Double-end tag-specific linker for blood micro cfDNA library, kit thereof, and construction method of library

A specific and kit-based technology, applied in the biological field of molecular biology, can solve the problems of difficulty in detecting cfDNA tumor gene mutations, inability to accurately analyze base mutations, and inability to determine original mutations, and achieves reduction of cfDNA loss, high cost performance, The effect of reducing material wastage and time

Inactive Publication Date: 2019-02-01
刘强
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, there are huge difficulties in realizing accurate detection of tumors by cfDNA, especially the detection of ultra-low frequency mutations in tumors in cfDNA
First of all, the content of cfDNA in the blood is extremely low, and it is highly fragmented, with a length of only 170bp, so it is difficult to extract; at the same time, the DNA fragments secreted by tumor cells only account for one percent of cfDNA, so it is extremely difficult to detect tumor gene mutations in cfDNA
In the process of library construction and sequencing, PCR amplification will produce a series of mutations, so the possibility of accurately detecting tumor gene mutations becomes very small
At present, there are many kits on the market that can use small amount of DNA to build a library, but the DNA needs to be amplified before connecting the adapter, and the original mutation cannot be determined; the stem-loop structure of the adapter used to build a library later, although it can be identified in the sequencing. False positive results, but it is impossible to accurately analyze the base mutations that may occur during PCR in library construction

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  • Double-end tag-specific linker for blood micro cfDNA library, kit thereof, and construction method of library
  • Double-end tag-specific linker for blood micro cfDNA library, kit thereof, and construction method of library
  • Double-end tag-specific linker for blood micro cfDNA library, kit thereof, and construction method of library

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Experimental program
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Effect test

Embodiment 1

[0048] The invention provides a cfDNA library construction kit product, which includes:

[0049] Double-end label-specific adapters DA1 and DA2 for connecting cfDNA, the single-stranded primer of the adapter DA2 has 5' phosphorylation, biotin labeling and restriction endonuclease sites;

[0050] Inner primers inner A and inner B for primary amplification and primers BC* and MUP for adding sequencing adapters;

[0051] 5× End Repair Buffer for cfDNA end repair;

[0052] Ligation buffer for ligation of cfDNA and adapters.

[0053] Wherein the double-end label-specific adapter is prepared by the following method:

[0054] (1) Mix the DA1 primer with 4.5 μL of each DA2 primer in equal amounts, add 1 μL of 10× primer binding buffer, the reaction condition is 95°C for 5 minutes, and then cool down to 16°C at a rate of 1°C / min to obtain Preliminary ligation linker for complementary moiety binding. The reaction system is as follows:

[0055] Primer RA1 4.5 μL

[0056] Primer RA2...

Embodiment 2

[0082] A method for preparing a double-end tag-specific library for cervical cancer blood trace cfDNA comprises the following steps:

[0083] (1) Perform end repair on cfDNA to obtain cfDNA with an A tail at the end, the steps are as follows:

[0084] (a) According to the instruction manual of the QIAamp Circulating Nucleic Acid Kit (50) kit provided by QIAGEN, use this kit to extract free cfDNA in plasma;

[0085] (b) Add the following components to the PCR tube (5×End-repair buffer components are 250mM NaCl, 50nMMgCl 2 , 0.5mM dNTP, 250mM Tris-HCl, 50mM DTT, 5mM ATP, pH7.5; End-repair enzyme composition is obtained by mixing T4 DNA polymerase and Taq DNA polymerase. ):

[0086]

[0087] Use a PCR instrument, react at 37°C for 30min, be careful not to heat the lid, and store at 4°C.

[0088] (2) Take out the cfDNA centrifuge tube with A tail at the end, mix it evenly with the synthesized linker with random sequence tag, and carry out the ligation reaction. The compositi...

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Abstract

The invention discloses a double-end tag-specific linker for a blood micro cfDNA library, a kit thereof, and a construction method of the library. The sequence of the double-end tag-specific linker isrepresented by SEQ ID NO.1-5. The sequence of the primer set is represented by SEQ ID NO.6-9. The invention also discloses the construction method for constructing the cfDNA double-end tag-specific genome library based on random sequences. The construction method of the library is simple to operate, and does not need tube replacement or purification in preliminary PCR, so the loss of cfDNA in thelibrary construction process is avoided, and the linkage efficiency of the library is greatly improved; and experimental steps are simplified, and restriction enzyme cutting sites and biotin labelingsites are added to the linker sequence, so the efficiency of the linker is greatly improved, thereby the sensitivity of the library construction is enhanced.

Description

technical field [0001] The invention relates to the field of biotechnology in molecular biology. More specifically, it relates to a method for preparing a blood trace cfDNA double-end tag-specific linker, a kit and a genomic library, which is suitable for the detection of ultra-low frequency mutations in blood trace cfDNA. Background technique [0002] Next-generation sequencing technology is currently the most commonly used technology in high-throughput sequencing research. On the basis of first-generation Sanger sequencing that can only measure one piece of DNA at a time, next-generation sequencing has achieved high-throughput sequencing, deep sequencing and resequencing. cfDNA (cellfree DNA), that is, the DNA circulating in the blood, is the fragmented DNA released into the blood after the body's cells, white blood cells and tumor cells die; the cfDNA high-throughput sequencing technology is a kind of liquid biopsy, it only needs a few With just one milliliter of venous ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10C40B50/06C40B40/08
CPCC12N15/1013C12N15/1065C12N15/11C40B40/08C40B50/06
Inventor 刘强李婷婷李志广
Owner 刘强
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