METHOD OF ACQUIRING INFORMATION REGARDING BASE SEQUENCE AND INFORMATION READING DEVICE FOR THE SAME

• US 20090233280A1
• Filed: 12/26/2006
• Published: 09/17/2009
• Est. Priority Date: 12/28/2005
• Status: Abandoned Application

• Current Assignee: Canon Kabushiki Kaisha (Canon Inc.)
• Original Assignee: Canon Kabushiki Kaisha (Canon Inc.)
• Inventors: Nomoto, Tsuyoshi, Kubo, Wataru
• Application Number:
  US12/093,138
• Publication Number:
  US 20090233280A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels

Abstract

Information regarding a base sequence of a target nucleic acid is acquired by preparing a sample comprising the target nucleic acid hybridized with a primer or a sample comprising the target nucleic acid containing a promoter sequence, a polymerase, a nucleotide derivative having an electrochemically convertible moiety; allowing the three components to coexist in a solvent; and detecting whether the nucleotide derivative is introduced into the primer or a transcription product of the target nucleic acid or not using an electrochemical reaction.

Claims

1. A method of acquiring information regarding a base sequence, comprising:

preparing multiple kinds of nucleotide derivatives each having a removable moiety which is electrochemically removable from the nucleotide derivatives, a double-stranded sample made of a target nucleic acid and a primer, and a polymerase;

elongating the primer by one base with one kind of the nucleotide derivatives by allowing the sample, the polymerase, and the nucleotide derivatives to coexist in a solvent;

applying a voltage to the sample; and

detecting an electric signal due to removal of the removable moiety from the nucleotide derivative which has been incorporated in the primer,

wherein the nucleotide derivative is capped with the removable moiety such that it acts as a chain terminator on its incorporation into a growing nucleic acid strand.

18. The method of acquiring information regarding a base sequence according to claim 1, wherein the removable moiety is removed from the nucleotide derivative as a result of an electrochemical reaction caused by applying the voltage.

2. A method of acquiring information regarding a base sequence, comprising:

preparing a sample comprising a target nucleic acid hybridized with a primer or a sample comprising a target nucleic acid containing a promoter sequence, a polymerase, and a nucleotide derivative having an electrochemically convertible moiety capable of undergoing a reaction of removal from the nucleotide derivative, substitution in the nucleotide derivative, or addition to the nucleotide derivative owing to the electrochemical conversion;

allowing the sample, the polymerase, and the nucleotide derivative to coexist in a solvent; and

detecting whether the nucleotide derivative is introduced into the primer or a transcription product of the target nucleic acid or not by electrochemically converting the electrochemically convertible moiety,

wherein the nucleotide derivative is capped with the electrochemically convertible moiety such that it acts as a chain terminator on its incorporation into a growing nucleic acid strand.

16. The method of acquiring information regarding a base sequence according to claim 2, wherein the allowing step and the detecting step are repeated successively using the same electrode.

17. The method of acquiring information regarding a base sequence according to claim 2, wherein the electrochemically convertible moiety is converted by applying a voltage to a working electrode as swept in a negative direction.

3. A method of acquiring information regarding a base sequence, comprising:

preparing a sample comprising a target nucleic acid hybridized with a primer or a sample comprising a target nucleic acid containing a promoter sequence, a polymerase, and multiple kinds of nucleotide derivatives each of which has an electrochemically convertible moiety capable of undergoing a reaction of removal from the nucleotide derivative, substitution in the nucleotide derivative, or addition to the nucleotide derivative owing to the electrochemical conversion and which are electrochemically distinguishable from one another;

allowing the sample, the polymerase, and the multiple kinds of the nucleotide derivatives to coexist in a solvent; and

identifying, among the multiple kinds of the nucleotide derivatives, a nucleotide derivative which has been introduced into the primer or a transcription product of the target nucleic acid by electrochemically converting the electrochemically convertible moiety thereof,

wherein each of the nucleotide derivatives is capped with the electrochemically convertible moiety such that it acts as a chain terminator on its incorporation into a growing nucleic acid strand.

4-7. -7. (canceled)

8. A base sequence information acquiring method for acquiring information regarding a base at an information acquiring position in a target nucleic acid, comprising the steps of:

(1) preparing;

the target nucleic acid;

a primer which recognizes at least a part of a 3′

-side region including a base prior in a 3′

-side direction to a base at an information acquiring position in the target nucleic acid, and which hybridizes with the 3′

-side region;

a polymerase; and

a nucleotide derivative having an electrochemically convertible moiety capable of undergoing a reaction of removal from the nucleotide derivative, substitution in the nucleotide derivative, or addition to the nucleotide derivative owing to the electrochemical conversion;

(2) hybridizing the 3′

-side region including the base adjacent in a 3′

-direction to the base at the information acquiring position in the target nucleic acid, with the primer;

(3) allowing the target nucleic acid having the hybridized region to coexist with the nucleotide derivative under a presence of the polymerase; and

(4) acquiring information regarding the base at the information acquiring position by detecting occurrence or absence of incorporation of the nucleotide derivative into a 3′

-terminal position of a strand including the primer, which position corresponds to the information acquiring position, based on electrochemical conversion of the electrochemically convertible moiety of the nucleotide derivative,

wherein the nucleotide derivative is capped with the electrochemically convertible moiety such that it acts as a chain terminator on its incorporation into a growing nucleic acid strand.

9. The base sequence information acquiring method according to claim 8, wherein the hybridization is performed using a primer which recognizes the entire 3′

-side region.

10. The base sequence information acquiring method according to claim 8, wherein the primer recognizes and hybridizes with a part of the 3′

-side region, and the entire 3′

-side region is made double-stranded by elongating a 3′

-terminal of the primer hybridized with the nucleic acid.

11. The base sequence information acquiring method according to claim 8, wherein:

the target nucleic acid is a DNA;

the polymerase to be used is a DNA polymerase;

the nucleotide derivative to be used comprises at least one of a 2′

-deoxyadenosine 5′

-triphosphate derivative, a 2′

-deoxycytidine 5′

-triphosphate derivative, a 2′

-deoxyguanosine 5′

-triphosphate derivative, and a 2′

-deoxythymidine 5′

-triphosphate derivative each of which has an electrochemically convertible moiety and which are electrochemically distinguishable from one another.

12. A base sequence analyzing method for analyzing a base sequence of a subject region to be analyzed in a target nucleic acid, comprising the steps of:

(1) preparing;

the target nucleic acid;

a primer which recognizes a 3′

-side region including a base adjacent in a 3′

-side direction to the subject region to be analyzed in the target nucleic acid, and which hybridizes with the 3′

-side region;

a DNA polymerase; and

a set of nucleotide derivatives including a 2′

-deoxyadenosine 5′

-triphosphate derivative, a 2′

-deoxycytidine 5′

-triphosphate derivative, a 2′

-deoxyguanosine 5′

-triphosphate derivative, and a 2′

-deoxythymidine 5′

-triphosphate derivative each of which has an electrochemically convertible moiety capable of undergoing a reaction of removal from the nucleotide derivative, substitution in the nucleotide derivative, or addition to the nucleotide derivative owing to the electrochemical conversion and which are electrochemically distinguishable from one another;

(2) hybridizing the target nucleic acid with the primer;

(3) elongating the primer by one base by allowing the target nucleic acid hybridized with the primer, the set of nucleotide derivatives, and the DNA polymerase to coexist so that a nucleotide derivative selected from the set of nucleotide derivatives is incorporated into a 3′

-terminal of the primer hybridized with the target nucleic acid;

(4) identifying a base in the subject region to be analyzed which is present at a position corresponding to the nucleotide derivative which has been incorporated into the 3′

-terminal of the elongated primer, by identifying the incorporated nucleotide derivative based on electrochemical conversion of the electrochemically convertible moiety in the incorporated nucleotide derivative; and

(5) repeating the steps (3) and (4) depending on the number of bases when the number of bases in the subject region to be analyzed is 2 or more, wherein

an additional elongation reaction of the elongated primer by the DNA polymerase is inhibited after the primer is elongated by one base in the step (3) owing to a presence of the electrochemically convertible moiety, and an additional elongation reaction of the elongated primer by the DNA polymerase is allowed to proceed after the electrochemical conversion of the electrochemically convertible moiety in the step (4) owing to a reaction of removal from, substitution in, or addition to the incorporated nucleotide derivative,

wherein each of the nucleotide derivatives is capped with the electrochemically convertible moiety such that it acts as a chain terminator on its incorporation into a growing nucleic acid strand.

14. The base sequence analyzing method according to claim 12, wherein the polymerase is immobilized onto an electrode, and the electric signal is determined using the electrode.

13. A base sequence analyzing method for analyzing a base sequence of a subject region to be analyzed in a target nucleic acid, comprising the steps of:

(1) preparing;

an RNA polymerase;

the target nucleic acid containing a promoter sequence for the RNA polymerase; and

a set of nucleotide derivatives including an adenosine 5′

-triphosphate derivative, a cytidine 5′

-triphosphate derivative, a guanosine 5′

-triphosphate derivative, and a uridine 5′

-triphosphate derivative each of which has an electrochemically convertible moiety capable of undergoing a reaction of removal from the nucleotide derivative, substitution in the nucleotide derivative, or addition to the nucleotide derivative owing to the electrochemical conversion and which are electrochemically distinguishable from one another;

(2) transcribing by one base the target nucleic acid which is located downstream of the promoter sequence by allowing the target nucleic acid, the set of nucleotide derivatives, and the RNA polymerase to coexist so that a nucleotide derivative selected from the set of nucleotide derivatives is incorporated into the target nucleic acid;

(3) identifying a base in the target nucleic acid, which is present at a position corresponding to the nucleotide derivative which has been incorporated into a 3′

-terminal of the transcription product, by identifying the incorporated nucleotide derivative based on electrochemical conversion of the electrochemically convertible moiety in the incorporated nucleotide derivative;

(4) repeating the steps (2) and (3) depending on the number of bases when the number of bases in the subject region to be analyzed is 2 or more, wherein

an additional transcription reaction by the RNA polymerase is inhibited after the target nucleic acid is transcribed by one base in the step (2) owing to a presence of the electrochemically convertible moiety, and an additional transcription reaction by the RNA polymerase is allowed to proceed after the electrochemical conversion of the electrochemically convertible moiety in the step (3) owing to a reaction of removal from, substitution in, or addition to the incorporated nucleotide derivative,

wherein each of the nucleotide derivatives is capped with the electrochemically convertible moiety such that it acts as a chain terminator on its incorporation into a growing nucleic acid strand.

19. The base sequence analyzing method according to claim 13, wherein the polymerase is immobilized onto an electrode, and the electric signal is determined using the electrode.

15. (canceled)