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Bridge type fluorescent probe with bridge type sequence zone doping into mismatched bases and application and method

A technology of mismatched bases and fluorescent probes, which is applied in the application field of bridge fluorescent probes, can solve the problems of low sensitivity and false positives of low-abundance mutant genes

Inactive Publication Date: 2016-01-27
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In view of this, in order to solve the problem that existing probes detect low-abundance mutant genes with low sensitivity and are prone to false positives, the present invention firstly provides a bridge-type fluorescent probe that incorporates mismatched bases in the bridge-type sequence region. The bridge-type fluorescent probe is simple in design, has high specific recognition ability for single-base variation, can completely inhibit non-specific hybridization, and can detect low-abundance single-base variation with high specificity. At the same time, the probe technology It is also especially suitable for the detection of nucleic acid target molecules with high homology

Method used

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  • Bridge type fluorescent probe with bridge type sequence zone doping into mismatched bases and application and method
  • Bridge type fluorescent probe with bridge type sequence zone doping into mismatched bases and application and method
  • Bridge type fluorescent probe with bridge type sequence zone doping into mismatched bases and application and method

Examples

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Effect test

Embodiment 1

[0040] Example 1. Detection of BRAF gene V600E gene mutation with a single tube of ipsilateral competitive bridge fluorescent probe

[0041] 1. Design of ipsilateral competitive probes targeting BRAF gene V600E gene mutation and its amplification primers

[0042] According to the nucleic acid sequence characteristics of BRAF gene V600E ( Image 6 ) Design ipsilateral competitive bridge fluorescent probes (Table 1), including wild-type bridge fluorescent probe one (SW-01) targeting BRAF gene wild-type V600E and mutant bridge targeting BRAF gene mutant V600E Formula fluorescent probe one (SW-03). The structural and sequence characteristics of the above-mentioned bridge fluorescent probes are as follows: they all target the sense strand of the BRAF gene; the identified mutant bases are located in the middle of the recognition sequence region (the corresponding sequences are the bases marked in bold and underlined in Table 1) , the 3'-terminus is modified with a phosphate group ...

Embodiment 2

[0074] Example 2. Detection of BRAF gene V600E gene mutation with a single tube of contralateral non-competitive bridge fluorescent probe

[0075] 1. Design of contralateral non-competitive probe and its amplification primers targeting BRAF gene V600E mutation

[0076] According to the nucleic acid sequence characteristics of BRAF gene V600E ( Image 6 ) Design contralateral non-competitive bridge fluorescent probes (Table 1), including wild-type bridge fluorescent probe one (SW-01) targeting BRAF gene wild-type V600E and mutant type targeting BRAF gene mutant V600E Bridge fluorescent probe II (SW-07). The structure and sequence characteristics of the above-mentioned bridge fluorescent probe are as follows: respectively target the sense strand and the antisense strand of the BRAF gene; the identified mutant base is located in the middle of the recognition sequence region (the corresponding sequence is the bold and underlined position in Table 1) , the 3'-terminus is modified...

Embodiment 3

[0093] Example 3, bridge fluorescent probe technology detection KRAS 12th codon gene mutation

[0094] The 12th and 13th codons of KRAS gene ( Figure 9 ) missense mutation is a common mutation site in a variety of malignant tumors including colorectal cancer, and its mutation status is directly related to the molecular classification of malignant tumors and the effect of individualized treatment.

[0095] 1. Design of ipsilateral competitive probe targeting KRAS gene position 12 and its amplification primers

[0096] According to the nucleic acid sequence characteristics of the 12th and 13th codons of the KRAS gene ( Figure 9 The shaded bases are the nucleic acid sequence of the 12th and 13th codons of KRAS) bridge fluorescent probes targeting the antisense strand of the 12th codon of the KRAS gene, including wild-type bridge fluorescent probes (SW- 16), c.34G>A mutant bridge fluorescent probe (SW-17), c.34G>C mutant bridge fluorescent probe (SW-18), c.34G>T mutant bridge ...

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Abstract

The present invention discloses a bridge type fluorescent probe with a bridge type sequence zone doping into mismatched bases and application and a method, the bridge type fluorescent probe 5'-end to 3'-end nucleic acid sequence successively comprises a recognition sequence zone complementary to a nucleic acid sequence of a to-be-tested mutant gene, the bridge type sequence zone, and an anchor sequence zone, wherein the anchor sequence zone is complementary to the outer side sequence zone of 3'-end of the to-be-tested mutant gene, the 3'-end of the anchor sequence is connected with an extension blocking group ; the bridge type sequence zone is a nucleotide derivative sequence containing at least one mismatched bases, an interactiveness fluorescent marking system is marked between the recognition sequence zone and the bridge type sequence zone, the bridge type fluorescent probe is simple in design, can specifically recognizes the gene, is high in sensitivity, and can be used to detect single base mutation and identify highly homologous species and subtypes.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection, and in particular relates to a bridge fluorescent probe in which mismatched bases are incorporated into a bridge sequence region, and also relates to the application and method of the bridge fluorescent probe. Background technique [0002] Single base mutations include common single nucleotide polymorphisms (SNPs) and gene mutations in disease states (such as cancer gene mutations that lead to malignant tumors). [0003] There are many existing detection techniques, mainly fluorescent probe detection techniques for single base variation, such as hydrolysis probes (also known as TaqMan probes), hybridization probes (hybridization probes), molecular beacons (molecular beacons), etc. It is the most common and most commonly used detection technique. However, the above-mentioned probes are not ideal for single-base variation detection. On the one hand, it is very difficult to design the above-m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C09K11/06
Inventor 黄庆府伟灵黄君富夏涵赵娜
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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