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Method for synthesizing nucleic acid under constant temperature condition, kit and application

A synthesis method and kit technology, applied in the field of genetic engineering, can solve the problems of samples and reaction solutions being susceptible to external contamination, cumbersome processes, etc.

Active Publication Date: 2021-08-03
国科宁波生命与健康产业研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main problems of the PCR method are: a special program temperature control system must be used in actual operation; samples and reaction solutions are susceptible to external contamination, and the problem of false positives is more prominent
One of the limitations of this technology is that because the method’s high specificity and sensitivity depend on the properties of the four primers, the acquisition of the best primers usually requires sequence alignment, online primer design, primer screening, and specificity tests. A very cumbersome process

Method used

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  • Method for synthesizing nucleic acid under constant temperature condition, kit and application
  • Method for synthesizing nucleic acid under constant temperature condition, kit and application
  • Method for synthesizing nucleic acid under constant temperature condition, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] Example 1 For the amplification of fragments in influenza A virus H1N1

[0117] Influenza A (H1N1) virus belongs to Orthomyxoviridae family (Orthomyxoviridae) and Influenzavirus A (Influenzavirus A). Symptoms of Influenza A (H1N1) are similar to common cold. H1N1 was widespread in 2009, which caused a certain degree of panic. Because of the nucleic acid detection of H1N1, the cDNA is generally detected by PCR technology after reverse transcription. Therefore, the new primers designed by the method of the present invention can also be applied to the detection of H1N1 virus. The nucleic acid of the present invention is tried using artificially designed H1N1 (from GenBank: GQ290690.1) inserted with restriction sites as a template. The two primers used in the experiment are N1-MF (the nucleotide sequence is shown in SEQ ID NO.1) and N1-MR (the nucleotide sequence is shown in SEQ ID NO.2). These are designed to anneal into ringed regions by exploiting the proximity packin...

Embodiment 2

[0134] Embodiment 2 confirms the reaction product among the embodiment 1 by the digestion of restriction enzyme

[0135] In order to verify that the nucleic acid obtained in Example 1 of the present invention has a structural form in which complementary nucleotide sequences are linked in a single strand in a circular structure, the product was digested with restriction enzymes. If theoretical fragments can be generated by digestion, and there are no unclear band patterns and unelectrophoresed bands at high molecular weights, it can be inferred that the synthetic product of Example 1 has complementary sequences alternately linked in single strands nucleic acid within.

[0136] The reaction solution after the termination of the reaction in Example 1 is deposited and purified by precipitation with ethanol, and the resulting precipitate is recovered and re-dissolved in ultrapure water, and the restriction enzymes XbaI and XhoI are used separately and then used in combination with ...

Embodiment 3

[0137] Example 3 Application of EvaGreen to Verify H1N1 Gene Amplification Reaction Products

[0138] Similar to SYBR Green I, EvaGreen is a dye with a green excitation wavelength that binds to the minor groove region of all dsDNA double helices, and its inhibition on nucleic acid amplification reactions such as PCR is much smaller than that of the latter. In the free state, EvaGreen emits weak fluorescence, but once bound to double-stranded DNA, the fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of EvaGreen is related to the quantity of double-stranded DNA, and the quantity of double-stranded DNA existing in the nucleic acid amplification system can be detected according to the fluorescent signal.

[0139] By primer N1-MF (nucleotide sequence as shown in SEQ ID NO.1) and N1-MR (nucleotide sequence as shown in SEQ ID NO.2), the combination of the reaction solution of the method for synthesizing nucleic acid of the present invention is as follows...

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Abstract

The invention discloses a method for synthesizing nucleic acid under a constant temperature condition, a kit and application. The method comprises the steps that a nucleic acid is provided, the nucleic acid is sequentially composed of an M1c region, an F1c region, an M region, an R1 region and an M2c region in the 3'to 5 'direction, the M region comprises an M1 region and an M2 region, and annealing of the M2c region at the 5'end and the M1c region at the 3' end of the nucleic acid and the M region on the same chain can form a closed ring structure; a first oligonucleotide I and a second oligonucleotide II of a primer are respectively annealed with an F1c region and an R1c region of the nucleic acid, and the nucleic acid is used as a template to react so that a nucleic acid chain continuously extends. According to the nucleic acid synthesis method disclosed by the invention, the length of the core primer is about 30bp and is shortened by 10bp compared with the core primer which is about 40bp and is used by LAMP (Loop-Mediated Isothermal Amplification) and CAMP (Cyclic Amplified Polymorphism), so that about 1 / 4 of primer cost can be saved, and meanwhile, the reaction rate of nucleic acid synthesis is also improved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular, the invention relates to a method, kit and application for synthesizing nucleic acid under constant temperature conditions. Background technique [0002] Based on the complementary base pairing proposed by Watson and Crick in the 1950s, the analysis method of nucleotide sequence complementarity can directly analyze the genetic characteristics carried by genes. This analysis is a very powerful method for identifying genetic diseases, cancerous changes, microbes, and more. However, when the target gene content in the sample is very small, it is generally difficult to detect, and the target gene must be amplified or the detection signal must be amplified. As a method for amplifying target genes, the PCR method is considered to be the most classical method (Saiki, Gelfand et al. 1988), and it is also the most commonly used technique for in vitro nucleic acid sequence ampli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12P19/34C12N15/11
CPCC12Q1/6844C12P19/34C12Q2531/119C12Q2527/125C12Q2521/107C12Q2563/107
Inventor 毛瑞王天祚蔡挺
Owner 国科宁波生命与健康产业研究院
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