Nucleotide derivative and DNA microarray

• US 20040186281A1
• Filed: 12/24/2003
• Published: 09/23/2004
• Est. Priority Date: 12/26/2002
• Status: Active Grant

• Current Assignee: Isao Saito, NGK Insulators Limited
• Original Assignee: Isao Saito, NGK Insulators Limited
• Inventors: Yoshida, Yasuko, Okamoto, Akimitsu, Saito, Isao
• Granted Patent: US 7,323,555 B2
• Time in Patent Office: Days
• US Class Current: 536/25.32
• 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 purine base, which is in case of existing as a member of a single-stranded sequence:

(1) a thymine/uracil derivative of which fluorescent dye emits light most intensely when the confronting base in the partner strand with which the single-stranded nucleotide sequence is hybridized is adenine;

(2) a cytosine derivative of which fluorescent dye emits light most intensely when the confronting base is guanine;

(3) an adenine derivative of which fluorescent dye emits light most intensely when the confronting base is cytosine; and

,

(4) a guanine derivative of which fluorescent dye emits light most intensely when the confronting base is cytosine or thymine/uracil.

2. The nucleotide derivative of claim 1, wherein the thymine/uracil derivative is represented by Formula (1):

3. The nucleotide derivative of claim 1, wherein the cytosine derivative is represented by Formula (2):

4. The nucleotide derivative according to claim 1, wherein the adenine derivative is represented by Formula (3):

5. The nucleotide derivative according to claim 1, wherein the guanine derivative is represented by Formula (4):

10. A single-stranded nucleotide sequence having as member at least one of the nucleotide derivatives (1) to (4) of claim 1.

11. A method for determining a single base type X in the partner strand with which the single-stranded nucleotide sequence of claim 10 is hybridized, wherein the base type X is determined as:

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

(ii) guanine when the fluorescent dye of the cytosine derivative (2) in the sequence emits light most intensely;

(iii) cytosine when the fluorescent dye of the adenine derivative (3) in the sequence emits light most intensely; and

,

(iv) thymine/uracil when the fluorescent dye of the guanine derivative (4) in the sequence emits light most intensely.

12. The method for determining a base type of claim 11, 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 the base type X in the partner strand is determined as:

(v) cytosine when the fluorescent dyes of the both of the adenine derivative (3) and the guanine derivative (4) emits light most intensely; and

,

(vi) thymine/uracil when the fluorescent dye only of the guanine derivative (4) emits light most intensely.

6. A nucleoside derivative, which is a precursor of a thymine/uracil derivative and is represented by Formula (5):

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

14. The DNA microarray of claim 13, 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 each of the nucleotide derivatives (1) to (4).

15. The DNA microarray of claim 13, which is a DNA microarray for determining the sequence of unknown one with n-nucleotides (n is 3 to 100), wherein the capture probes are a set of at least 4ⁿ whose nucleotide sequences are all different from each other, 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.

16. The DNA microarray of claim 13, 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) or not, 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.

17. The DNA microarray of claim 13, 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 the capture probes is a set of at least 4ⁿ 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).

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

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

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