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Method for inspecting nucleic acid signal amplification of ligation nucleic acid intrusive reaction and cutting endonuclease reaction

A nucleic acid invasion reaction and nicking endonuclease technology, applied in the field of molecular biology, can solve the problems of increasing detection cost, low sensitivity, and failing to meet the requirements of nucleic acid detection, and achieve the effect of low cost and high specificity

Inactive Publication Date: 2011-02-16
HUADONG RES INST FOR MEDICINE & BIOTECHNICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the B-DNA method and the nucleic acid invasion signal amplification experiment have high sensitivity, but they all need to synthesize probes with special structures or special fluorescent labels, which increases the detection cost; while the signal amplification method based on electrochemical detection Often the sensitivity is low and cannot meet the requirements of nucleic acid detection

Method used

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  • Method for inspecting nucleic acid signal amplification of ligation nucleic acid intrusive reaction and cutting endonuclease reaction
  • Method for inspecting nucleic acid signal amplification of ligation nucleic acid intrusive reaction and cutting endonuclease reaction
  • Method for inspecting nucleic acid signal amplification of ligation nucleic acid intrusive reaction and cutting endonuclease reaction

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1 Detection of Artificially Synthesized Oligonucleotide Fragments Using a Nucleic Acid Signal Amplification Detection Method Using a Ligation Reaction Coupled with a Nucleic Acid Invasion Reaction and a Nicking Endonuclease Reaction

[0033] In this embodiment, artificially synthesized single-stranded DNA templates of different concentrations are detected to verify the feasibility of the nucleic acid detection method stated in the present invention and to examine the sensitivity of the method. Another unrelated oligonucleotide fragment is used to verify the sequence specificity of the method stated in the present invention.

[0034] Reaction conditions:

[0035] 1) Nucleic acid invasion signal amplification

[0036] The nucleic acid invasion reaction system consists of 10 mM MOPS, pH 7.5, 0.05% Tween-20, 0.05% Nonidet P40, 0.1 μM upstream probe (sequence: 5'-ATG TCA CTT CCC CTT GGT TCT CTC C-3', namely SEQ ID NO.1), 1μM downstream probe (sequence: 5'-AGC AGG A...

Embodiment 2

[0042] Example 2 A nucleic acid signal amplification detection method using a ligation reaction coupled with a nucleic acid invasion reaction and a nicking endonuclease reaction to realize the differential detection of single-base differential templates

[0043] In this example, six artificially synthesized mutation templates (a: 5'-CTATTG CAC CAG GCC AGA AGA GAG AAC CAA GGG GAA GTG ACAT-3', which have one base difference with the target nucleic acid at different positions, are detected, namely SEQ ID NO. 7; b: 5'-CTA TTG CAC CAG GCC AGT TGA GAG AAC CAA GGG GAA GTG ACA T-3', namely SEQID NO.8; c: 5'-CTA TTG CAC CAG GCG AGA TGA GAG AAC CAA GGG GAA GTG ACA T-3', namely SEQ ID NO.9; d: 5'-CTA TTG CAC CAG GCC AGA TCA GAG AAC CAA GGG GAA GTGACAT-3', namely SEQ ID NO.10; e: 5'-CTA TTG CAC CAG GCC AGA TGT GAG AAC CAA GGGGAA GTG ACA T-3', namely SEQ ID NO.11; f: 5'-CTA TTG CAC CAG GCC AGA TGA GAG AACCAA CGG GAA GTG ACA T-3', namely SEQ ID NO. 12), the sequence of each mutation templa...

Embodiment 3

[0052] Example 3 PEG 6000, Nonidet P-40, Tween-20 and antioxidant DTT improve the reaction efficiency of nicking endonuclease

[0053] The invention provides a method for improving the reaction efficiency of nicking endonuclease, by respectively introducing surfactant polyethylene glycol 6000 (PEG 6000), ethylphenyl polyethylene glycol (Nonidet P-40), polyoxyethylene-20 sorbitan monolaurate (Tween-20) or antioxidant dithiothreitol (DTT) can significantly improve the efficiency of nicking endonuclease signal amplification. This example only focuses on the nicking endonuclease reaction, and investigates the improvement effect of the additive on the reaction efficiency of the nicking endonuclease reaction, without performing step 1) nucleic acid invasion signal in the three steps in the method provided by the present invention Amplification and step 2) flap connection, but adding the molecular beacon and an artificially synthesized flap connection product complementary to the cir...

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Abstract

The invention discloses a method for detecting nucleic acid signal amplification of ligation nucleic acid intrusive reaction and cutting endonuclease reaction, which belongs to the field of molecular biology. The method comprises the following steps: 1) during nucleic acid intrusive signal amplification, accumulating flap fragments; 2) during flap fragment connection, connecting the flap fragments with another oligonucleotide on a molecular beacon to form a cutting endonuclease site; 3) during cutting endonuclease signal amplification, only cutting a molecular beacon chain so that the fluorescent group of the molecular beacon is separated from a quenching group, and a new integrated molecular beacon is hybridized with a flap fragment connected with the molecular beacon, thus amplifying a target nucleic acid signal; and 4) inspecting the target nucleic acid signals. The method is capable of inspecting nucleic acid target sequences and respectively inspecting nucleic acid sequences with single base difference near intrusive sites.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a nucleic acid signal amplification detection method in which a ligation reaction is coupled with a nucleic acid invasion reaction and a nick endonuclease reaction. Background technique [0002] Nucleic acid detection has been widely used in clinical diagnosis, environmental monitoring, prevention and control of infectious diseases and many other aspects. At present, most commonly used nucleic acid detection technologies are based on the principle of template amplification, that is, the amplification product is identical to the target nucleic acid sequence, such as polymerase chain reaction technology (Polymerase Chain Reaction, PCR), loop-mediated nucleic acid isothermal amplification technology (Loop- Mediated Isothermal Amplification (LAMP), Rolling Cycle Amplification (RCA), Nucleic Acid Sequence Based Amplification (NASBA), Transcriptase Mediated Amplification (TMA) , Helicas...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64C12Q1/44
Inventor 周国华邹秉杰武海萍马寅姣
Owner HUADONG RES INST FOR MEDICINE & BIOTECHNICS
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