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Method for the enrichment of circulating tumor dna

A tumor, purpose technology, applied in the field of circulating tumor DNA, which can solve problems such as interference

Active Publication Date: 2017-09-26
벨지언볼리션에스알엘
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the DNA is from the same subject and therefore has a similar sequence and would interfere in any method used to quantify or analyze ctDNA

Method used

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  • Method for the enrichment of circulating tumor dna
  • Method for the enrichment of circulating tumor dna
  • Method for the enrichment of circulating tumor dna

Examples

Experimental program
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Embodiment 1

[0102]Antibodies involved in specific binding to histone isoforms H3.1, H3.2, or H3t were biotinylated by the manufacturer's recommended method and immobilized on streptavidin-coated magnetic beads (Dynal). The beads were washed several times with loading buffer using a magnetic separation system. Serum or plasma obtained from a cancer patient is diluted with loading buffer and applied to the beads. Nucleosomes containing histone H3 variants were adsorbed to the beads. Other serum / plasma components remain in solution and are removed by magnetic separation. Wash the beads with buffer. At this point the histone H3 variant-containing nucleosomes are isolated onto beads. The ctDNA associated with the isolated nucleosomes is extracted by the phenol / chloroform method or other standard extraction methods. The extracted DNA can be analyzed for genetic or epigenetic characteristics of the cancer.

Embodiment 2

[0104] Antibodies involved in specific binding to histone isoforms H3.1, H3.2, or H3t were biotinylated by the manufacturer's recommended method and immobilized on streptavidin-coated magnetic beads (Dynal). The beads were washed several times with loading buffer using a magnetic separation system. Serum or plasma obtained from a cancer patient is diluted with loading buffer and applied to the beads. Nucleosomes containing histone H3 variants were adsorbed to the beads. Other serum / plasma components remain in solution and are removed by magnetic separation. Wash the beads with buffer. At this point the histone H3 variant-containing nucleosomes are isolated onto beads. The isolated nucleosomes are removed from the beads using elution buffer and analyzed by proteomic methods including mass spectrometry.

Embodiment 3

[0106] Circulating cell-free nucleosome levels were determined using a commercial (total) nucleosome ELISA from Roche in serum samples obtained from six patients with CRC preoperatively and at 6, 24, 48, 72 and 96 hours postoperatively , which uses common anti-nucleosomal core epitopes and tagged anti-dsDNA antibodies. The samples were then re-assayed but using anti-histone variants H3.1, H3.2 or H3t capture antibodies. Using a commercial ELISA, measured (total) nucleosome levels increased following surgical trauma, but for histone H3 variant-containing variants measured using assays employing anti-histone H3.1, H3.2 or H3t antibodies Nucleosomes are altered and diminished in response to surgery. result in image 3 displayed in .

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Abstract

The invention relates to the use of histone binding agents for detecting, isolating and / or purifying cell free nucleosomes of tumor originor circulating tumor DNA from a biological sample. The invention also relates to methods and kits using said histone binding agents.

Description

field of invention [0001] The present invention relates to a method for purifying or enriching tumor-derived circulating cell-free nucleosomes and associated circulating tumor DNA from blood, serum or plasma. Background of the invention [0002] Cellular DNA exists as a protein-nucleic acid complex called chromatin. The nucleosome is the basic unit of chromatin structure and consists of double-stranded DNA (dsDNA) wrapped around a protein complex. The DNA is wound around successive nucleosomes in what is often referred to as a "string of beads" like structure, which forms the basic structure of open chromatin, or euchromatin. In compact chromatin or heterochromatin, the strands are helical and supercoiled in a compact and complex structure. [0003] Each nucleosome in chromatin is composed of eight highly conserved core histones (containing each pair of histones H2A , H2B , H3 with H4 ) of protein complexes. Around this complex wraps about 146 base pairs (bp) of DNA....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N27/62
CPCG01N33/6875G01N27/622C12N15/1003G01N2560/00G01N2800/7028
Inventor J.V.米卡莱夫M.赫佐格M.E.埃克莱斯顿
Owner 벨지언볼리션에스알엘