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Method for constructing double-stem-loop structure DNA template to detect nucleic acid based on ligation reaction

A ligation reaction and template detection technology, applied in the field of biological analysis, can solve problems such as difficult to distinguish single base differences, false positive amplification signals of primer dimers, specificity difficult to meet mutations, etc., and achieve the goal of reducing cross-contamination of reagents Risk, Avoidance of Radiation Hazards, Effect of Improving Sensitivity

Inactive Publication Date: 2016-08-03
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, PCR not only requires precise thermocycling, but it is also prone to false positive amplification signals caused by primer dimers, and it is difficult to distinguish single-base differences in DNA or RNA
Although allele-specific PCR is used to detect single-base differences in DNA, the specificity of DNA polymerases to recognize single-base mismatches is difficult to satisfy a small number of mutations among a large number of unmutated bases

Method used

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  • Method for constructing double-stem-loop structure DNA template to detect nucleic acid based on ligation reaction
  • Method for constructing double-stem-loop structure DNA template to detect nucleic acid based on ligation reaction
  • Method for constructing double-stem-loop structure DNA template to detect nucleic acid based on ligation reaction

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Experimental program
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Effect test

Embodiment 1

[0027] Taking let-7a as the microRNA analysis model, according to the let-7a sequence 5′-UGAGGUAGUAGGUUGUAUAGUU-3′, probe A and probe B with stem-loop structure were designed and synthesized. The base sequence of probe A is 5′-CGACAGCAGAGGATTTGTTGTGTGGAAGTGTGAGCGGATTTTCCTCTGCTGTCGTTTTAACTATACAAC- 3' (provided by Treasure Bioengineering (Dalian) Co., Ltd.); the base sequence of probe B is 5'-CTACTACCTCATTTTATCGTCGTGACTGTTTGTAATAGGACAGAGCCCCGCACTTTTCAGTCACGACGAT-3' (provided by Treasure Bioengineering (Dalian) Co., Ltd.), wherein the 5' of probe B Modified phosphate groups. Schematic diagram of the structure of probe A and probe B as figure 1 shown.

[0028] Such as figure 2 As shown, when there is let-7a, the detection and recognition regions of probe A and probe B are specifically hybridized with let-7a respectively, using let-7a as a template, under the catalysis of T4RNA ligase 2, probe A and probe B Probe B is connected to form a double-stem-loop structure DNA, which ca...

Embodiment 2

[0035] The human let-7miRNA family is a highly homologous similar sequence with a difference of only one or several bases. Use probe A and probe B designed for let-7a in Example 1 to detect let-7b (5'-UGAGGUAGUAGGUUGUGUGGUU-3') and let-7c (5'-UGAGGUAGUAGGUUGUAUGGUU-3') in the let-7 family , let-7d (5′-AGAGGUAGUAGGUUGCAUAGUU-3′), let-7e (5′-UGAGGUAGGAGGUUGUAUAGUU-3′), the detection method is to use let-7b, let-7c, let-7d, let- 7e to replace let-7a, other experimental steps were the same as in Example 1, and the real-time fluorescence curves were measured respectively. Let-7b and let-7d differ from the let-7a sequence by two bases, and let-7c and let-7e differ from the let-7a sequence by one base. Test results such as Figure 5 shown. Depend on Figure 5 It can be seen that let-7a can be clearly distinguished from other miRNAs in the let-7 family, the non-specific interference of let-7b is 4.6%, the non-specific interference of let-7c is 12.9%, the non-specific interference ...

Embodiment 3

[0037] Taking the mutant DNA fragment of exon 8 of the human P53 gene as an example, the mutant DNA (5′-TGTTTGTGCCTGTCCTGGGAGAGACTGGCGCACAGAGGAAGAGAATCTC-3′) was used as the detection target sequence, and probe A and probe B with a stem-loop structure were designed and synthesized. The base sequence of needle A is 5′-CGACAGCAGAGGATTTGTTGTGTGGAAGTGTGAGCGGATTTTCCTCTGCTGTCGCTCTTCCTCTGTGCGCCA-3′ (provided by Bao Bioengineering (Dalian) Co., Ltd.); the base sequence of probe B is 5′-GTCTCTCCCAGGACAGGCATCGTCGTGACTGTTTGTAATAGGACAGAGCCCCGCACTTTCAGTCACGACGAT-3′ (provided by Bao Bioengineering (Dalian) Co., Ltd.), wherein the 5' end of probe B is modified with a phosphate group.

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Abstract

The invention discloses a method for constructing a double stem loop structure DNA template to detect nucleic acid based on a ligation reaction. The method includes the steps that ligase is used for connecting a probe containing a stem-loop structure and complemented with nucleic acid to be detected to form a double-stem-loop structure DNA template, the template can guide fast and efficient loop-mediated isothermal amplification reaction to achieve high-sensitivity nucleic acid detection, and meanwhile the method can specifically distinguish nucleic acid with single-base difference. It is provided for the first time that double stem loop structure DNA is constructed through the high-specificity ligation reaction, the specificity template is provided for the loop-mediated isothermal amplification reaction in the next step, fluorescence labeling is not needed in the method, cost is low, the precise heat cycle process in the PCR process is avoided, and fast amplification of the nucleic acid to be detected can be realized at constant temperature. The method can be used for quantitative analysis, methylation detection, SNP detection and the like on RNA or DNA and provides a new strategy for high-sensitivity nucleic acid analysis, early cancer diagnosis and other researches.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, and in particular relates to a method for constructing a DNA template with a double-stem-loop structure based on a ligation reaction, and the application of the method in RNA quantitative detection, DNA quantitative detection, methylation detection and single base discrimination. Background technique [0002] With the completion of the Human Genome Project and the continuous development of next-generation high-throughput sequencing technology, the research on variation detection, DNA methylation analysis, and microRNA expression analysis of specific sequence nucleic acids (DNA or RNA) has become a research field in the field of molecular biology. hotspots. They are used as gene markers for disease pathological research, early diagnosis and drug development. Therefore, DNA or RNA detection and quantitative analysis methods that are simple, fast, sensitive, and capable of single-base r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2531/119C12Q2521/501C12Q2563/107
Inventor 李正平王辉王洪红孙圆圆
Owner SHAANXI NORMAL UNIV
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