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

• US 8,012,684 B2
• Filed: 06/04/2007
• Issued: 09/06/2011
• Est. Priority Date: 09/09/2000
• Status: Expired due to Term

• Current Assignee: Goodgene, Inc.
• Original Assignee: Goodgene, Inc.
• Inventors: 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, Moon, Woo-chul
• Primary Examiner(s): SITTON, JEHANNE SOUAYA
• Application Number:
  US11/757,689
• Publication Number:
  US 20080312094A1
• Time in Patent Office: 1,555 Days
• Field of Search: None
• 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 method of diagnosing cancer in a human subject comprising obtaining a sample containing nucleic acid from the subject, and detecting in the sample, the presence of at least one mutation of SEQ ID NO:

1 present within the subject'"'"'s AQP5 gene, wherein the presence of at least one mutation of SEQ ID NO;

1 is detected by at least one method selected from the group consisting of dot blotting, RT-PCR, nucleic acid sequencing restriction enzyme analysis, northern blotting, southern blotting, RNase protection assay, in situ hybridizatiaon, SSCP (single strand conformational polymorphism) analysis, MSO hybridizatiaon (Mutant specific oligonucleotide-hybridization) assay, ARMS (amplification refractory mutation system) and DNA chip (microarray) analysis.

2. The method as set forth in claim 1, wherein the cancer is selected from the group consisting of lung cancer, stomach cancer, colon cancer, prostatic cancer, and head and neck cancer.

3. The method as set forth in claim 2, wherein the cancer is lung cancer.

4. The method as set forth in claim 1, wherein the MSO hybridization assay comprises the steps of:

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

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

end;

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

     1) and eliminating unbound DNA by washing; and

4) treating the washed membrane with alkaline phosphatase-labeled streptavidin, wherein locations of mutations are identified by color development induced by the treatment with alkaline phosphatase-labeled streptavidin.

5. The method as set forth in claim 1, wherein the DNA chip analysis comprises the steps of:

1) synthesizing oligonucleotide primers so that each base of AQP5 cDNA is analyzed by two different oligonucleotides such that one is for sense and the other is for antisense, modifying the 5′

end of primers with a chemical linker, and spotting the primers onto a slide to provide an oligo DNA chip;

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

3) adding the fragmented PCR products of

     2) and 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 the results of the arrayed primer extension reaction in

     3) by using a four color fluorescence DNA scanner.

6. The method as set forth in claim 5, wherein the chemical linker comprises an amine.