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Non-enzymatic SNP detection method based on DNA self-assembly

A self-assembly and DNA chain technology, applied in the field of non-enzyme detection based on DNA self-assembly, can solve the problems of difficulty in popularizing laboratory applications and expensive instruments, and achieve the effects of shortening operation time, high sensitivity, and reducing background signals

Inactive Publication Date: 2015-02-18
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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Problems solved by technology

Although compared with traditional methods, these new technologies are easy to operate and have high detection sensitivity, but they all require laboratory-specific instruments and professional technicians, and the instruments are expensive, making it difficult to be extended to all laboratory applications

Method used

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  • Non-enzymatic SNP detection method based on DNA self-assembly
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  • Non-enzymatic SNP detection method based on DNA self-assembly

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Embodiment 1

[0059] Primer design: Design and synthesize CP, MP, positive template T1, negative template WT1, AP1 and AP2, and couple biotin at the 3' end of CP. The specific nucleic acid sequence is as follows:

[0060] Among them, CP is completely complementary to the 5' end of T1, MP is completely complementary to the 3' end of T1, and the underlined A in T1 is the proto-oncogene BRAF T1799A For the SNP sites, AP1 and AP2, the underlined nucleic acids are complementary, and the parts marked in italics are also complementary.

[0061] and self-assembly of AP2:

[0062] DNA agarose gel electrophoresis was used to verify whether AP1 and AP2 could self-assemble into long double strands. In the second and third lanes, 5 μL of 1 μM AP1 and 1 μM AP2 were added, and in the fourth lane, 5 μL (AP1+AP2) mixture was added, electrophoresis was performed at a constant voltage of 100 V for 30 min, and the final results were observed with an imaging system.

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Abstract

A non-enzymatic SNP detection method based on DNA self-assembly is disclosed by the invention, and comprises the following steps: designing and separating a capture probe CP, a single chain MP, a probe AP1 and a probe AP2; adding the CP, the MP, a nucleic acid ligase and a sample into a reaction solution, mixing uniformly and completing a reaction; making the double-stranded DNA generated in the reaction solution to unwind into a single-stranded DNA; separating the single-stranded DNA connected with a separation mark, washing to remove other free DNA strands; adding the washed single-stranded DNA with the separation mark, the AP1 and the AP2 into the reaction solution to form a self-assembly double strand after a complete reaction; separating the DNA strand connected with the separation mark, washing to remove other free DNA strands; detecting the amount of double-stranded DNA in the washed DNA strand, and determining the amount of SNP. The method has advantages of being high in sensitivity, good in specificity, simple in operation, short in time, low in sample requirement and the like.

Description

technical field [0001] The invention relates to a nucleic acid detection method, in particular to a non-enzymatic detection method based on DNA self-assembly. Background technique [0002] Single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) refers to the polymorphism caused by the replacement of a single nucleotide (A, G, C, T) in the genomic DNA sequence, which is a single nucleotide variation. [0003] SNP has high genetic stability, abundant loci and wide distribution, and is easy to realize automatic analysis. SNPs located in the internal coding region of genes may lead to abnormal protein function and are closely related to genetic diseases and lesions. Therefore, SNP research has attracted more and more attention, and has been widely used in many fields such as biology, agriculture, medicine, and biological evolution. It is currently the most promising molecular marker. [0004] In medical research, if certain SNPs or specific combinations of certain...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 曾令文吴薇陈俊华
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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