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Method for reducing nucleic acid and detection method of nucleic acid

A nucleic acid and nucleic acid molecule technology, applied in the field of gene sequencing, can solve problems such as large fluctuations and instability, and achieve the effects of stable recovery, lower detection costs, and fewer purification steps.

Active Publication Date: 2022-01-18
SHENZHEN GENEPLUS CLINICAL LAB +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the prior art, after the TET enzyme oxidation step is completed, the oxidized product needs to be purified with 1.8x magnetic beads, but the median recovery rate after oxidation is only about 70%, and it is very unstable, ranging from 30% to 90%. volatile

Method used

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  • Method for reducing nucleic acid and detection method of nucleic acid
  • Method for reducing nucleic acid and detection method of nucleic acid
  • Method for reducing nucleic acid and detection method of nucleic acid

Examples

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Embodiment 1

[0092] Example 1: Same-pipe reduction

[0093] In this embodiment, unless otherwise specified, the magnetic beads used are magnetic beads purchased from Axygen Corporation of the United States, also known as Axygen 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] In this example, nucleic acid is oxidized and then ...

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Abstract

The invention relates to a method for reducing nucleic acid and a detection method of the nucleic acid. The method for reducing the nucleic acid comprises the step of directly reducing oxidized nucleic acid without purifying the oxidized nucleic acid. The oxidized nucleic acid is directly reduced, purification after oxidation and before reduction is not needed, same-tube reduction is achieved, the conversion recovery rate is remarkably increased, and the recovery rate is more stable. A purification step between an oxidation step and a reduction step is reduced, the experimental operation steps are simplified, the time is shortened, the detection cost is reduced, and meanwhile, subsequent library amplification efficiency and the conversion rate are not influenced.

Description

technical field [0001] The invention relates to the technical field of gene sequencing, in particular to a method for reducing nucleic acid and a detection 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C12N15/10C12Q1/6844C12Q1/6869
CPCC12P19/34C12N15/1013C12N15/1003C12Q1/6844C12Q1/6869C12Q2523/32C12Q2563/143C12Q2563/149
Inventor 沈璐刘佳慧姜叶钟文星赵美茹易鑫
Owner SHENZHEN GENEPLUS CLINICAL LAB
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