequence of the target double-stranded nucleic acid molecule, and a carrier a modified with a first ligand capable of specifically binding and/or adsorbing to the first linker molecule such that a mixed solution is produced, (2) forming a complex A by binding the carrier a to the first PI polyamide with which the target double-stranded nucleic acid molecule is bound in the mixed solution, and (3) separating the complex A from the mixed solution.

First claim

What is claimed is:

1. A method of separating a target double-stranded nucleic acid molecule from a sample, comprising:
mixing a sample, a first PI polyamide, and a carrier such that a mixed solution comprising the sample, the first PI polyamide, and the carrier is produced;
forming a complex A by binding the carrier to the first PI polyamide with which a target double-stranded nucleic acid molecule in the sample is bound in the mixed solution; and
separating the complex A from the mixed solution,
wherein the sample includes the target double-stranded nucleic acid molecule and a non-target double-stranded nucleic acid molecule, the first PI polyamide is a pyrrole-imidazole-containing polyamide which is modified with a first linker molecule and specifically binds to a sequence of the target double-stranded nucleic acid molecule in the sample, the carrier is modified with a first ligand which specifically binds or adsorbs to the first linker molecule, and a content of the target double-stranded nucleic acid molecule for separating the target double-stranded nucleic acid molecule from the sample is less than 1% in the sample.

2. The method of claim 1 , wherein the mixing includes mixing the sample and the first PI polyamide to form a complex a1 by binding the first PI polyamide with the target double-stranded nucleic acid molecule in the mixed solution, and the forming of the complex A includes binding the carrier to the first PI polyamide in the complex a1.

3. The method of claim 2 , wherein the mixing further includes mixing a second PI polyamide with the sample to form a complex b1 by binding the second PI polyamide with the non-target double-stranded nucleic acid molecule in the mixed solution.

4. The method of claim 1 , wherein the mixing includes mixing the first PI polyamide and the carrier to form a complex a2 by binding the first PI polyamide with the carrier in the mixed solution, and the forming of the complex A includes binding the target double-stranded nucleic acid molecule to the first PI polyamide of the complex a2.

5. A method of removing a non-target double-stranded nucleic acid molecule from a sample including a target double-stranded nucleic acid molecule and the non-target double-stranded nucleic acid molecule, comprising:
(1) mixing the sample,
a pyrrole-imidazole-containing polyamide (second PI polyamide) modified with a second linker molecule and capable of specifically binding to a sequence of the non-target double-stranded nucleic acid molecule, and
a carrier b modified with a second ligand molecule capable of specifically binding and/or adsorbing to the second linker molecule such that a mixed solution is produced;
(2) forming a complex B by binding the carrier b to the second PI polyamide with which the non-target double-stranded nucleic acid molecule is bound in the mixed solution; and
(3) removing the complex B from the mixed solution wherein a content of the target double-stranded nucleic acid molecule is less than 1% in the sample.

6. The method of claim 5 , wherein the mixing includes mixing the sample and the second PI polyamide to form a complex b1 by binding the second PI polyamide with the non-target double-stranded nucleic acid molecule in the mixed solution, and further the carrier b to form the complex B by binding the carrier b to the second PI polyamide of the complex b1.

7. The method of claim 5 , wherein the mixing includes mixing the second PI polyamide and the carrier b to form a complex b2 by binding the second PI polyamide with the carrier b in the mixed solution, and further the sample to form the complex B by binding the non-target double-stranded nucleic acid molecule to the second PI polyamide of the complex b2.

8. The method of claim 6 , wherein the mixing further includes mixing a first PI polyamide together with the sample and the second PI polyamide to form a complex a1, by binding the first PI polyamide with the target double-stranded nucleic acid molecule in the mixed solution, and the complex b 1,
and further:
(4) mixing a carrier a modified with a first ligand capable of specifically binding and/or adsorbing to the first linker molecule;
(5) forming a complex A by binding the carrier a to the first PI polyamide of a complex a1 in which the target double-stranded nucleic acid molecule is bound with the first PI polyamide in the mixed solution; and
(6) separating the complex A from the mixed solution.

9. A method of separating a target double-stranded nucleic acid molecule from a sample, comprising:
mixing a sample, a first PI polyamide, a first carrier, a second PI polyamide, and a second carrier such that a mixed solution comprising the sample, the first PI polyamide, the first carrier, the second PI polyamide, and the second carrier is obtained;
forming a complex A by binding the first carrier to the first PI polyamide with which a target double-stranded nucleic acid molecule in the sample is bound in the mixed solution and a complex B by binding the second carrier to the second PI polyamide with which a non-target double-stranded nucleic acid molecule in the sample is bound in the mixed solution;
removing the complex B from the mixed solution; and
separating the complex A from the mixed solution,
wherein the first PI polyamide is a pyrrole-imidazole-containing polyamide which is modified with a first linker molecule and specifically binds to a sequence of the target double-stranded nucleic acid molecule, the first carrier is modified with a first ligand molecule which specifically binds or adsorbs to the first linker molecule, the second PI polyamide is a pyrrole-imidazole-containing polyamide which is modified with a second linker molecule and specifically binds to a sequence of the non-target double-stranded nucleic acid molecule, the second carrier is modified with a second ligand molecule which specifically binds or adsorbs to the second linker molecule, and a content of the target double-stranded nucleic acid molecule for separating the target double-stranded nucleic acid molecule from the sample is less than 1% in the sample.

10. The method of claim 9 , wherein the mixing includes mixing the sample and the first PI polyamide to form a complex a1 by binding the first PI polyamide with the target double-stranded nucleic acid molecule in the mixed solution, and the forming of the complex A includes binding the first carrier to the first PI polyamide in the complex a1 the mixing includes; or mixing the sample and the second PI polyamide to form a complex hi by binding the second PI polyamide with the non-target double-stranded nucleic acid molecule in the mixed solution, and the forming of the complex B includes binding the second carrier to the second PI polyamide the complex b1.

11. The method of claim 9 , wherein the mixing includes mixing the sample and the first PI polyamide to form a complex a1 by binding the first PI polyamide with the target double-stranded nucleic acid molecule in the mixed solution, and the forming of complex A includes binding the first carrier to the first PI polyamide of the complex a1; or the mixing includes mixing the second PI polyamide and the second carrier to form a complex b2 by binding the second PI polyamide with the second carrier in the mixed solution, and the forming of complex B includes binding the non-target double-stranded nucleic acid molecule to the second PI polyamide of the complex b2.

12. The method of claim 9 , wherein the mixing includes mixing the first PI polyamide and the first carrier to form a complex a2 by binding the first PI polyamide with the first carrier in the mixed solution, and the forming of complex A includes binding the target double-stranded

Assignees

• Toppan Printing Co Ltd

Inventors

• Nagase Hiroki
• Tsukamoto Kei
• Kitano Shiro

Classifications

• C07H1/06

  Separation; Purification · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title

• C07H21/00

  Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids · CPC title

• C08G73/0611

  with only one nitrogen atom in the ring, e.g. polypyrroles (polysuccinimides C08G73/1092) · CPC title

• C08G73/0616

  with only two nitrogen atoms in the ring · CPC title