Methods for analysis of nucleic acid methylation status and methods for fragmentation, labeling and immobilization of nucleic acids

a nucleic acid and methylation status technology, applied in the field of methods for analysis of nucleic acid methylation status, and methods for fragmentation, labeling and/or immobilization of nucleic acids, can solve the problems of increased cancer risk, increased methylation of dna, and associated risks, and achieve the effect of reducing the number of incubations

Inactive Publication Date: 2005-09-22
NUGEN TECH
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  • Claims
  • Application Information

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Benefits of technology

[0034] As is evident to one skilled in the art, aspects that refer to combining and incubating the resultant mixture also encompasses method embodiments which comprise incubating the various mixtures (in various combinations and/or subcombinations) so that the desired products are formed. The reaction mixtures may be combined (thus reducing the number of incubations) in any way, with one or more reaction mixtures above combined. It is understood that any combination of these incubation steps, and any single incubation step, to the extent that the incubation is performed as par...

Problems solved by technology

DNA methylation has also been associated with increased risk of cancer, as well as cancer development itself.
Alteration in DNA methylation is one manifestation of the genome instability characteristic of human tumors.

Method used

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  • Methods for analysis of nucleic acid methylation status and methods for fragmentation, labeling and immobilization of nucleic acids

Examples

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example 1

Acid-Catalyzed Fragmentation and Labeling of cDNA

[0274] Single stranded cDNA was prepared from universal human total RNA (Stratagene, La Jolla Calif.). Pooled purified cDNA product at a concentration of 145 μg / mL in water was aliquoted into each of five 200 μL PCR tubes, dispensing 13 μL or 1.89 μg into each tube. 12 μL water was then added to each tube, followed by 2.5 μL of 0.5 M glycolic acid buffer (prepared by dissolving glycolic acid in water at a concentration of 1.0 M, adjusting pH with 1 M NaOH, then diluting to 0.5 M with water). Three tubes received buffer at a pH of 3.0, and two received buffer of pH 3.5. The pH 3 tubes were heated at 95° C. for 5 minutes and 65° for 5 minutes or 30 minutes. The pH 3.5 tubes were heated at 65° C. for 5 minutes or 30 minutes, all in a MJ Research Peltier Effect thermal cycler. Tubes were then held briefly on ice for the next step.

[0275] To each tube was added 5 μL of 0.5 M acetate buffer pH 4.33 (prepared by pH adjustment of acetic acid...

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Abstract

The invention relates to methods for analysis of nucleic acid methylation status, and fragmentation and / or labeling and / or immobilization of nucleic acids. More particularly, the invention relates to methods for fragmentation and / or labeling and / or immobilization of nucleic acids comprising labeling and / or cleavage and / or immobilization at abasic sites.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 60 / 533,381, filed on Dec. 29, 2003, the disclosure of which is incorporated herein by reference in its entirety.TECHNICAL FIELD [0002] The invention relates to methods for analysis of nucleic acid methylation status, and methods for fragmentation and / or labeling and / or immobilization of nucleic acids. More particularly, the invention relates to methods for fragmentation and / or labeling and / or immobilization of nucleic acids comprising labeling and / or cleavage and / or immobilization at abasic sites. BACKGROUND [0003] Methylation of DNA is involved in both normal and abnormal cellular processes. For example, DNA methylation has been implicated in X-inactivation, genomic imprinting, and differential gene expression (such as by upregulation or silencing of genetic loci). DNA methylation plays a role in gene inactivation, cell differentiation, tumorigenesis, X-chromosome ...

Claims

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Application Information

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IPC IPC(8): C12P19/34C12Q1/68
CPCC12P19/34C12Q1/6827C12Q2525/119C12Q2521/531
Inventor KURN, NURITHDAFFORN, GEOFFREY A.
Owner NUGEN TECH
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