Mutated aqp, method for detecting cancer using the same, dna chip having oligonucleotides of said mutated aqp sequence

• US 20050069872A1
• Filed: 09/10/2001
• Published: 03/31/2005
• Est. Priority Date: 09/09/2000
• Status: Active Grant

• Current Assignee: Goodgene, Inc.
• Original Assignee: Goodgene, Inc.
• Inventors: Moon, Woo-Chul, Kim, Byung-gu, Moon, Chul-so, Um, Tae-han, Song, Mi-kyung, Song, Seok-beom, Kim, Hyeung-jae, Moon, Young-ho, Shin, Chan-jae, Kim, Hwa-su, Kim, Dong-hwan
• Granted Patent: US 7,470,534 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The present invention relates to mutation genes of the AQP(aquaporin), a method for detecting cancer using mutations and expressions of the AQP and a DNA chip possessing oligonucleotides of mutated AQP base sequence. In case of the present method for detecting cancer and DNA chip using the AQP'"'"'s mutations and expressions, it is highly accurate, rapid and effective in cancer diagnosis.

Claims

1. A mutant AQP5 gene, in which at least single base was changed from SEQ ID NO:

1.

2. A method for diagnosing cancer comprising detecting presence or absence of mutation of AQP5 gene in claim 1 and expression of AQP5, AQPL1, AQP3 and/or AQP 4.

3. The method as set forth in claim 2, wherein the cancer comprises lung cancer, stomach cancer, colon cancer, prostatic cancer, and head and neck cancer.

4. The method as set forth in claim 3, wherein the cancer comprises lung cancer.

5. The method as set forth in claim 2, wherein presence or absence of mutation of AQP5 gene is detected by at least one method selected from the group consisting of immunohistochemical study, western blotting, dot blotting, ELISA (enzyme-linked immunosorbent assay), RT-PCR, nucleic acid sequencing, restriction enzyme analysis, nothern blotting, Southern blotting, RNase protection assay, in situ hybridization, SSCP (single strand conformational polymorphism) analysis, MSO hybridization (Mutant specific oligonucleotide-hybridization) assay, ARMS (amplification refractory mutation system) and DNA chip (microarray) analysis.

6. The method as set forth in claim 5, wherein the MSO hybridization assay comprises the steps of:

1) arraying oligonucleotides to nitrocellulose membrane or nylon membrane in parallel;

2) preparing biotin labeled probe DNA by using PCR with oligonucleotide primer labeled by biotin at 5′

end;

3) denaturing biotin labeled probe, followed by hybridization of probe with the membrane in

     1) and eliminating unlabelled probe by washing; and

4) treating the washed membrane with alkaline phosphatase-labeled streptavidin, combining the formed biotin labeled hybrid, and inducing the color reaction, in particular comprising the use of strip in hybridization.

7. The method as set forth in claim 5, wherein the DNA chip analysis comprises the steps of:

1) synthesizing oligonucleotide primer for each base of AQP5 cDNA, modifying 5′

end of primers with chemical linker, spotting the primers onto slide treated by special coating and thus producing oligo DNA chip;

2) preparing target DNAs by PCR amplification of coding sequence of AQP5 followed by fragmentation of the PCR products into nucleotides with 50 to 100-bp in length;

3) adding fragmented PCR products of

     2), four fluorescence-labelled dideoxynucleotides and DNA polymerase onto the oligo DNA chip in

     1), mixing them, and then performing arrayed primer extension reaction; and

4) analyzing automatically the results of arrayed primer extension reaction in

     3) by using 4 color fluorescence DNA scanner.

8. A DNA chip comprising one of nucleotide sequences of mutant AQP5 gene in claim 1.

9. An oligonucleotide chip wherein oligonucleotide primers sequences of FIG. 20 are arrayed.