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Protective agent, preservation method and application of low-concentration DNA reference material

A standard substance and storage method technology, applied in the direction of DNA preparation, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problem of low concentration, achieve good stability, realize the traceability of the value, and ensure the effect of accuracy and reliability

Active Publication Date: 2019-11-05
SHANGHAI INST OF MEASUREMENT & TESTING TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] CN106011133A discloses a small DNA molecular weight standard, standard substance plasmid and preparation method thereof, utilizing overlapping polymerase chain reaction, restriction endonuclease digestion And DNA ligation and other methods to construct small DNA sequences of 100bp, 200bp, 300bp, 400bp, 500bp, 600bp and 700bp on a plasmid, which can be cut out after being completely digested by a single restriction enzyme. Seven bands with uniform brightness, the present invention is used to prepare DNA standard reference substance, which is used to indicate the relative size of DNA molecular weight in agarose electrophoresis, but the above-mentioned DNA standard reference substance does not have the characteristics of low concentration, and cannot be used as a target sequence for study ctDNA

Method used

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  • Protective agent, preservation method and application of low-concentration DNA reference material
  • Protective agent, preservation method and application of low-concentration DNA reference material
  • Protective agent, preservation method and application of low-concentration DNA reference material

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Effect test

Embodiment 1

[0055] Example 1 Preparation of TP53 gene fragment

[0056] This embodiment adopts the artificial synthesis method to prepare the TP53 gene fragment standard substance, the method is as follows:

[0057] (1) Determine the TP53 gene sequence (NCBI Gene ID: 7157) according to the GeneBank database, select a 300bp gene sequence with a specific mutation as the target fragment, first synthesize a short-chain DNA containing the ctDNA sequence, and then pass PCR The short-chain DNA is connected to obtain a 300bp long-chain DNA as shown in SEQ ID NO:4;

[0058] (2) Cloning the 300bp DNA shown in SEQ ID NO:4 into the PUC57 vector to construct a plasmid DNA containing the target sequence;

[0059] (3) Using the plasmid DNA as a template, a double-stranded DNA with a length of 300 bp was amplified by PCR, which was a candidate for the standard substance of the TP53 fragment.

Embodiment 2

[0060] Example 2 Sequence verification of TP53 gene fragment

[0061] In this example, the first-generation sequencing method was used to sequence and verify the constructed plasmid DNA to identify whether the target gene fragment of TP53 had been cloned into the plasmid. Sequencing results showed that the correctness of the inserted target sequence was 100%, and the constructed plasmid could be used as a template to synthesize a double-stranded PCR product.

[0062] Subsequently, independent bidirectional sequencing was performed on the PCR amplification product to verify whether the sequence of the amplified fragment was correct. The results proved that the correctness of each amplified target sequence was 100%, completely consistent with the designed gene sequence.

Embodiment 3

[0063] Example 3 Verification of the purity of the TP53 gene fragment

[0064] This embodiment uses three methods to jointly verify the purity of the TP53 gene fragment:

[0065] The first method uses agarose gel electrophoresis for identification, specifically: take 10 μL of TP53 gene fragment samples and load them, and use 3% agarose gel electrophoresis with a voltage of 90V and a time of 30 minutes. After the electrophoresis is completed, stain the gel. The imager analyzes the DNA bands, and judges the purity of the nucleic acid according to whether there are any bands in the picture;

[0066] The results of agarose gel electrophoresis are shown in Figure 1(A). A single DNA band can be clearly seen in lane 1 without RNA and protein contamination, which proves that the prepared TP53 gene fragment standard substance has a high purity.

[0067] The second method uses ultraviolet spectrophotometry for identification. By detecting the ultraviolet light absorbance of the sample ...

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Abstract

The invention provides a protective agent, a preservation method and application of a low-concentration DNA reference material. The protective agent comprises salmon sperm DNA. A sequence of a TP53 gene with the length of 300 bp is selected as a target fragment to develop a DNA reference material with a low-concentration level, the salmon sperm DNA with the concentration of 20 [mu]g / mL to 100 [mu]g / mL is taken as a protective agent for protecting, a the DNA reference material is stored in a low-absorption centrifuge pipe and placed in a freezing condition of -15 DEG C to -20 DEG C, and the concentration stability of the DNA reference material is maintained within 6 months; the DNA reference material has good uniformity and stability, the accurate reliability of a biological frontier detection technology are ensured, the detection ability of clinical detection and early diagnosis of cancer is improved, and the reliable measurement support is provided for precision medical plans.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a protective agent, preservation method and application of a low-concentration DNA standard substance. Background technique [0002] Abnormal expression of genes is an important factor causing diseases. In the process of diagnosis and treatment of diseases, only by accurately targeting and quantifying abnormal genes can precise treatment of diseases be carried out. Due to the complexity of the human body environment and the low level of abnormal gene expression, accurate and reliable detection of low-concentration target genes is an important challenge for the clinical application of precision medicine. Circulating tumor DNA (ctDNA) is a class of tumor markers with broad application prospects, which can be used non-invasively for early diagnosis of tumors, detection of development process, prognosis judgment, and personalized drug guidance. However, the content of ctDNA is very low, ac...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6876
CPCC12N15/10C12Q1/6876
Inventor 闻艳丽刘刚梁文杨雪杨镇州李兰英王乐乐许丽李妍罗超
Owner SHANGHAI INST OF MEASUREMENT & TESTING TECH
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