A kind of restriction endonuclease-mediated primer self-generated rolling circle amplification method

Abstract

The present invention relates to a restriction endonuclease-mediated primer self-generating rolling circle amplification method, comprising the following steps: 1) thio-modifying single-stranded DNA at the restriction endonuclease cleavage site; 2) cyclizing the thio-modified single-stranded DNA to obtain a thio-modified single-stranded circular DNA; 3) using the thio-modified single-stranded circular DNA obtained in step 2) as an amplification template, the amplification template Add to the reaction system containing the target DNA primer to be detected, DNA polymerase with strand displacement and restriction endonuclease to carry out multiple rounds of rolling circle amplification reaction, and the molecular signal of the target DNA is amplified. The beneficial effects of the rolling circle amplification method of the present invention are: 1) using restriction endonucleases to initiate the self-generating reaction of primers, and constructing a restriction endonuclease-mediated rolling circle amplification technology, which can ensure high amplification efficiency and ensure high amplification efficiency. Widen its application range; simple operation, nucleic acid amplification reaction can be realized under constant temperature conditions.

Description

technical field [0001] The present invention relates to a DNA rolling circle amplification method, in particular to a rolling circle amplification method using restriction endonuclease for auxiliary cleavage reaction to trigger self-generation of primers, which can realize rapid and efficient amplification of nucleic acid, and belongs to nucleic acid Amplification technology field. Background technique [0002] Rolling circle amplification (RCA) originated in the 1890s and was inspired by the replication of pathogenic microorganisms in nature. The process of replicating DNA as a template. The emergence of this technology is widely used in genome amplification, biology and medical diagnosis because it does not require complicated heating and cooling procedures, is not limited by the requirements of precision instruments, and has the characteristics of good specificity, high sensitivity and easy operation. Over the years, rolling circle amplification technology has been cont...

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Problems solved by technology

However, they are all limited to a certain extent. For example, linear RCA amplification is less efficient than exponential amplification; although exponential amplification is significantly improved in amplification efficiency, even in the presence of sufficient enzymes and raw materials can reach 10 6 ~10 9 copy number, but the disadvantage is that the selection and design of two primer sequences need to be considered, which reduces the simplicity of experimental design

[0005] However, in the process of PG-RCA, exponential rolling circle amplification can only be performed if one strand of the double-stranded DNA is cut; if both strands of the double-stranded DNA are cut at the same time, the amplification cannot continue

In the prior art, nicking endonucleases are used to achieve the purpose of cutting only one strand of double-stranded DNA, but there are fewer types of nicking endonucleases that have been developed and used, and they are easily denatured and inactivated under high temperature conditions. There are strict requirements on the reaction temperature and other restrictions, which greatly restrict the practical promotion of this technology and limit its application range to a certain extent.

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