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Method for specific signal amplification and detection of DNA targeted sequence

A target sequence and signal amplification technology, which is applied in biochemical equipment and methods, microbial measurement/testing, material excitation analysis, etc., can solve the problems of insufficient sensitivity and indistinct distinction of single base mismatches

Inactive Publication Date: 2012-09-12
PEKING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the sensitivity of some signal amplification methods currently developed is not ideal, which is 4-5 orders of magnitude lower than that of PCR, and the performance of specificity is relatively general: the distinction between single base mismatches is not obvious

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  • Method for specific signal amplification and detection of DNA targeted sequence
  • Method for specific signal amplification and detection of DNA targeted sequence
  • Method for specific signal amplification and detection of DNA targeted sequence

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0039] Example 1

[0040] In this embodiment, the target is a DNA single strand, and the probe is completely matched with the target DNA single strand except for the abasic site. In the experiment, a series of concentration gradients of DNA single strands were set up, hoping to detect as few target strands as possible. For detection principle see figure 1 ,Specific steps are as follows:

[0041] 1. Design and synthesize a double-labeled fluorescent probe containing uracil deoxynucleotide residues, and then use UDG enzyme to obtain a single abasic probe;

[0042] 2. Mix the obtained single abasic probe with different concentrations of the target DNA sequence, then add endonuclease IV and Lambda exonuclease, and quickly measure the change of the fluorescence value of the mixed solution over time.

[0043] In this embodiment, the designed probe sequence is as follows:

[0044] 5'-TCGUCT(-FAM)CCACAGACACATACTCCA-BHQ 1-3'(SEQ ID No.1)

[0045] The 5' end of the probe is -OH, th...

Embodiment 2

[0052] Test results: Fluorescence value change curve such as figure 2 As shown, the curves a, b, c, d, e and f correspond to the addition of 5pmol, 1pmol, 0.1pmol, 10fmol, 1fmol and 0 of the target sequence amount respectively. After 30 minutes, the fluorescence value reached a plateau in the reaction system with more than 1 pmol of the target sequence. Example 2

[0053] In this example, the target is a single strand of DNA. A series of different types of DNA single strands were set up in the experiment: In addition to the abasic site, the single abasic double fluorescent label probe was mismatched with these single strands, 1 mismatch, perfect match, 2 mismatches, 3 mismatches Or 1, 3 mismatches. For detection principle see figure 1 ,Specific steps are as follows:

[0054] 1. Design and synthesize a double-labeled fluorescent probe containing uracil deoxynucleotide residues, and then use UDG enzyme to obtain a single abasic probe;

[0055] 2. Mix the obtained single ...

Embodiment 3

[0107] Example 3

[0108] In this embodiment, the target is a DNA single strand, and except for the abasic site, the probe is mismatched with the target DNA single strand at position 1. In the experiment, a series of concentration gradients of DNA single strands were set up, hoping to detect as few target strands as possible. For detection principle see figure 1 ,Specific steps are as follows:

[0109] 1. Design and synthesize a double-labeled fluorescent probe containing uracil deoxynucleotide residues, and use UDG enzyme to obtain a single abasic probe;

[0110] 2. Mix the obtained single abasic probe with different concentrations of target DNA sequences, then add endonuclease IV and Lambda exonuclease, and quickly measure the change of the fluorescence value of the mixed solution over time.

[0111] In this embodiment, the designed probe sequence is as follows:

[0112] 5'-TCGUCT(-FAM)CCACAGACACATACTCCA-BHQ 1-3'(SEQ ID No.1)

[0113] The 5' end of the probe is -OH, th...

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Abstract

The invention discloses a method for specific signal amplification and detection of a DNA targeted sequence of a system needing to be detected. The method is characterized in that through distinguishing effects of a single-abasic site dual-labeled fluorogenic probe and selective cutting effects of an incision enzyme 1V and a Lamdba excision enzyme, high-sensitivity, high-selectivity and fast detection of a DNA targeted sequence is realized under mild conditions. The method provided by the invention can distinguish a targeted sequence from a single-basic group difference interference sequence and is suitable for detection of single nucleotide polymorphism (SNP) genotyping and low-abundance mutation.

Description

technical field [0001] The invention relates to the field of DNA sequence detection, including the technical fields of nucleic acid signal amplification, single nucleotide polymorphism (SNP) typing, low-abundance point mutation detection and the like. Specifically, it involves an amplification system using endonuclease IV / Lambda exonuclease complex enzyme system and a single abasic probe to achieve high sensitivity, high selectivity, and rapid detection of target DNA sequences under mild conditions. Background technique [0002] Nucleic acid signal amplification technology has achieved rapid development in recent years, and signal amplification methods based on restriction endonuclease, exonuclease III and DNase have been established respectively. The detection tools of this type of technology are DNA probes, which are theoretically expected to be applied to in-situ and in vivo detection in living systems. On the other hand, the amplification process is generally constant t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 赵美萍肖先金张晨苏昕
Owner PEKING UNIV
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