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Composition, kit and purification method thereof

A composition and kit technology, applied in the field of biomedicine, can solve problems such as time-consuming and labor-intensive, inability to match automatic instruments, and inability to recycle, etc., to achieve the effect of improving recovery recovery rate, increasing sample throughput, and reducing reagent costs

Inactive Publication Date: 2022-01-18
SHENZHEN GENEPLUS CLINICAL LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the existing non-bisulfite sequencing process, the product after pyridine borane reduction contains high concentration of NaAC (about 600mM) and pyridine borane (about 1M), plus a lower pH (about 4.3), NGS magnetic beads cannot recover the reduced DNA under conventional purification operating conditions
The prior art uses commercial spin column purification methods to purify the reduced DNA, such as the IC-Spin column of Zymo Company and the Micro Bio-Spin 6 column of Bio-rad Company. The median recovery rate is only about 45%, and it is very unstable , the range is as wide as 30-70%, and the fluctuation is large
In addition, column method purification requires high-speed centrifugation equipment, and the sample throughput is limited by the centrifuge throughput. When the sample volume is large, it cannot match the automatic instrument, which is very time-consuming and labor-intensive.

Method used

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  • Composition, kit and purification method thereof
  • Composition, kit and purification method thereof
  • Composition, kit and purification method thereof

Examples

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Embodiment 1

[0093] In this embodiment, unless otherwise specified, the magnetic beads used are magnetic beads purchased from Axygen Corporation in the United States, also known as Axygen en magnetic beads, and the product name is AxyPrep Magnetic Bead Purification Kits, Cat. No. MAG-PCR-CL-250. The beads are in suspension.

[0094] In this embodiment, unless otherwise specified, "room temperature" refers to 23°C±2°C.

[0095] In this embodiment, unless otherwise specified, "80% ethanol" refers to ethanol with a concentration of 80% by volume, also known as "80% V / V ethanol", which is composed of ethanol and NF-H 2 O (nuclease-free water, also known as Nuclease-Free Water) is prepared according to the volume ratio of 80:20.

[0096] In this example, TE buffer (TE Buffer) was purchased from Invitrogen, Cat. No. 12090015. The composition is as follows: 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA.

[0097] The operation steps of this embodiment are as follows:

[0098] (1) Take 90 cases of hum...

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Abstract

The invention provides a composition, a kit and a purification method thereof. The composition comprises magnetic beads and polyethylene glycol. The composition can be used for purifying nucleic acid molecules reduced by an acidic reducing reagent, and the addition of polyethylene glycol can significantly improve the reduction recovery rate of nucleic acid and make the recovery rate more stable. As a purification system, the composition can be more conveniently matched with an automated instrument to improve the sample flux, the purified and recovered product does not affect the subsequent library amplification and the detection process of modified bases, the reagent cost is effectively reduced, and special equipment such as a high-speed centrifuge is not needed.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a composition, a kit and a purification method thereof. Background technique [0002] As early as 1925, before the identification of the DNA double helix structure, DNA methylation modification had been discovered. The most important form of DNA methylation modification is 5mC and its derivative modification, which is considered as the "fifth base" of DNA. DNA methylation plays an important role in physiological and pathological processes such as gene regulation, genetic imprinting, aging, inflammation, and tumors. Recent studies have shown that the methylation signature of cell-free DNA (hereinafter referred to as cfDNA) is an important marker for early screening of tumors. [0003] Bisulfite can deaminate cytosine (C) into uracil (U). In the subsequent PCR process, U-tolerant polymerase is used to recognize U as thymine (T), realizing C to T Transformation, so as to achieve the pu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013C12Q2525/117C12Q2527/125C12Q2563/143C12Q2563/149C12Q2523/10C12Q2523/115
Inventor 吴炜刘佳慧赵美茹沈璐钟文星易鑫伍佳豪
Owner SHENZHEN GENEPLUS CLINICAL LAB