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A circular blocking probe, an amplification hindering mutation system containing the circular blocking probe and its application

A technology for amplifying impeding mutations and probes, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve problems such as the inability to significantly improve linear performance and the inability to effectively reduce non-specific amplification. , to avoid non-specific amplification, improve amplification specificity and linear range, and reduce non-specific amplification

Active Publication Date: 2021-02-05
QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to differences in template sequences, traditional blockers often cannot effectively reduce non-specific amplification, and cannot significantly improve linear performance

Method used

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  • A circular blocking probe, an amplification hindering mutation system containing the circular blocking probe and its application
  • A circular blocking probe, an amplification hindering mutation system containing the circular blocking probe and its application
  • A circular blocking probe, an amplification hindering mutation system containing the circular blocking probe and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Detection Principle of the Amplification-Hindered Mutation System Containing a Circular Blocking Probe

[0055] The blocking principle of the circular blocking probe is as follows: figure 1 As shown, under non-denaturing conditions, the first binding sequence and the second binding sequence of the circular blocking probe complement each other to form a circular structure, which does not bind to the template, which reduces the interference to the PCR reaction to a certain extent; Under these conditions, the first binding sequence and the second binding sequence of the circular blocking probe melt, and the circular structure opens, and the blocking probe specifically binds to the wild-type site corresponding to the point mutation and its downstream, preventing the wild-type template from forming. Amplify.

[0056] The detection principle of the fluorescent quantitative amplification hindered mutation system containing the above-mentioned circular blocking probe...

Embodiment 2

[0057] Example 2 Quantitative detection of JAK2-1849G / T mutation

[0058] In this example, using human JAK2 genomic DNA (NCBI: NG_009904.1) as a template, PCR primer pairs, fluorescent probes and circular blocking probes were designed for the 1849G / T mutation site.

[0059] Among them, the 3' end of the specific downstream primer is located at the mutation site and is complementary to the mutant type (T). Considering that the 3' end of the downstream primer and the wild-type template are strongly A / G mismatched, the 3' end of the downstream primer is strongly mismatched with the wild-type template. The penultimate position of the ' end introduces a weak A / C mismatch; the 3' end of the circular blocker probe is located at the mutation site and is complementary to the wild type (G), considering that the 3' end of the circular blocker probe is compatible with If the mutant template is a strong C / T mismatch, a weak A / C mismatch is introduced at the penultimate third position of th...

Embodiment 3

[0077] Embodiment 3 result analysis

[0078] The fluorescent quantitative PCR curve of the experimental results is as follows: figure 2 As shown, compared with the reaction system without adding the blocking probe, the wild-type non-specific amplification was significantly reduced after adding the blocking probe; while the specificity of the reaction system adding the circular blocking probe was better than that adding Reaction system for non-circular retardation probes. In addition, from the amplification curve, after adding the circular blocking probe, the repeatability of the low-frequency samples is good, and the amplification curves of samples with different frequencies are significantly different, indicating that the variance of the Ct value is small and the detection results are more accurate.

[0079] The standard curve of the experimental results is as image 3 As shown, when the mutation frequency of the sample is as low as 0.05%, the linearity of the standard cur...

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Abstract

The invention provides a circular blocking probe, an amplification hindering mutation system containing the circular blocking probe and its application. The circular blocking probe is the first binding sequence according to the 5'-3' direction, A circular sequence and a second binding sequence, the first binding sequence and the second binding sequence are mutually inverted repeat sequences; the 1 base at the 3' end of the circular blocking probe is complementary to the wild-type site of the template ; The 3' end of the circular blocking probe is modified with a blocking group; the circular sequence of the circular blocking probe is complementary to the template. The invention adds a circular blocking probe to the amplification hindering mutation system, and the circular blocking probe is preferentially combined with the wild-type template before the PCR starts, to block the wild-type template, and then use specific primers to perform PCR on the mutant template Amplification, which significantly improves the specificity of PCR amplification, expands the linear range, and realizes the accurate quantification of low-frequency mutation samples.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a circular blocking probe, an amplification blocking mutation system containing the circular blocking probe and applications thereof. Background technique [0002] Myeloproliferative neoplasms (MPN) are usually accompanied by a mutation at position 1849 (G / T) of the JAK2 gene, resulting in a mutation of valine (V) at position 617 of the protein to phenylalanine (F), thereby causing a series of lesions. In polycythemia vera (PV) patients, the mutation rate of JAK2-V617F is about 90%~95%, while in essential thrombocythemia (ET) and primary myelofibrosis (PMF), JAK2-V617F The mutation rate is about 50% to 60%. The World Health Organization (WHO) clearly regards the JAK2-V617F mutation as one of the main diagnostic indicators for MPN. With the launch of the new drug ruxolitinib (Jiekewei), there is gradually a demand for quantitative detection of JAK2-V617F in the market. Through quantit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6858
CPCC12Q1/6858C12Q2531/113C12Q2535/137C12Q2537/163C12Q2525/307C12Q2563/107
Inventor 程雪涛王洁焦柔张亚飞
Owner QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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