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A method for synthesizing nucleic acid under constant temperature conditions

A nucleic acid and conditional technology, applied in the field of genetic engineering, can solve problems such as instability, errors, contamination of samples and reaction solutions, etc.

Active Publication Date: 2020-07-17
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the PCR method obviously has the following problems: a special program temperature control system must be used in actual operation; the exponential rise of the amplification reaction makes it difficult to quantify; samples and reaction solutions are easily contaminated by external sources, and the problem of false positives is more prominent
However, foreign genes are introduced into the primer sequences of this method, which will greatly increase the possibility of mismatching and lead to erroneous results
In addition, in the closed loop structure in the initial structure, the overlapped ring portion is only about 20 base pairs, which will weaken the intermolecular hydrogen bonds and make the helical loop unstable. At the same time, if the length of the foreign gene is too long, it will Affects the helical loop formation process

Method used

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  • A method for synthesizing nucleic acid under constant temperature conditions
  • A method for synthesizing nucleic acid under constant temperature conditions
  • A method for synthesizing nucleic acid under constant temperature conditions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1 For the amplification of fragments in MERS-orf1b

[0113] In the present invention, the nucleic acid with complementary strands connected into a single strand in the form of a helical loop was attempted using MERS-orf1b (from GenBank: NM_001012270.1) as a template. Four primers were used in the experiment, namely Mo1bHF, Mo1bHR, Mo1bF2 and Mo1bR2. Mo1bF2 and Mo1bR2 are outer primers that replace the first nucleic acid obtained from Mo1bHF and Mo1bHR respectively. Because the outer primer after Mo1bHF (or Mo1bHR) synthesis is used as the primer for the synthesis origin of the complementary chain. These are designed to anneal into ringed regions by exploiting the proximity packing phenomenon. In addition, setting these primers at high concentrations allows the annealing of Mo1bHF (or Mo1bHR) to occur preferentially.

[0114] The nucleotide sequences constituting each primer are shown in the sequence listing, and the structural features of the primers are summ...

Embodiment 2

[0145] Example 2 Confirmation of Reaction Products by Restriction Enzyme Digestion

[0146] In order to elucidate the structure of the nucleic acid obtained in Example 1 of the present invention having complementary nucleotide sequences linked in a single strand in a circular structure, the product was digested with restriction enzymes. Any of these products would be expected to be native if the theoretical fragments would be produced by digestion, while disappearing as observed in Example 1 at high molecular weights resulting in an unclear smeared band pattern and bands that were not electrophoresed. The invention has nucleic acids in which complementary sequences are alternately linked within a single strand.

[0147] In Example 1, the reaction solution was deposited and purified by treatment with phenol and precipitation with ethanol, and the resulting precipitate was recovered and redissolved in ultrapure water, digested with restriction enzyme HindIII at 37° C. for 2 hour...

Embodiment 3

[0151] Embodiment 3 application EvaGreen verification reaction product

[0152] 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.

[0153] Combinations of reaction solutions for the method of synthesizing the nucleic acid of the present invention using these primers are shown below.

[0154] Reaction solution mix (25 μL)

[0155] 20mM Tris-HCl pH8.8

[0156] 10mM KCl

[0157] 10mM (NH 4 ) 2 SO ...

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Abstract

The invention relates to the technical field of gene engineering and in particular relates to a method for synthesizing nucleic acid under a constant-temperature condition. The method comprises the following steps: 1) providing a nucleic acid, wherein the 3' end of the nucleic acid has an F1r region for annealing with an F1c region on the same chain, and simultaneously annealing the F1r region and the F1c region to form a spiral ring; 2) synthesizing an own complementary chain by taking the nucleic acid of the step 1) as a template; 3) carrying out complementary chain synthesis through a polymerase catalytic chain replacement type complementary chain synthesis reaction, so as to replace the complementary chain synthesized in the step 2). According to the method provided by the invention, a nucleotide sequence provided at a 5'-side of a primer of oligonucleotide is basically the same as a region which is synthesized by taking the primer as a synthesis starting point. The nucleic acid synthesis based on a constant-temperature reaction is realized on the basis of the composition of a pure reagent.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering. Specifically, the present invention relates to a method for synthesizing nucleic acids under constant temperature conditions, in particular to a method for synthesizing nucleic acids with specific nucleotide sequences that can form special structures, and based on the specific nucleic acid A useful method of amplifying nucleic acids from sequences. Background technique [0002] The most fundamental difference of organisms carrying genetic information, the analysis method based on 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, etc. When the target gene content in the sample is very small, it is generally difficult to detect, so the target gene must be amplified or the detection signal must be amplified. As a method for ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/6806
CPCC12Q1/6806C12Q2531/113
Inventor 杜昱光毛瑞刘洪涛王倬
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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