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Method for detecting DNA single base mutant color by using nuclease reaction

A single-base mutation and color detection technology, applied in the field of DNA single-base mutation color detection, can solve the problems of reduced stability, difficult detection, cumbersome experimental steps, etc., and achieve the effect of improving selectivity

Active Publication Date: 2012-12-26
SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The experimental steps are relatively cumbersome, and the amount of DNA, DNase, RNase or nucleic acid aptamer is large
The second type of approach is the non-crosslinked nanoparticle aggregation method, which takes advantage of the reduced stability of the nanoparticles upon addition of the analyte.
This is similar to the traditional SNP detection method that relies on precise temperature control and gradient elution, and the selectivity of single base mutation detection is not high
Especially for mutation sites off-center, it is not easy to detect
And can only be used to detect targets of shorter length, usually shorter than 20bp

Method used

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  • Method for detecting DNA single base mutant color by using nuclease reaction
  • Method for detecting DNA single base mutant color by using nuclease reaction
  • Method for detecting DNA single base mutant color by using nuclease reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Preparation of unmodified gold nanoparticles.

[0056] An aqueous solution of trisodium citrate (25ml, 38.8mM) was quickly added to a boiling auric acid HAuCl4 solution (250ml, 1mM). After a few minutes, the color of the solution changed from light yellow to dark red. The solution continued to reflux and stir for 15 min to complete the reaction. Then cool slowly to room temperature. Store at 4°C. According to the UV absorption intensity of gold nanoparticles at 520nm ( figure 1 (C)), the concentration of prepared nano-gold is 12 ± 1nM, and the size is 13nm (see figure 1 (A) and (B)).

Embodiment 2

[0057] Example 2: Differences in the stabilizing effects of dNMP, single-stranded DNA and double-stranded DNA on unmodified gold nanoparticles

[0058] The purpose of this example is to confirm that dNMP, the nuclease degradation product of nucleic acid, can stabilize gold nanoparticles better than single-stranded and double-stranded DNA, which is the basis of the present invention.

[0059] Double-stranded DNA sample PM / A: 0.25 microliters of 100 μM single-stranded DNA probe A (see Table 1), 0.25 microliters of 100 μM target DNA PM that is completely complementary to single-stranded DNA probe A (see Table 1), 1 µl of buffer (0.6M NaCl, 10 mM phosphate, pH 7.4), and 0.5 µl of water, and kept at room temperature for 10 minutes.

[0060] Single-stranded DNA sample PM: composed of 0.25 μl of 100 μM single-stranded DNA probe PM, 1 μl of buffer (0.6M NaCl, 10 mM phosphate, pH 7.4), and 0.75 μl of water, and stored at room temperature 10 minutes.

[0061] ssDNA sample A: Consists ...

Embodiment 3

[0064] Example 3: The use of S1 nuclease for color detection of gold nanoparticles for single base mutations.

[0065] Reagent preparation: Prepare the solutions of probe A (Table 1), target PM (Table 1), target AC (Table 2), and target N (Table 1) with ultrapure water, and calibrate with a UV-visible spectrophotometer The concentration of the probe and each target solution was 100 μM. Prepare 5U μL with ultrapure water -1 S1 nuclease solution.

[0066] Take 1 μL each of probe A, target PM, AC or N, and mix with 1 μL 10× enzyme reaction buffer (containing 300 mM CH 3 COONa, 2800mM NaCl, and 10mM ZnSO 4 , pH 4.6) and 6 μL of ultrapure water were mixed and reacted at room temperature for 5 min. Take 1μL 5U μL -1 The S1 nuclease solution was added to the above mixture, mixed evenly, and reacted at 30°C for 30min. Take 2.5 μL of the above enzyme reaction mixture, add 50 μL of nano-gold solution with a concentration of 12±1 nM, and immediately add 40 μL of 0.5M NaCl phosphate...

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Abstract

The invention provides a method for detecting DNA single base mutant color by using nuclease reaction. The method is characterized in that: single-chain or double-chain DNA is selectively degraded into mononucleotide (dNMP) and short-chain DNA capable of better stabilizing nano gold by using nuclease (taking S1 nuclease special for single-chain DNA and DSN nuclease special for double-chain DNA for example) with special structure, so that single base mutant color detection is conveniently realized, and accurate temperature control, hydrolysis or ultrasonic treatment is not needed. The detection method provided by the invention can detect single base mutant at random position in a 16bp synthetic target with high selectivity, the length of the detectable target exceeds 80bp, and the method has broad application prospect.

Description

technical field [0001] The invention relates to a method for detecting the color of DNA single base mutation by using nuclease reaction, which belongs to the field of nano-biotechnology. Background technique [0002] Studies have shown that single base mutations (SNPs) are directly related to the occurrence of various diseases and drug responses. Accurate detection of mutations in a very small number of genes in a large number of DNA base sequences is very important for the prevention of major diseases, early diagnosis, research on gene damage, and pharmaceuticals. There is an urgent need for technologies capable of sensitive detection of SNPs. The selectivity of single-base mutation detection (that is, the difference in detection signals between fully complementary targets and single-base mutation targets) is the most important indicator of SNP detection technology. The higher the selectivity of this technology for single-base mutations, the lower the chance of false negat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/44
Inventor 娄新徽刘美英袁敏赵建龙
Owner SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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