A nucleic acid isothermal amplification method based on positioning probe-mediated cleavage

A technology for isothermal amplification and positioning probes, applied in the field of thermal amplification, to achieve the effects of broadening the application range, strong versatility, high sensitivity and specificity

Active Publication Date: 2019-01-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The invention provides a nucleic acid isothermal amplification method based on positioning probe-mediated cleavage, which can not only overcome the dependence of endonucleases on specific recognition sequences in the target nucleic acid sequence, but also solve the problem of arbitrary cleavage of the target nucleic acid sequence and subsequent amplification. It can also reduce the difficulty of primer design, and at the same time improve the generality, sensitivity and specificity of the method

Method used

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  • A nucleic acid isothermal amplification method based on positioning probe-mediated cleavage
  • A nucleic acid isothermal amplification method based on positioning probe-mediated cleavage
  • A nucleic acid isothermal amplification method based on positioning probe-mediated cleavage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] In this example, artificially synthesized target nucleic acid sequences were used to verify the feasibility of the method. According to the cleavage site and length requirements of the target nucleic acid sequence, corresponding positioning probes and primers are designed, and the amplified products are analyzed by agarose gel (3%) electrophoresis. Reaction principle see image 3 ,Specific steps are as follows:

[0062](1) Take 2 EP tubes, add 2.5μL reaction buffer (100mMNaCl, 50mM Tris-HCl, 60μg / mLBSA), 0.6μL endonuclease Nt.BstNBI (concentration: 10U / μL), 0.1μL Bst DNA polymerase (concentration is 8U / μL), 1μL dNTPs (concentration is 10mM), 4μL MgCl 2 (concentration is 25mM), 0.25 μL upstream localization probe P1 (concentration is 10 μM), 0.25 μL downstream localization probe P2 (concentration is 10 μM), 0.25 μL upstream primer L1 (concentration is 10 μM), 0.25 μL downstream primer L2 (concentration 10 μM) and 12.8 μL water, labeled a, b;

[0063] (2) Add 2.5 μL o...

Embodiment 2

[0078] This example is used to verify the amplification specificity of the method of the present invention. In this example, the analyte is an artificially synthesized target nucleic acid sequence (completely matched with the positioning probe / primer) and a mismatched nucleic acid sequence (only one base mismatch), according to the cleavage site and length of the target sequence It is required to design corresponding upstream and downstream positioning probes and upstream and downstream primers, and use SYBR Green I, a specific fluorescent dye dependent on double-stranded DNA, to display signal changes. For detection principle see image 3 ,Specific steps are as follows:

[0079] (1) Take 5 EP tubes, add 2.5μL reaction buffer (100mMNaCl, 50mM Tris-HCl, 60μg / mLBSA), 0.6μL endonuclease Nt.BstNBI (concentration: 10U / μL), 0.1μL Bst DNA polymerase (concentration is 8U / μL), 1μL dNTPs (concentration is 10mM), 4μL MgCl 2 (concentration is 25mM), 0.25 μL upstream localization probe ...

Embodiment 3

[0097] This example is used to verify the amplification sensitivity of the method of the present invention. In this example, artificially designed, synthetic target sequences were used to test the detection sensitivity of the method. According to the cleavage site and length requirements of the target sequence, the corresponding positioning probes and primers are designed, and the specific fluorescent dye SYBR Green I dependent on double-stranded DNA is used to display the signal change. For detection principle see image 3 ,Specific steps are as follows:

[0098] (1) Take 6 EP tubes, add 2.5μL reaction buffer (100mMNaCl, 50mM Tris-HCl, 60μg / mLBSA), 0.6μL endonuclease Nt.BstNBI (concentration: 10U / μL), 0.1μL Bst DNA polymerase (concentration is 8U / μL), 1μL dNTPs (concentration is 10mM), 4μL MgCl 2 (concentration is 25mM), 0.25 μL upstream localization probe P5 (concentration is 10 μM), 0.25 μL downstream localization probe P6 (concentration is 10 μM), 0.25 μL upstream prime...

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Abstract

The invention discloses a nucleic acid isothermal amplification method based on positioning probe mediated shearing. The method comprises an index amplified reaction for an enzyme digestion target nucleic acid sequence, wherein a reagent adopted by the enzyme digestion target nucleic acid sequence comprises a positioning probe and an incision enzyme which are at the upper stream and the lower stream; the positioning probe comprises a section of double chains and at least one section of single chain connected with the double chains; the single chain is provided with a recognition zone capable of being crossed with the specificity of the target nucleic acid sequence; the double chains are provided with a bonding zone for the incision enzyme to be combined for the adjacent recognition zone. The method disclosed by the invention has the advantages of being simple, high in specificity, sensitivity and universality, and the like, the dependency of the incision enzyme on specific recognition sequence in the target nucleic acid sequence is thoroughly overcome, the problem of amplification caused by any shearing of the target nucleic acid sequence is solved, the difficulty of primer design in a present isothermal amplification method is reduced, and the application range of isothermal index amplification technology is expected to be expanded.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a nucleic acid isothermal amplification method based on positioning probe-mediated shearing. Background technique [0002] Nucleic acid in vitro amplification technology is one of the most rapidly developing technical fields in clinical chemistry. The amplification detection of nucleic acid is more practical than other detection techniques in terms of operation and timeliness, so it is favored by researchers and instrument developers. Polymerase chain reaction (PCR), as a revolutionary technology for nucleic acid amplification, has firmly occupied a leading position after more than 30 years of development, and a variety of derivative technologies have emerged such as reverse transcription PCR (RT-PCR), real-time PCR (Real-time PCR) -time PCR), multiplex PCR (Multiplex PCR), nested PCR (Nested PCR), etc. However, these technologies rely heavily on temperature cycling t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/686C12Q2531/119C12Q2521/301
Inventor 张涛丁雄范宏亮吴望华韩达牟颖
Owner ZHEJIANG UNIV
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