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Epigenetic markers of colorectal cancers and diagnostic methods using the same

A large intestine and coordinate technology, applied in biochemical equipment and methods, microbial measurement/testing, nucleotide library, etc., can solve problems such as expensive, low patient willingness to swallow, invasiveness, etc.

Active Publication Date: 2013-09-18
CLINICAL GENOMICS PTY LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the more sensitive tests are quite invasive and expensive, so patients are less willing to swallow

Method used

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  • Epigenetic markers of colorectal cancers and diagnostic methods using the same
  • Epigenetic markers of colorectal cancers and diagnostic methods using the same
  • Epigenetic markers of colorectal cancers and diagnostic methods using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[1475] Bisulfite labeling was employed to generate methylated and unmethylated genomic fractions as described below. Briefly, DNA was digested with the methylation-insensitive enzymes MspI and TaqI and treated with sodium bisulfite under non-denaturing conditions so that if it was unmethylated, the DNA left by each restriction enzyme Cytosines in the 5'-CG single-stranded overhang will be converted to uracil, but will remain unconverted if methylated. Separate adapters with 5'-CG or 5'-CA overhangs were ligated to provide the ligated methylated and unmethylated portions, respectively. After incorporation of the second primer by random priming copies of the reverse strand, this common primer is used in combination with the appropriate forward primer to amplify the methylated and unmethylated fractions.

[1476] In detail, tumor samples and matched normal DNA samples from 8 patients were processed and analyzed as described below.

[1477] 1. Using a Bioruptor UCD-200 sonicator...

Embodiment 2

[1523] Methylated DNA fractions were prepared from bisulfite-treated DNA of colorectal cancer cell lines HCT116, HT29 and SW480 and from DNA isolated from whole blood using the biotin capture method described below , and the library of methylated DNA was sequenced using the Applied Biosystems SOliD sequencing system. Briefly, DNA was cut and a modified SOLiD P2 adapter was ligated to the cut DNA. The DNA was then cleaved with Csp61 (cleavage site G'TAC) and ligated with a modified SOLiD P1 linker. The DNA is then denatured and treated with sodium bisulfite to convert all unmethylated cytosines to uracils. The bisulfite-treated DNA was copied using the modified P2 primer, and the original uracil-containing bisulfite-treated DNA strand was removed. The P1 forward primer was then used to prime forward strand synthesis in the presence of biotin-dCTP. Biotin dCTP is thus incorporated at positions in the original DNA that contained methylated cytosines and were thus not converted...

Embodiment 3

[1550] DNA methylation profiles of selected genes in colorectal cancer DNA and normal tissue DNA

[1551] Primers were designed for methylation status-independent amplification of promoter regions of genes and / or gene sets identified in previous examples. Gene coordinates, primer coordinates and chromosome coordinates of the amplicons are shown in Table 5.

[1552] Primers were used for PCR from 10 colorectal cancer specimens, their bisulfite-treated DNA from matched normal tissues, and normal blood DNA. Use Promega GoTaq master mix (without SybrGreen), 4mM MgCl 2 Amplification was performed with 200 nM primers and 10 ng input DNA. Cycling conditions were 95°C for 2 minutes (1 cycle), followed by 50 cycles of the following: 95°C for 15 seconds; N°C for 30 seconds; 72°C for 30 seconds, where the annealing temperature N for each amplicon is shown in Table 5. For some amplicons, an additional 200 μM dATP and dTTP were added to enable comparable amplification of methylated and un...

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Abstract

The present invention relates generally to nucleic acid molecules in respect of which changes to DNA methylation levels are indicative of the onset or predisposition to the onset of a neoplasm. More particularly, the present invention is directed to nucleic acid molecules in respect of which changes to DNA methylation levels are indicative of the onset and / or progression of a large intestine neoplasm, such as an adenoma or adenocarcinoma. The DNA methylation status of the present invention is useful in a range of applications including, but not limited to, those relating to the diagnosis and / or monitoring of colorectal neoplasms, such as colorectal adenocarcinomas. Accordingly, in a related aspect the present invention is directed to a method of screening for the onset, predisposition to the onset and / or progression of a neoplasm by screening for modulation in DNA methylation of one or more nucleic acid molecules.

Description

field of invention [0001] The present invention relates generally to nucleic acid molecules for which changes in DNA methylation levels are indicative of tumor initiation or a predisposition to tumor initiation. More specifically, the present invention relates to nucleic acid molecules for which changes in DNA methylation levels are indicative of colorectal neoplasia (eg, adenoma or adenocarcinoma) initiation and / or progression. The DNA methylation status of the invention is useful in a range of applications including, but not limited to, those involving the diagnosis and / or monitoring of colorectal neoplasms, such as colorectal adenocarcinoma. Accordingly, in a related aspect, the invention relates to a method of screening for tumor initiation, initiation propensity and / or progression by screening for modulation of DNA methylation of one or more nucleic acid molecules. Background technique [0002] Colorectal cancer includes cancerous growths in the colon, rectum, and cecu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6886C40B40/06C12Q2600/158C12Q2600/154
Inventor J·P·罗斯H·德鲁M·巴克利P·L·莫洛伊S·M·米切尔K·R·迪辛Z-Z·徐
Owner CLINICAL GENOMICS PTY LTD
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