Probe based on target triggering and supportive of secondary amplification and application thereof

A probe and target technology, applied in the field of molecular bioinformatics, can solve problems such as the inability to detect trace DNA sensitivity, and achieve the effects of wide dynamic detection range, enhanced signal, and simple operation.

Inactive Publication Date: 2015-11-25
JIANGSU INST OF NUCLEAR MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, colorimetric probes using the above principles for DNA analysis are relatively rare, and cannot achieve the sensitivity of detecting trace amounts of DNA.

Method used

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  • Probe based on target triggering and supportive of secondary amplification and application thereof
  • Probe based on target triggering and supportive of secondary amplification and application thereof
  • Probe based on target triggering and supportive of secondary amplification and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Probe design based on target-triggered secondary amplifiable probes

[0049] In this embodiment, the target-triggered secondary amplifiable probe includes: a first hairpin H1, a second hairpin H2 and a third hairpin H3, and the sequences of each are shown in Table 1. The target DNA to be detected has the following sequence: 5'-CAATATGTAGATCTAGTCACGCTA-3'.

[0050] Table 1 Probe sequences that can be amplified twice based on target triggering

[0051]

[0052] The first hairpin H1 includes: a recognition region I that recognizes target DNA and a first stem region II that partially hybridizes with the recognition region I to form a hairpin structure;

[0053] The second hairpin H2 includes: base complementary region III hybridized with the recognition region I part of the first hairpin;

[0054] The third hairpin H3 comprises: a second stem region II' hybridizable to a portion of the first stem region II of the first hairpin and a G-quadruplex region IV. ...

Embodiment 2

[0067] Example 2: Detection of target DNA at different concentrations based on target-triggered secondary amplifiable probes

[0068] Exonuclease III and hemin used in this example were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. Tris(hydroxymethyl)aminomethane (Tris), N-2-hydroxyethylpiperazine -N'-2-ethanesulfonic acid (HEPES), 2,2'-azino-bis(3-ethylbenzothiazole-6-sulfonic acid) diammonium salt (ABTS), hydrogen peroxide purchased from Aladdin reagents Company (Shanghai, China), there is no difference in the effect between the products of various commercially available models of the above materials. All solutions were prepared using redistilled water prepared by Milli-Q Purification System (Billerica, Massachusetts, USA).

[0069] The detection of the target DNA in embodiment 1 comprises the following steps:

[0070] First, take the solutions of the first hairpin, the second hairpin, and the third hairpin, heat them to 95° C., keep them for 5 minutes, then sl...

Embodiment 3

[0076] Embodiment 3: Signal amplification verification experiment based on target-triggered secondary amplifiable probes

[0077] In this example, the following solutions were prepared according to the method in Example 2, and numbered a, b, c, d in sequence:

[0078] Sample a: a solution in which the concentrations of the first hairpin H1, the second hairpin H2, and the third hairpin H3 are all 250 nM;

[0079] Sample b: a solution in which the concentrations of the first hairpin H1, the second hairpin H2, and the third hairpin H3 are all 250 nM, and the concentration of the exonuclease III is 2 U / uL;

[0080] Sample c: a solution in which the concentrations of the first hairpin H1, the second hairpin H2, and the third hairpin H3 are all 250 nM, and the concentration of the target DNA is 100 nM;

[0081] Sample d: a solution in which the concentrations of the first hairpin H1, the second hairpin H2, and the third hairpin H3 are all 250nM, the concentration of the exonuclease...

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Abstract

The invention provides a probe based on target triggering and supportive of secondary amplification and application thereof. The probe comprises a first hairpin, a second hairpin and a third hairpin.The probe can realize amplification twice when detecting target DNA, so that signals are enhanced remarkably, and sensitivity in target DNA detection is improved greatly; limit of detection can be lowered to be 0.36pM, and the probe has a wide dynamic detection range exceeding 5 orders of magnitude, can meet requirements on detecting trace DNA and is simple to operate and low in cost.

Description

technical field [0001] The invention belongs to the field of molecular bioinformatics, and in particular relates to a target-triggered probe capable of secondary amplification and its application. Background technique [0002] The detection of trace amounts of DNA plays a vital role in mutation analysis, clinical diagnosis, and gene therapy. In order to detect a trace amount of DNA, it is necessary to construct a DNA biosensor with high detection sensitivity. At present, there are sensors that use signal amplification strategies to achieve high-sensitivity detection. These high-sensitivity sensors are mainly concentrated in the field of nucleic acid research. The amplification methods used include polymerase chain reaction (PCR), ligase chain reaction (LCR) ), strand displacement amplification (SDA) and rolling circle amplification (RCA). Although highly sensitive analysis of target analytes can be achieved using the above methods, multiple analysis steps are usually invol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 吴昊邹霈刘娅灵王洪勇吴军诸飞帆
Owner JIANGSU INST OF NUCLEAR MEDICINE
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