Method for signal amplification and detection of dna target sequences

A target sequence and signal amplification technology, applied in biochemical equipment and methods, microbial measurement/inspection, material stimulation analysis, etc., can solve problems such as low detection temperature, loss of probe discrimination ability, and limited application range, and achieve detection low limit effect

Inactive Publication Date: 2011-12-28
PEKING UNIV
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Problems solved by technology

But they also have some defects: the restriction endonuclease amplification system uses a restriction endonuclease, so there is a certain sequence requirement for the target strand to be detected, which greatly limits the scope of application of this method in practice
However, the exonuclease III in the exonuclease III amplification system cannot work at a higher temperature (above 50°C), and the detection temperature of the system is lower than the melting temperature of the mismatched and matched double strands, so that the ability of the probe to distinguish is greatly lost

Method used

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  • Method for signal amplification and detection of dna target sequences
  • Method for signal amplification and detection of dna target sequences
  • Method for signal amplification and detection of dna target sequences

Examples

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Embodiment 1

[0030] Example 1

[0031]In this embodiment, the target is a perfectly matched DNA single strand, and a series of concentration gradients of the DNA single strand are set in the experiment, hoping to detect as few target strands as possible. For detection principle see figure 1 ,Specific steps are as follows:

[0032] 1. Use UDG enzyme to treat double-labeled fluorescent probes containing uracil deoxynucleotide residues to obtain single abasic probes;

[0033] 2. Mix the obtained single abasic probe with different concentrations of target DNA sequences, then add endonuclease IV, and quickly measure the change of fluorescence value of endonuclease IV in the mixed solution over time.

[0034] In this embodiment, the designed probe sequence is as follows:

[0035] 5'-FAM-TCGTCTCCACUGAAACATACTCCATAA-TAMRA-3' (SEQ ID No.1)

[0036] The base composition of the target sequence is as follows:

[0037] 5'-GTTTTAAATTATGGAGTATGTTTCTGTGGAGACGAGAGTAAG-3' (SEQ ID No. 2)

[0038] In 5...

Embodiment 2

[0043] Example 2

[0044] In this example, the system to be tested is a mixed system of the wild chain and the mutant chain, and the total amount of the two chains is 5 pmol, but a series of different mutation ratios are set, hoping to detect as low a mutation ratio as possible. For detection principle see image 3 , the specific implementation steps are as follows:

[0045] 1. Prepare a single abasic probe, the method is the same as step 1 in Example 1;

[0046] 2. Measure the melting temperature of the double strand formed by the single abasic probe, the wild strand and the mutant strand respectively, and determine the appropriate detection temperature;

[0047] 3. Mix the single abasic probe with the mixed target system containing different proportions of mutant chains, add endonuclease IV, rapidly raise the temperature to the detection temperature determined in step 2, and measure the change of fluorescence value with time.

[0048] In this embodiment, the designed prob...

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Abstract

The invention discloses a method for signal amplification and detection of a DNA target sequence, and prepares a single abasic double-labeled fluorescent probe. One end of the probe is labeled with a fluorescent group, and the other end is labeled with a quenching group. Outside the base site, its sequence completely matches the DNA target sequence; at a certain detection temperature, the probe specifically combines with the target sequence to form a double-strand gap, and the endonuclease IV is used to recognize and cut the double-strand gap, and the cleaved Probe fragments dissociate from the target sequence to generate fluorescent signals, and the target sequence released by dissociation can continue to bind to another probe, repeating the above process, thereby achieving signal amplification and detection of the target sequence. The method of the invention can realize high selectivity and high sensitivity low-abundance gene mutation detection.

Description

technical field [0001] The invention relates to the technical field of gene mutation detection. Specifically, it relates to an amplification system based on a single abasic fluorescent probe and endonuclease IV. The amplification system is used to realize high-selectivity, high-sensitivity detection of low-abundance gene mutations. Background technique [0002] Gene mutations are closely related to human cancers. Some important mutations can be used as biomarkers for cancer diagnosis, treatment and prognosis evaluation, so the detection of gene mutations has great clinical significance. However, due to factors such as tumors at different stages, distant metastasis, and fusion of tumor cells and normal cells, gene mutations usually show low abundance. Therefore, the detection method is required to have both high sensitivity (detection of a very low absolute amount) and high selectivity (selection of mutant chains among many wild-type chains). [0003] The most commonly used...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 赵美萍肖先金宋晨张晨苏昕
Owner PEKING UNIV
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