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Method for synthesizing nucleic acid under asymmetric loop-mediated constant temperature condition, kit and application

A synthesis method and kit technology, applied in the field of bioengineering, can solve problems such as cumbersome process and increased application cost

Active Publication Date: 2022-03-15
国科宁波生命与健康产业研究院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the main problem of the PCR method is that a special program temperature control system must be used in actual operation, which greatly increases the application cost.
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 asymmetric loop-mediated constant temperature condition, kit and application
  • Method for synthesizing nucleic acid under asymmetric loop-mediated constant temperature condition, kit and application
  • Method for synthesizing nucleic acid under asymmetric loop-mediated constant temperature condition, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1 For the amplification of fragments in influenza A virus H1N1

[0093] Type A H1N1 virus belongs to Orthomyxoviridae (Orthomyxoviridae) and Influenzavirus A (Influenzavirus A), patients will have fever, cough, fatigue, loss of appetite and so on. The nucleic acid detection of H1N1 generally uses PCR technology after reverse transcription to detect cDNA. Therefore, the new primers designed by the nucleic acid synthesis 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-TP (nucleotide sequence shown in SEQ ID NO.1) and N1-FP (nucleotide sequence shown in SEQ ID NO.2). These are designed to anneal into ringed regions by exploiting the proximity packing phenomenon.

[0094] Through the primers N1-TP and N1-FP, an asymmetric ring ...

Embodiment 2

[0110] Embodiment 2 confirms the reaction product among the embodiment 1 by the digestion of restriction enzyme

[0111] 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.

[0112] The reaction solution after the termination of the reaction in Example 1 was deposited and purified by precipitation with ethanol, and the precipitate produced was reclaimed and redissolved in ultrapure water, digested with restriction enzyme EcoRV at 37°C for 2 hours, and the sample was heated at 90mV in...

Embodiment 3

[0113] Example 3 Application of EvaGreen to Verify H1N1 Gene Amplification Reaction Products

[0114] 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.

[0115] By primer N1-TP (nucleotide sequence as shown in SEQ ID NO.1) and N1-FP (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 non-diagnostic asymmetric loop-mediated constant temperature condition, a kit and application. The method comprises the following steps: providing a nucleic acid which is sequentially composed of Rc, Fc, R, Dc and D regions in the 3'to 5 'direction, annealing the D region at the 5'end of the nucleic acid and the adjacent Dc, annealing the Rc region at the 3' end and the R region on the same chain, and thus forming an asymmetric ring structure; an Rc region and an R region at the 3'end of the nucleic acid are annealed, a complementary chain of the nucleic acid is synthesized by taking the nucleic acid as a template, and the synthesized nucleic acid sequence is called as nucleic acid A; performing a synthesis step by annealing the Fc region of the nucleic acid using a primer first oligonucleotide; the Dc region at the 3'end of the nucleic acid A and the adjacent D region are annealed and react by taking the nucleic acid as a template, so that a nucleic acid chain is continuously extended. By utilizing the nucleic acid synthesis method disclosed by the invention, the target fragment can be only about 40bp, and compared with other isothermal amplification methods, nucleic acid amplification under a constant-temperature condition can be easily realized.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular, the invention relates to a method and a kit for synthesizing nucleic acid under constant temperature conditions for non-diagnostic purposes. Background technique [0002] The polymerase chain reaction (PCR) method is considered to be the most classic method for amplifying target genes (Saiki, Gelfand et al. 1988), and is also the most commonly used technique for in vitro nucleic acid sequence amplification. However, the main problem of the PCR method is that a special program temperature control system must be used in actual operation, which greatly increases the application cost. [0003] LAMP technology (Notomi, Okayama et al.2000) can amplify the target gene under constant temperature conditions, and its core is to use four specific primers designed for six regions on the target gene to rely on a highly active strand to displace DNA polymerization Enzymes that make stran...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/6806
CPCC12Q1/6844C12Q1/6806C12Q2521/101C12Q2531/119C12Q2537/1376
Inventor 毛瑞吴欣瑶蔡挺缪青
Owner 国科宁波生命与健康产业研究院