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Method and kit for non-specifically amplifying natural short-fragment nucleic acid

A non-specific, short-segment technology, applied in the field of molecular biology, which can solve the problems that cannot replace natural short-segment nucleic acid test samples, standards, quality control substances and reference substances, and it is difficult to achieve the size and variability of natural short-segment nucleic acids. To improve detection sensitivity and accuracy, reduce preference, and increase content

Active Publication Date: 2021-02-02
BERRYGENOMICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the length distribution of the digested products is also significantly different from that of natural short-fragment nucleic acids, and limited by the restriction sites, it is generally difficult for the digested products to reach the size of natural short-fragment nucleic acids (for example, the average length of plasma cfDNA is only about 170bp ), and its ends are not as varied as natural short-segment nucleic acids
Therefore, neither sonicated nor enzyme-digested DNA fragments can replace natural short-segment nucleic acids as test items, standards, quality controls, and references
[0004] Therefore, there is a need for a method that can obtain a large amount of natural short-segment nucleic acids to solve the problems of research and detection of such DNA

Method used

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  • Method and kit for non-specifically amplifying natural short-fragment nucleic acid
  • Method and kit for non-specifically amplifying natural short-fragment nucleic acid
  • Method and kit for non-specifically amplifying natural short-fragment nucleic acid

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1: non-specific amplification of plasma cell-free DNA according to the method of the present invention

[0045] 1. Preparation of plasma cell-free DNA

[0046] Cell-free DNA in the peripheral blood of pregnant women was extracted using a plasma cell-free DNA extraction kit (Hangzhou Berry Hekang Gene Diagnostics Technology Co., Ltd., Cat. No. R0011), and 4 ng of plasma DNA was obtained.

[0047] 2. End repair and add A

[0048] Use the NEBNext End Repair / Add dA Tail Module Kit (NEB, E7442S) to prepare the reaction system shown in Table 1 for end repair and 3' end A treatment at the same time. The reaction program is: 37°C, 20 minutes; 72°C, 20 minutes; store at 4°C. No purification was required after the reaction, and the next experiment was carried out directly.

[0049] Table 1

[0050]

[0051]

[0052] 3. Connect the connector

[0053] (1) Preparation of joints

[0054] Synthesize the sequence shown in Table 2, wherein SEQ ID NO: 1 has a deoxy...

Embodiment 2

[0085] Embodiment 2: non-specific amplification of tumor plasma cell-free DNA according to the method of the present invention

[0086] The steps of this example are the same as Example 1, the only difference is that the original DNA samples are different. Specifically, agMAXCell-Free DNA Isolation Kit (Thermo Fisher, Cat. No. A29319) was used to extract free DNA from tumor plasma to obtain 6 ng of free DNA. After non-specific amplification, its yield was 132ng, which was 22 times higher than the original plasma DNA concentration.

Embodiment 3

[0087] Example 3: Quality assessment of non-specifically amplified natural short fragment nucleic acids

[0088] Get 4ng of natural plasma cell-free DNA and the non-specific amplification product prepared according to the method of Example 1 respectively, according to the manufacturer's instructions, use the fetal chromosomal aneuploidy (T13 / T18 / T21) detection kit (Beijing Berry and Kang Company, Cat. No. R1000) to construct a sequencing library. Then, the Nextseq CN500 sequencer was used for sequencing, and the sequencing type was 36SE, and 5M data were measured for each sample. The quality of library sequencing data is shown in Table 8. Among them, Total Rds is the total number of sequences measured, Uniq% is the percentage of DNA sequences uniquely aligned to the human genome (hg19) in Total Rds, Redundancy% is the percentage of redundant reads in Total Rds, and Unimap_GC% is The GC percentage of the DNA sequence uniquely aligned to the human genome (hg19).

[0089] Table ...

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Abstract

The invention relates to a method for non-specifically amplifying natural short-fragment nucleic acid. The method comprises the following steps: (1) performing terminal repair on the natural short-fragment nucleic acid to obtain terminal-repaired nucleic acid; (2) connecting the terminal-repaired nucleic acid with a double-chain connector to obtain a connection product, wherein each chain of the double-chain connector only comprises three basic groups; (3) performing PCR amplification on the connection product by using a PCR primer with a deoxyuridine marker to obtain a PCR product, wherein the PCR primer is completely or partially complementary to one chain of the double-chain connector and only comprises three basic groups; and (4) performing enzyme digestion on the PCR product by usingan enzyme with a deoxyuridine cutting function, and performing enzyme digestion by using an enzyme with 5'->3' polymerase activity and 3'->5' exonuclease activity in the presence of a deoxynucleotidesolution to obtain a non-specific amplification product of the natural short-fragment nucleic acid, wherein the deoxynucleotide solution only comprises complementary basic groups of the basic groups which are lacked by the primer. The invention also relates to a kit for implementing the method.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method and a kit for non-specifically amplifying natural short-segment nucleic acids. Background technique [0002] Natural short-segment nucleic acid is extracellular free DNA (cell-freeDNA, cfDNA) existing in animal, plant and human body fluids, and its length is generally less than 500bp. In the process of apoptosis, the DNA in human cells is broken and secreted to the outside of the cell, thus forming cfDNA. Currently, oncology and prenatal diagnostic studies have demonstrated the use of cfDNA as a marker. However, on the one hand, the content of cfDNA in body fluids is very low, for example, the content in plasma is even lower than 10 ng / mL, and it is easy to lose and difficult to extract during the extraction process. On the other hand, cfDNA is widely used in liquid biopsy and non-invasive prenatal screening. This makes it difficult for the directly extrac...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2521/301C12Q2525/191C12N15/10C12N15/11C12Q1/6806C12Q2521/319C12Q2521/501C12Q2525/121C12Q2525/173
Inventor 田婕吴娜拉胡杨雪艳张扬张建光
Owner BERRYGENOMICS CO LTD
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