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Nicking endonuclease-based netted rolling cycle amplification system and use thereof

A technology of nicking endonuclease and rolling circle amplification, which is applied in the field of network rolling circle amplification system based on nicking endonuclease

Inactive Publication Date: 2014-12-17
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, conventional PCR technology also has certain defects: (1) It depends on high-quality thermal cyclers in application, so it is difficult to popularize and apply it at the grassroots level; (2) Many factors affect the amplification effect; (3) Often cause non-specific amplification. (4) The amplification reaction time is long, and an updated method is urgently needed to replace

Method used

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  • Nicking endonuclease-based netted rolling cycle amplification system and use thereof
  • Nicking endonuclease-based netted rolling cycle amplification system and use thereof
  • Nicking endonuclease-based netted rolling cycle amplification system and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1: Electrophoretic characterization of target DNA amplification products with different concentrations

[0033] 100 nM circular DNA template, different concentrations of target DNA, 1 μM primer, 400 μM dNTP, 8 U Bst DNA polymerase large fragment, 10 U nicking endonuclease Nb. BsrDI, 1×Bst DNA polymerase large fragment buffer solution (20 mM Tris-HCl, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, pH 8.8) were mixed, and double-distilled water was added to make the total volume of the reaction system reach 50 μL. After the reaction was placed in a metal bath at 65 °C for 1 hour, the reaction was terminated at 95 °C for 10 minutes. The main difference from the existing hyperbranched rolling circle amplification reaction system is the use of the nicking endonuclease Nb.BsrDI. Mix 5 μL sample and loading buffer solution 6×loading buffer (1 μL) evenly, and pour into the sample well. The voltage was set to 80 V and the time was 30 min for agar...

Embodiment 2

[0035] Example 2: Electrophoretic characterization of target DNA amplification specificity

[0036] 100 nM circular DNA template, 1 μM target DNA, DNA with 1, 3, 5 base mismatches with the target DNA, and DNA completely irrelevant to the target DNA, 1 μM primer, 400 μM dNTP, 8 U of Bst DNA polymerase large fragment, 10 U of nicking endonuclease Nb. BsrDI, 1×Bst DNA polymerase large fragment buffer solution (20 mMTris-HCl, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, pH 8.8) were mixed, and double-distilled water was added to make the total volume of the reaction system reach 50 μL. After the reaction was placed in a metal bath at 65 °C for 1 hour, the reaction was terminated at 95 °C for 10 minutes. The main difference from the existing hyperbranched rolling circle amplification reaction system is the use of the nicking endonuclease Nb.BsrDI. Mix 5 μL sample and loading buffer solution 6×loading buffer (1 μL) evenly, and pour into the sample well. Th...

Embodiment 3

[0037] Example 3: Atomic Force Microscopy Characterization of Amplified Products of Linear Rolling Circle Amplification, Hyperbranched Rolling Circle Amplification, and Network Rolling Circle Amplification

[0038] The amplification products of linear rolling circle amplification, hyperbranched rolling circle amplification and network rolling circle amplification were characterized by atomic force microscopy.

[0039] attached by Image 6 A It can be seen that the amplification product of the linear rolling circle amplification reaction is several linear single strands, which is different from the theoretical rolling circle replication reaction, that is, the extension product generated by hybridizing the target DNA to a circular single-stranded DNA molecule It is the multiple connections of the complementary pairing products of this circular DNA template molecule to form a certain length of linear single-stranded DNA.

[0040] attached by Image 6 B It can be seen that the a...

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Abstract

The invention relates to a nicking endonuclease-based netted rolling cycle amplification system and a use thereof. Based on the existing second-generation hyperbranched rolling cycle amplification technology, the amplification system utilizes a special endonuclease which is nicking endonuclease Nb.BsrDI so that a third-generation rolling cycle amplification technology which is a netted rolling cycle amplification (NGCA) technology is produced. Intensity of fluorescence signals produced by the netted rolling cycle amplification is increased with increasing of a target DNA concentration, is obviously stronger than that of fluorescence signals produced by linear rolling cycle amplification and hyperbranched rolling cycle amplification and has a detection limit of 0.1fM. Compared with the first-generation linear rolling cycle amplification and the second-generation hyperbranched rolling cycle amplification, the nicking endonuclease-based netted rolling cycle amplification system retains the prior art advantages in operationality, use cost and amplification time, further realizes signal amplification based on the prior art thereby providing good technical conditions for ultralow-abundance nucleic acid sample analysis and detection, and has a wide application prospect.

Description

technical field [0001] The present invention relates to a networked rolling circle amplification system and its application, in particular to a networked rolling circle amplification system based on a nicking endonuclease and its application, which can realize efficient DNA amplification and signal amplification, and Ultra-low concentrations of target DNA are detected. Background of the invention [0002] With the continuous heating up of gene detection and research, various corresponding molecular biology techniques are emerging. In order to realize the rapid and sensitive detection of genes, it is necessary to develop corresponding detection methods for its material basis, that is, nucleic acids. Existing micro-nucleic acid detection mainly relies on nucleic acid amplification technology, a common technique in the field of molecular biology. Polymerase chain reaction (PCR) is currently the most widely used DNA amplification technology. It can not only amplify and isolat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 李根喜朱小立冯畅佟蕙张斌
Owner SHANGHAI UNIV
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