Method of modifying nucleotide chain

• US 8,129,115 B2
• Filed: 06/04/2007
• Issued: 03/06/2012
• Est. Priority Date: 06/06/2006
• Status: Expired due to Fees

• Current Assignee: PHC Holdings Corporation
• Original Assignee: Panasonic Corporation (Panasonic Holdings Corporation)
• Inventors: Joko, Shigeki
• Primary Examiner(s): Marschel, Ardin
• Assistant Examiner(s): Mahatan, Channing S
• Application Number:
  US12/303,313
• Publication Number:
  US 20100015670A1
• Time in Patent Office: 1,737 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C07H 21/00 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 Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2525/117 incorporating modified base

Abstract

A nucleotide chain to be modified, a nucleotide having a particular base that is different from bases constituting the nucleotide chain, an enzyme catalyzing addition of the nucleotide to the 3′-terminus of the nucleotide chain, a degrading enzyme acting specifically on the nucleotide, and a desired modifier for modifying the nucleotide chain are allowed to coexist in a buffer solution as a mixture solution such that: the nucleotide is added to the 3′-terminus of the nucleotide chain; the sequence of the added nucleotide is degraded to form, at the 3′-terminus of the nucleotide chain, a functional group capable of binding to the modifier; and the 3′-terminus of the nucleotide chain having the functional group thus formed is directly modified with the modifier. The reactions at three stages continuously proceed in the mixture solution. As a result, simplified procedures and reduced reaction time can be achieved.

Claims

1. A method of modifying a nucleotide chain, comprising:

providing an initial DNA nucleotide chain having at least two consecutive 2′

-deoxyuridine 5′

-monophosphate at the 3′

terminus;

preparing a reaction mixture solution comprising;

a buffer solution, the initial DNA nucleotide chain, terminal deoxynucleotidyl transferase, a divalent metal cation other than a cobalt ion, and a buffer component selected from the group consisting of 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, 2 morpholinoethanesulfonic acid, and 3,3-dimethylglutaric acid;

adding uracil-DNA glycosylase to the reaction mixture solution to degrade the at least two 2′

-deoxyuridine 5′

-monophosphate at the 3′

-terminus of the nucleotide chain, thereby forming an aldehyde group capable of binding to a modifier having an —

NH₂ group; and

modifying directly the 3′

-terminus of the nucleotide chain having the aldehyde group, with a modifier having an —

NH₂ group.

2. The method of modifying a nucleotide chain according to claim 1, wherein the divalent metal cation is a manganese or magnesium ion.

3. The method of modifying a nucleotide chain according to claim 1, wherein the modifier having an —

NH₂ group is a substance for labeling and/or conjugating the nucleotide chain.

4. The method of modifying a nucleotide chain according to claim 1, wherein the modifier having an —

NH₂ group is a substance via which the nucleotide chain is bound to a substrate.

5. The method of modifying a nucleotide chain according to claim 4, wherein the having an —

NH₂ group further comprises a thiol group.

6. The method of modifying a nucleotide chain according to claim 4, wherein the having an —

NH₂ group further comprises an alkoxysilyl group that is hydrolyzable to a silanol group.

7. The method of modifying a nucleotide chain according to claim 1, wherein the reaction mixture solution is heat-treated or alkali-treated after the step of adding but before the step of modifying.