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Settling agent for improving extraction rate of free DNA (Deoxyribonucleic Acid) in blood plasma sample

A sedimentation aid and a technology in samples, which are applied in the direction of DNA preparation and recombinant DNA technology, can solve the problems of not being able to meet the detection of micro-gene mutations, the yield of free DNA is not high enough, and the content of circulating DNA in plasma is low. Improve the detection rate, improve the extraction rate, and have a wide range of applications

Active Publication Date: 2019-04-26
麦凯(浙江)医疗器械有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, even in patients with advanced tumors, the plasma circulating DNA (cfDNA) content of most patients is very low, which brings great challenges to the subsequent detection of ctDNA
The more commonly used plasma cfDNA extraction methods in current research usually include several steps of plasma separation from whole blood, lysing, column binding, washing and elution, and free nucleic acids can be extracted from plasma. The yield of cell-free DNA extracted by the plasma cell-free DNA extraction kit QIAamp Circulating Nucleic Acid Kit (QIAGEN) is still not high enough to meet the needs of trace gene mutation detection

Method used

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  • Settling agent for improving extraction rate of free DNA (Deoxyribonucleic Acid) in blood plasma sample
  • Settling agent for improving extraction rate of free DNA (Deoxyribonucleic Acid) in blood plasma sample

Examples

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Embodiment 1

[0022] A sedimentation aid for increasing the extraction rate of free DNA in plasma samples provided in this example is composed of the following formula, that is, added to each milliliter of plasma samples:

[0023] Human Cot-1 DNA 4.3mg;

[0024] Chelex-100 cation exchange resin 1.8mg.

[0025] 1) Take 10 cases of normal separated plasma samples, 4mL for each case; divide each case of plasma into 2 parts, 2mL respectively;

[0026] 2) Add the sedimentation aid of the present invention (namely: 8.6 mg of human Cot-1 DNA, 3.6 mg of Chelex-100 cation exchange resin) to one plasma sample of each case according to the above ratio, mix well and heat at 90°C 10min, then cooled down to room temperature;

[0027] 3) Cell-free DNA was extracted from two plasma samples at the same time. The extraction kit used the QIAamp Circulating Nucleic Acid Kit nucleic acid extraction reagent (product number: 55114) from Kaiser, Germany. The specific extraction operation was as follows:

[0028...

Embodiment 2

[0039] A sedimentation aid for increasing the extraction rate of free DNA in plasma samples provided in this example is composed of the following formula, that is, added to each milliliter of plasma samples:

[0040] Human Cot-1 DNA 4.5mg;

[0041] Chelex-100 cation exchange resin 2.0mg.

[0042] 1) Take plasma samples from 10 patients with EGFR gene mutation-positive non-small cell lung cancer, 4 mL for each case; divide each case of plasma into 2 parts, 2 mL each;

[0043] 2) Add the sedimentation aid of the present invention (i.e.: 9.0 mg of human Cot-1 DNA, 4.0 mg of Chelex-100 cation exchange resin) to one plasma sample of each case according to the above ratio, mix well and heat at 90°C 10min, then cooled down to room temperature;

[0044] 3) Cell-free DNA was extracted from two plasma samples at the same time. The extraction kit used the QIAamp Circulating Nucleic Acid Kit nucleic acid extraction reagent (product number: 55114) from Kaiser, Germany. The specific extra...

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Abstract

The invention discloses a settling agent for improving the extraction rate of free DNA (Deoxyribonucleic Acid) in a blood plasma sample. The settling agent is prepared according to the following formula: 3.2 to 5.3 mg of human Cot-1 DNA and 1.4 to 2.2 mg of Chelex-100 cation exchange resin are added into each milliliter of the blood plasma sample. An experiment shows that the settling agent provided by the invention can be used for effectively removing interfering substances in blood plasma and remarkably improving the extraction yield of the free DNA, so that the detection rate of gene mutation of the free DNA of tumors can be improved (the extraction yield of the free DNA can reach 99 percent or more under the same extraction condition, so that the detection rate of the gene mutation ofthe free DNA of the tumors can be improved to 90 percent from 70 percent); and furthermore, the settling agent disclosed by the invention is convenient to use, and the extraction rate of the free DNAin the blood plasma sample can be remarkably improved only if the settling agent is added into the blood plasma sample and an existing extraction kit is used.

Description

technical field [0001] The invention relates to a sedimentation aid for improving the extraction rate of free DNA in plasma samples, and belongs to the technical field of nucleic acid extraction. Background technique [0002] As a branch of in vitro diagnosis, liquid biopsy is to diagnose the genetic status of tumor patients by collecting blood from patients, and has broad application prospects in the field of clinical medicine. Among them, tumor cell-free DNA (ctDNA), as a non-invasive tumor marker, is considered to have a good development prospect. However, even in patients with advanced tumors, the plasma circulating DNA (cfDNA) content of most patients is very low, which brings great challenges to the subsequent detection of ctDNA. The more commonly used plasma cfDNA extraction methods in current research usually include several steps of plasma separation from whole blood, lysis, column binding, washing and elution, and free nucleic acids can be extracted from plasma, b...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 鹿亚超齐民强
Owner 麦凯(浙江)医疗器械有限公司
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