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A low-sequence-dependent high-order isothermal exponential amplification method for the detection of microRNAs

A constant-temperature exponential amplification and sequencing technology, which is applied in biochemical equipment and methods, and the determination/inspection of microorganisms, can solve problems such as sequence dependence, and achieve small sequence dependence, simple amplification system, and high amplification efficiency. Effect

Active Publication Date: 2021-11-16
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is an obvious sequence-dependent problem in the existing constant temperature exponential amplification reaction, and the amplification templates designed for different target molecules miRNA will show different amplification efficiencies

Method used

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  • A low-sequence-dependent high-order isothermal exponential amplification method for the detection of microRNAs
  • A low-sequence-dependent high-order isothermal exponential amplification method for the detection of microRNAs
  • A low-sequence-dependent high-order isothermal exponential amplification method for the detection of microRNAs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 A low-sequence-dependent high-order constant temperature exponential amplification template for detecting microRNA

[0046] Such as figure 1 As shown, two partially complementary amplification templates T1 and T2 are designed according to the target miRNA; the 5′-terminal sequences of the templates T1 and T2 are the same, and both contain two identical sequences x (14-25 nt in length), and the two sequences There is a specific site recognized by a nicking endonuclease between x, which is denoted as x-specific site-x;

[0047] The 3' end sequence of the template T2 can be reverse complementary to the 5' end sequence of the target microRNA (the length of the 5' end sequence accounts for 8 / 21-12 / 21 of the total length of the target microRNA);

[0048] The 3' end sequence of the template T1 can be reverse complementary to the middle sequence of the template T2 (length is 8-12bp), and the middle sequence of the T2 is located between the above-mentioned 3' end sequ...

Embodiment 2

[0052] Example 2 A method for detecting microRNA by low-sequence-dependent high-order constant temperature exponential amplification

[0053] Taking the detection of let-7b in the let-7 family as an example, according to the sequence of the target let-7b to be detected, templates T1 and T2 that are partially complementary to it were designed, and the specific sequences are shown in Table 1.

[0054] Table 1 Sequences of let-7b and its template T1 and template T2

[0055]

[0056] Prepare A and B reaction solutions on ice, and the A reaction solution is divided into four types: A1, A2, A3, and A4:

[0057] A1 contains endonuclease buffer, template T1, template T2, dNTP, target let-7b;

[0058] A2 contains endonuclease buffer, template T1, dNTP, target let-7b;

[0059] A3 contains endonuclease buffer, template T2, dNTP, target let-7b;

[0060] A4 contains endonuclease buffer, template T1, template T2, dNTP.

[0061] B reaction solution contains DNA polymerase buffer solut...

Embodiment 3

[0064] Example 3 A low sequence-dependent high-order constant temperature exponential amplification method for detecting microRNA

[0065] Taking the detection of let-7b in the let-7 family as an example, according to the sequence of the target let-7b to be detected, templates T1 and T2 that are partially complementary to it were designed, and the specific sequences are shown in Table 1.

[0066] Prepare the A and B reaction solutions of the high-order constant temperature exponential amplification technology (TEXPAR) of the present invention on ice, the A reaction solution includes endonuclease buffer, template T1, template T2, dNTP, target let-7b; the B reaction solution includes Polymerase buffer solution, Vent(exo-) polymerase, endonuclease Nt.BstNBI, SYBR Green I fluorescent dye. Mix A and B (the final concentration of T1 in the mixed system is 0.1μM, the final concentration of T2 is 0.075μM, the final concentration of endonuclease is 0.4U / μL, and the final concentration ...

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Abstract

The invention discloses a method for detecting microRNA by low-sequence-dependent high-order constant temperature exponential amplification. The method designs specific templates T1 and T2 capable of detecting target microRNA, and incises the templates T1 and T2, DNA polymerase, and nick endo The enzyme and dNTP are mixed and reacted at 35-50°C, the amplification curve is detected, and whether the sample contains the target microRNA is determined according to the amplification curve and the standard curve. The method of the present invention can further calculate the specific content of the target microRNA in the sample. The method of the present invention has high amplification efficiency and high sensitivity, and shortens the analysis time; the method has little sequence dependence on the target microRNA and has strong versatility; the detection linear range is wide and the specificity is good; the amplification system is simple and can be developed into a kit and promote it to the market.

Description

technical field [0001] The invention belongs to the field of nucleic acid quantitative detection, and in particular relates to a method for detecting microRNA (hereinafter referred to as miRNA) by low sequence dependence and high-order constant temperature exponential amplification. Background technique [0002] miRNA is a type of endogenous non-coding small molecule RNA, about 18-24 nucleotides in length, it has a regulatory effect on gene expression, and actively participates in cell growth, differentiation and apoptosis in vivo. A large number of research results show that the expression level of miRNA is closely related to cancer or certain diseases. Therefore, the quantitative detection of miRNA not only helps to understand its mechanism of action, but also has great significance for the diagnosis and treatment of diseases and the development of related gene medicines. However, due to the short sequence of miRNAs and high sequence similarity, the difference between hom...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6876C12Q1/6844
CPCC12Q1/6844C12Q1/6876C12Q2521/301C12Q2521/101C12Q2563/107
Inventor 戴宗郑旭玲李裕陈俊邹小勇
Owner SUN YAT SEN UNIV
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