Nucleotide derivative and DNA microarray

• US 7,323,555 B2
• Filed: 12/24/2003
• Issued: 01/29/2008
• Est. Priority Date: 12/26/2002
• Status: Expired due to Fees

• Current Assignee: Isao Saito, NGK Insulators Limited
• Original Assignee: Isao Saito, NGK Insulators Limited
• Inventors: Saito, Isao, Yoshida, Yasuko, Okamoto, Akimitsu
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/746,157
• Publication Number:
  US 20040186281A1
• Time in Patent Office: 1,497 Days
• Field of Search: 546/23.1, 546/26.6, 435/6, 436/809
• US Class Current: 536/23.1
• CPC Class Codes: C07H 19/067 with ribosyl as the saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: Y10S 436/809 Multifield plates or multicontainer arrays

Abstract

A novel nucleotide derivative, in case of existing as a member of a single-stranded sequence, undergoing a change in the fluorescent signal intensity depending on the corresponding base type in the partner strand with which the single-stranded sequence is hybridized, and which is (1) a thymine/uracil derivative emitting light most intensely when a confronting base in the partner strand with which the single-stranded nucleotide sequence is hybridized is adenine; (2) a cytosine derivative emitting light most intensely when the confronting base is guanine; (3) an adenine derivative emitting light most intensely when the confronting base is cytosine; and, (4) a guanine derivative emitting light most intensely when the confronting base is cytosine or thymine/uracil.

Claims

1. A nucleotide derivative having a fluorescent dye intercalator bound via a linker to a pyrimidine base or a purine base, which, when existing as a member of a single-stranded sequence is selected from the group consisting of:

(1) a thymine/uracil derivative represented by Formula (1);

6. A single-stranded nucleotide sequence comprising at least one of the nucleotide derivatives (1) to (4) of claim 1.

7. A method for determining a single base type X in a partner strand hybridized with the single-stranded nucleotide sequence of claim 6, comprising:

(i) determining that the base type X is adenine when the fluorescent dye of the thymine/uracil derivative (1) in the single-stranded nucleotide sequence emits light most intensely;

(ii) determining that the base type X is guanine when the fluorescent dye of the cytosine derivative (2) in the sequence emits light most intensely;

(iii) determining that the base type X is cytosine when the fluorescent dye of the adenine derivative (3) in the sequence emits light most intensely; and

,

(iv) determining that the base type X is thymine/uracil when the fluorescent dye of the guanine derivative (4) in the sequence emits light most intensely.

8. The method of claim 7, wherein two single-stranded nucleotide sequences having the adenine derivative (3) and the guanine derivative (4) in the respective identical positions are hybridized with the respective identical partner strands and wherein the method further comprises:

(v) determining that the base type X is cytosine when the fluorescent dyes of the both of the adenine derivative (3) and the guanine derivative (4) emits light most intensely; and

,

(vi) determining that the base type X is thymine/uracil when the fluorescent dye only of the guanine derivative (4) emits light most intensely.

9. A DNA microarray having as a capture probe the single-stranded nucleotide sequence of claim 6.

10. The DNA microarray of claim 9, which is a DNA microarray for detecting a single nucleotide polymorphism (SNP) in a target nucleotide sequence, wherein a set of capture probes is complementary at least with a region containing the SNP nucleotide in the target nucleotide sequence, and in each capture probe the nucleotide in the position corresponding to the SNP nucleotide in the target nucleotide sequence is selected from the group consisting of nucleotide derivatives (1), (2), (3), and (4).

11. The DNA microarray of claim 9, which is a DNA microarray for determining an unknown sequence with n-nucleotides (n is 3 to 100), wherein there is a set of at least 4 capture probes with n-nucleotides, and wherein each probe has a different nucleotide sequence, and in each capture probe each of the nucleotide derivatives (1) to (4) is in at least one of the 1st to the ‘

n’

th positions.

12. The DNA microarray of claim 9, which is a DNA microarray for detecting whether a target nucleotide sequence contains a region homologous to a known sequence region consisting of n-nucleotides (n is 3 to 100), wherein a set of capture probes is complementary with the known sequence region in the target nucleotide sequence, and in each capture probe each of the nucleotide derivatives (1) to (4) is in at least one of the 1st to the ‘

n’

th positions.

13. The DNA microarray of claim 9, which is a DNA microarray for determining the sequence of an unknown sequence region consisting of n-nucleotides (n is 3 to 100) of a target nucleotide sequence having the unknown sequence region and a known sequence region, wherein a set of at least 4 capture probes with n-nucleotides having sequences complementary with the known sequence region of the target nucleotide sequence and probe sequence regions whose nucleotide sequences are all different from each other, and at least one of the 1st to the ‘

n’

th positions in each probe sequence region is any of the nucleotide derivatives (1) to (4).

2. A nucleoside derivative that is a precursor of a thymine/uracil derivative and is represented by Formula (5):

3. A nucleoside derivative that is a precursor of a cytosine derivative and is represented by Formula (6):

4. A nucleoside derivative that is a precursor of an adenine derivative and is represented by Formula (7):

5. A nucleoside derivative that is a precursor of a guanine derivative and is represented by Formula (8):