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Aptamer specifically binding to reverse transcriptase and application thereof

A nucleic acid aptamer and reverse transcriptase technology, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve problems such as reverse transcriptase enzyme antibody inactivation

Active Publication Date: 2021-09-07
ZYBIO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the ambient temperature of reverse transcriptase is not high (about 50°C), the reverse transcriptase antibody cannot be inactivated at this temperature, which also leads to few products on the market that block reverse transcriptase activity at low temperature. Commercial raw materials, reverse transcriptase hot start has become a technical problem

Method used

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  • Aptamer specifically binding to reverse transcriptase and application thereof
  • Aptamer specifically binding to reverse transcriptase and application thereof
  • Aptamer specifically binding to reverse transcriptase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Screening of nucleic acid aptamers specifically binding to reverse transcriptase

[0028] 1. ssDNA random library and enrichment library:

[0029] The DNA random library and primer sequences used were synthesized by Sangon Bioengineering (Shanghai, China) Co., Ltd. and purified by High Performance Liquid Chromatography (HPLC).

[0030] 2. Screening process of nucleic acid aptamers

[0031] The 200pm random ssDNA library was denatured in Hank’s solution at 95°C for 5 minutes, and ice-bathed for 10 minutes. In order to reduce non-specific binding, 0.1 mg / ml salmonspermDNA and 1 mg / ml BSA were added to Hank’s solution as binding buffer. Then, the random library and the magnetic beads coupled with 2ug target polypeptide were reacted in 200ulbinding buffer at 37°C for 30min, and Hank’s washed 3 times. Finally, the magnetic beads bound with ssDNA were used as the template, and the dsDNA was amplified with primers labeled with FITC and biotin. Double-stranded prod...

Embodiment 2

[0049] The using method of embodiment 2 reverse transcriptase nucleic acid aptamers

[0050] 1. Reaction system

[0051] In this embodiment, the reverse transcriptase nucleic acid aptamer of the present invention is used in the following reaction system, which includes reagent R1 and reagent R2 that are independent of each other.

[0052] (1) Reagent R1 is prepared according to the following formula:

[0053] components Serving per person (μL) unit / concentration 10×Buffer for Taq 3.2 / MgCl2 0.2 100mM dATP 0.01 100mM dCTP 0.01 100mM dGTP 0.01 100mM dUTP 0.02 100mM h 2 o

3.54 /

[0054] (2) Reagent R2 is prepared according to the following formula:

[0055] Enzyme composition Per serving (µl) unit / concentration Taq 0.3 5U / μL Anti-Taq 0.3 5U / μL RNase Inhibitor 0.05 40U / μL UNG, heat-labile 0.02 2U / μL RT 0.005 40U / μL aptamer 0.01 1OD / mL h ...

Embodiment 3

[0061] Example 3 Performance Verification of Reverse Transcriptase Nucleic Aptamer

[0062] A total of 3 sets of experiments were set up, and the difference between the kit used in each set of experiments and Example 2 was only the reverse transcriptase nucleic acid aptamer in reagent R2:

[0063] Group A: select the nucleic acid aptamer with the sequence shown in SEQ ID NO.1 to prepare a PCR reaction system;

[0064] Group B: no reverse transcriptase nucleic acid aptamer was added, and 0.01 μL ultrapure water was added at the same time.

[0065] (1) Application of reverse transcriptase nucleic acid aptamer in PCR warm start:

[0066] Using ultrapure water as a template, the PCR reaction was carried out according to the reaction procedure described in Example 2. The result of the reaction is as figure 1 As shown, the results show that: in the reaction system without template, the CT value of group B is significantly lower than that of group A, indicating that more primer di...

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Abstract

The invention discloses an aptamer specifically binding to reverse transcriptase and application thereof in inhibiting the activity of the reverse transcriptase. The sequence of the aptamer is at least one of the following four sequences: (1) a DNA sequence shown as SEQ ID No. 1; and (2) a DNA sequence which has 60% or more homology with the DNA sequence shown as SEQ ID No. 1 and can specifically bind to the reverse transcriptase. The aptamer provided by the invention can bind to the reverse transcriptase with extremely high specificity and can bind to the reverse transcriptase at low temperature, thereby achieving the goal of warm start of the reverse transcriptase. The dissolution curve method proves that the start temperature of the reverse transcriptase can reach 37 DEG C, and does not influence the original functions of the reverse transcriptase. The aptamer provided by the invention has the advantages of simple sequence and low synthesis difficulty, and can be matched with different reverse transcription temperatures by setting the number of bases in the sequence complementary part.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nucleic acid aptamer specifically binding to reverse transcriptase and its application. Background technique [0002] In the field of molecular diagnosis, PCR technology is still the main method used for nucleic acid amplification. In the PCR process, there are two main methods for identifying nucleic acid amplification signals. One is to use specific binding to target nucleic acid sequences. Fluorescent probes (such as TaqMan probes), which release fluorescent signals during the dissociation process to indicate nucleic acid amplification results, and the other is released by fluorescent dyes (such as SYBR) that bind non-specifically to double-stranded amplification products The former is more suitable for nucleic acid detection of specific diseases. Before nucleic acid amplification, heat or other denaturing methods are usually used to separate double-stranded RNA or DNA targets ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12Q1/686
CPCC12N15/115C12Q1/686C12N2310/16C12Q2521/107Y02A50/30
Inventor 陈威董璐张天张越于琼洋李达
Owner ZYBIO INC
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