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Nucleic acid signal amplification sequence and amplification method

A signal amplification and sequence technology, applied in the field of molecular biology, can solve the problems of increased management costs, false positives, aerosol pollution, etc., and achieve the effects of short detection process, high repeatability and good stability

Active Publication Date: 2019-08-06
成都诺森医学检验有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the stability and repeatability of PCR technology are poor, and there are serious aerosol pollution defects. Although the follow-up closed-tube fluorescent PCR solved the problem of cross-contamination, it still failed to solve the natural defect of PCR technology.
Secondly, the cost of PCR is relatively high. The PCR reaction needs to build a special laboratory with a high level of air purification, and the experiment must be managed in different areas, which greatly increases the management cost; and the reaction time process is too long, which increases the time cost of each test.
In addition, the Taq enzyme or polymerase in the PCR reaction is also easily affected by the substances in the sample, resulting in false positive or false negative results.

Method used

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  • Nucleic acid signal amplification sequence and amplification method
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  • Nucleic acid signal amplification sequence and amplification method

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Effect test

preparation example Construction

[0042] The present invention also provides a method for preparing the above-mentioned nucleic acid signal amplification sequence, which specifically includes the following steps:

[0043] A. Design a nucleic acid signal leader sequence and multiple repeat sequences according to the target target, and add a restriction endonuclease I cutting site at the 5' end of the leader sequence, and add a restriction endonuclease I at the 3' end of several multiple repeat sequences The enzyme cutting site of restriction endonuclease II; the enzyme cutting site of the restriction endonuclease I is connected with the leading sequence of the nucleic acid signal amplification sequence, and becomes a cohesive end after enzyme cutting; the restriction endonuclease II The enzyme cutting site is connected to the repeat sequence of the nucleic acid signal amplification sequence, and the end is blunt after enzyme cutting;

[0044] B. Cloning and replication after the synthetic sequence is inserted i...

Embodiment 1

[0064] Embodiment 1, the preparation of nucleic acid signal amplification sequence

[0065] Design the leader sequence according to the primer sequence 1, and design the repeat sequence of different multiples at the same time, and add the HindII enzyme cutting site at the 5' end of the leader sequence, and the EcoR V enzyme cutting site at the 3' end of the repeat sequence, and synthesize After different conformational sequences were inserted into the pGEM-3ZF(-) vector, clone and replicate; HindII single-digested the vector sequence to generate 5' sticky ends, and used Kelnow large fragment polymerase and adenosine thiotriphosphate to fill in the 5' sticky ends, and then used EcoR V excises the target band from the vector, and exonuclease III enzyme cuts the single-stranded DNA probe. Each sequence is shown in Table 1.

[0066] Table 1 each sequence

[0067] sequence name sequence target ATCGTTAGGCATTCAGGGA leader sequence TCCCTGAATGCCTAACGAT ...

Embodiment 2

[0068] Embodiment 2, primary signal amplification

[0069] 1. Coat the target target in Table 1 with a concentration of 100pmol on a microwell plate;

[0070] 2. According to the method of Example 1, each sequence was synthesized and prepared into a single-stranded probe, and the probes prepared by Synthetic Sequence 1, Synthetic Sequence 2, and Synthetic Sequence 3 were respectively named as pre-amplification 1, pre-amplification 2, and pre-amplification 3;

[0071] 3. Control the hybridization temperature between 50°C and 55°C, add pre-amplification 1, 2, and 3 to each well at a concentration of 100 pmol / μL, and hybridize for 40 to 50 minutes;

[0072] 4. Add the reporter probe (AAAAGAATATTGGTGGTAAAGACCT-AP) respectively, and react at 51℃~53℃ for 30~40min;

[0073] 5. Add the chemiluminescent substrate lumi-Phos Plus (Beckman Inc.), and read the experimental results.

[0074] The layout design is shown in Table 2, and the corresponding data results are shown in Table 3.

...

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Abstract

The invention relates to the field of molecular biology, in particular to a nucleic acid signal amplification sequence and a nucleic acid signal amplification method. The nucleic acid signal amplification sequence comprises a leader sequence and a repetitive sequence in a plurality of times; the leader sequence and an objective target drone sequence complement each other; and the repetitive sequence and a reporter probe complement each other. An objective target drone can be captured and hybridized by the leader sequence of the nucleic acid signal amplification sequence, and then by the repetitive sequence in a plurality of times and corresponding reporter probes, signals can be amplified, so that nucleic acid signals of the objective target drone are detected. Compared with a PCR technology which needs enzyme to maintain a hybrid system, the nucleic acid signal amplification sequence can detect nucleic acid substances on the premise of not increasing concentration of a detected object, and is good in stability, high in repeatability, low in cost and short in detection technological process.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a nucleic acid signal amplification sequence and a nucleic acid signal amplification method. Background technique [0002] Polymerase Chain Reaction (Polymerase Chain Reaction, PCR) is a molecular biology technique used to amplify specific DNA fragments. It can be regarded as special DNA replication outside the body, which can greatly increase the amount of DNA . Therefore, whether it is ancient organisms in fossils, the remains of historical figures, or the hair, skin or blood left by the murderer in a murder case decades ago, as long as a little bit of DNA can be isolated, it can be amplified by PCR for comparison. right. [0003] The basic principle of PCR technology is similar to the natural replication process of DNA, and its specificity depends on oligonucleotide primers complementary to both ends of the target sequence. PCR consists of three basic reaction steps: denatu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/682C12N15/10
CPCC12Q1/6806C12Q1/682
Inventor 张鹭鹭李先坤张爱国
Owner 成都诺森医学检验有限公司
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