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Detecting method of low-frequency DNA (Deoxyribonucleic Acid) mutation

A detection method and DNA library technology, applied in the field of detection of low-frequency DNA mutations, can solve problems such as detection data errors

Inactive Publication Date: 2019-04-26
北京中源维康基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection data errors caused by other types of DNA oxidative damage should not be underestimated ( figure 1 )

Method used

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  • Detecting method of low-frequency DNA (Deoxyribonucleic Acid) mutation
  • Detecting method of low-frequency DNA (Deoxyribonucleic Acid) mutation
  • Detecting method of low-frequency DNA (Deoxyribonucleic Acid) mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1D

[0057] Example 1DNA sample preparation

[0058] Take 30ml of whole blood from a patient with non-small cell lung cancer, and transport it at room temperature with 3 BCT tubes from Streck Company, and the transport time is about 46 hours.

[0059] Plasma was separated by a two-step centrifugation method, that is, the whole blood was centrifuged at 1600g for 10 minutes to obtain the supernatant; the supernatant of the previous step was centrifuged at 16000g for 10 minutes, and the obtained supernatant was plasma.

[0060] Two parts of the separated plasma were taken and immediately used the QIAamp Circulating Nucleic Acid Kit from Qiagen to extract the total free DNA respectively. The extraction amount is shown in Table 1.

[0061] Table 1

[0062] sample name

Embodiment 2

[0063] Example 2 DNA Oxidative Damage Repair

[0064] One of the DNA samples prepared in Example 1 was taken, and the DNA oxidative damage repair reaction mixture was prepared referring to the ratio in Table 2.

[0065] Table 2

[0066]

[0067] DNA repair buffer and enzyme mixture are FFPE DNA Repair Mix system of NEB Company.

[0068] The above system was transferred into a PCR machine, at 20° C. for 15 minutes, to repair the oxidative damage of DNA. Then, 186 μl of AMPure XP magnetic beads (purchased from Beckman) were added for purification, and 50 μl of nuclease-free water was used for elution.

Embodiment 3

[0069] Example 3 End completion of DNA fragments and addition of A bases at the 3' end

[0070] Take the total free DNA fragments repaired in Example 2, and prepare the reaction mixture according to the ratio in Table 3. In this example, KAPA Hyper Prep Kit was used for end repair and A buffer and end repair and A enzyme mixture.

[0071] table 3

[0072]

[0073] Move the above system into the PCR machine, and set up the program according to Table 4 to carry out the reaction.

[0074] Table 4

[0075]

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Abstract

The invention discloses a joint mixture for DNA (Deoxyribonucleic Acid) library construction and a detecting method for low-frequency DNA (Deoxyribonucleic Acid) mutation by utilizing the joint mixture. According to the detecting method disclosed by the invention, false positive mutation generated by oxidative damage, PCR (Polymerase Chain Reaction) guiding or sequencing can be removed by repairing the oxidative damage of DNA fragments before library construction, guiding specific internal reference sequences to each DNA fragment and supplementing by a unique data analysis method, so that thesensitivity and the specificity of low-frequency DNA mutation detection can be increased.

Description

technical field [0001] The invention belongs to the technical field of gene detection, in particular to a detection method for low-frequency DNA mutations. Background technique [0002] With the deepening of molecular biology research, DNA carrying genetic information has been proved to be closely related to human health, especially more and more diseases have been confirmed to be related to gene or protein variation. In this context, the clinical application prospect of detecting human DNA by molecular biological methods is very broad. At present, widely used DNA mutation detection methods include polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH), gene chip (gene chip), Sanger sequencing and so on. [0003] Low-frequency mutations refer to extremely rare gene mutations that exist in the background of a large number of wild gene sequences. The most common example of low-frequency mutations is gene mutations that occur in tumor cells. Many somatic m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6876C12Q1/6869
CPCC12Q1/6876C12Q1/6869C12Q2600/166C12Q2525/191C12Q2521/501
Inventor 李同恩张振宇朱兵刘宇飞陈兴京刘明明林鑫
Owner 北京中源维康基因科技有限公司
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