Quenching group for multicolor melting curve analysis, probe and application

Abstract

The invention relates to a quenching group for multicolor melting curve analysis, a probe and application, and belongs to the field of gene detection.The GT quenching group is obtained through modification by introducing a long-chain alkyl group, so that the signal intensity and the signal-to-noise ratio of the GT quenching group in multiple melting curve analysis are improved. Through a specific modification strategy, the quenching group disclosed by the invention shows higher sensitivity and accuracy in application of genotyping, mutation detection, pathogen identification and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of gene detection, relates to a quenching gene, and specifically relates to a quenching group, a probe and an application thereof for multicolor melting curve analysis. Background Art

[0002] Cervical cancer is one of the common gynecological malignancies, and its incidence ranks second among female malignant tumors in my country. The occurrence and development of cervical cancer are closely related to infection with human papillomavirus (HPV). Different HPV subtypes have different pathogenicity. More than 120 HPV subtypes have been isolated, of which 18 are common high-risk types. In addition to the traditional risk factors such as persistent infection with high-risk HPV, multiple sexual partners, and early first sexual intercourse, epigenetic changes, such as DNA methylation, are found in cervical lesions and increase accordingly with the increase in the severity of lesions, which are considered to be factors leading to the occurrence and development of cervical cancer. Therefore, the analysis of related gene methylation can be used as a method to predict and evaluate cervical precancerous lesions. Different genes, such as PAX1, SOX1, DAPK1, ZNF582, etc., have different sources, gene expressions, and the degree of their influence on cervical lesions. HPV DNA genotyping tests commonly used in clinical practice can reflect the type and load of viruses in cervical epithelial cells, but cannot tell the activity of the virus. Studies have shown that EP300 (16%), FBXW7 (15%), PIK3CA (14%), HLA-B (9%) and p53 (9%) gene mutations are associated with the occurrence of cervical squamous cell carcinoma, while PIK3CA (16%), ELF3 (13%), KRAS (8%) and CBFB (8%) gene mutations are associated with the occurrence of cervical adenocarcinoma. These potential gene mutations can not only serve as markers for the early diagnosis of cervical cancer, but also play a certain role in the prognosis of cervical cancer. For example, PIK3CA gene mutations can increase the risk of distant metastasis of cervical cancer.

[0003] PAX1 gene: Paired box PAX gene family usually contains paired box domain and paired type homeodomain, which is essential in fetal development and plays a key role in spinal development. PAX gene family plays a regulatory role in multiple signal transduction pathways. Then it is continuously expressed in stem cells and mature cells. Specific PAX expression proteins can maintain stem cell characteristics, thereby affecting the occurrence and progression of tumors.

[0004] SOX1 gene: The SOX gene family is a conservative sequence with 80 amino acid sequences. Current research divides the SOX gene family into 9 subfamilies (SOXB1, SOXB2) from SOXA to SOXH. Its functions include participating in testicular development, central nervous system neurogenesis, oligodendrocyte development, cartilage formation and neural crest cell development. SOX1 (sex determining region Y-box1) belongs to the SOXB1 subfamily. Several key experiments have shown that the SoxB1 subfamily genes can maintain the state of progenitor cells. The expression of SOX1 in a certain conformation in progenitor cells can prevent their differentiation, so the SOX1 gene plays a role in inhibiting tumor differentiation and proliferation. Low expression of the SOX1 gene was found in tumor cells, which is related to its methylation. It has now been found that SOX1 gene methylation is related to the occurrence of cervical cancer, prostate cancer, liver cancer, etc.

[0005] ZNF582 gene: zinc finger protein 582 (ZNF582) is located at chromosome 19q13.43. In 1991, Bellefroid et al. first discovered the KRAB-ZNF family (encoding proteins containing KRAB). The KRAB-ZNF family may be involved in many physiological processes, including cell differentiation, cell proliferation, cell apoptosis and tumor transformation. Animal experiments have found that the KRAB protein encoded by the KRAB-ZNF family plays a regulatory role in cell morphogenesis.

[0006] DAPK1 gene: The death-associated protein kinase 1 (DAPK1) gene is located at the human chromosome 9q34.1 site and is a Ca 2+ / CaM-dependent serine / threonine protein kinase, as a positive apoptosis regulator, widely participates in the apoptosis process through multiple pathways and plays a role in cell signal transduction pathways. It participates in regulating cell survival, apoptosis and autophagy. In the current research on squamous cell carcinoma and myelodysplastic syndrome, DAPK1 gene is considered to be a biomarker for squamous cell carcinoma. Related studies have also shown that abnormal methylation of CpG island in the promoter region of DAPK gene in tumor cells is an important reason for the loss of DAPK gene expression.

[0007] Multiple melting curve analysis is an analytical method based on the melting characteristics of nucleic acid amplification products, which is used to distinguish DNA or RNA of different sequences. In this technology, fluorescent groups and quenching groups are usually used to label probes to monitor the melting behavior of target sequences. However, existing fluorescent groups and quenching groups may not provide sufficient signal intensity or signal-to-noise ratio in some cases, limiting the sensitivity and accuracy of the analysis.

[0008] my country is in a critical period of accelerating the elimination of cervical cancer. Using high-quality screening methods to increase screening coverage is one of the main means to eliminate cervical cancer.

[0009] Multiple melt curve analysis (MMR) is a technique widely used in molecular biology and clinical diagnosis, which can detect multiple targets simultaneously. Traditional probes modified with fluorescent groups and quenching groups have problems such as low sensitivity, poor specificity and high background signal in multiple melt curve experiments. Therefore, it is urgent to develop new fluorescent groups and quenching groups and their modification methods to improve the performance of multiple melt curve experiments.

[0010] In multiple melting curve analysis, the quencher works as follows: 1. Energy transfer: The quencher group binds to the fluorescently labeled probe through the fluorescence resonance energy transfer (FRET) mechanism. When the fluorophore (reporter) is excited, it can transfer energy to the adjacent quencher group, causing the fluorescence of the fluorophore to be quenched.

[0011] 2. Avoid background signals: The presence of quenching groups can reduce the fluorescence signal of unbound probes, thereby reducing background noise and improving the specificity and sensitivity of detection.

[0012] 3. Signal release: When the probe specifically binds to the target sequence, the distance between the quencher and the fluorophore increases, resulting in a decrease in energy transfer efficiency and the fluorescence of the fluorophore is restored, thereby generating a detectable signal. Summary of the invention

[0013] In view of the above problems, the purpose of the present invention is to provide a quencher group, a probe and an application for multicolor melting curve analysis. The present invention prepares a quencher group through a specific modification strategy, improves its signal intensity and signal-to-noise ratio in multiple melting curve analysis, and makes it show higher sensitivity and accuracy in applications such as genotyping, mutation detection and pathogen identification.

[0014] In order to achieve the above object, the specific scheme adopted by the present invention is: In the first aspect, the present invention claims a GT quenching group, wherein the GT quenching group is a conventional quenching group by introducing a long-chain alkyl group (C 16H 33 -) is modified by changing its structure. Further, the carbon chain length of the long-chain alkyl is C 6 H 13 —C 10 H 21 Furthermore, the conventional quenching group is BHQ, and the long-chain alkyl group is C 16 H 33 .

[0015] In a second aspect, the present invention claims a probe, wherein the 3' end of the probe is labeled with the above-mentioned GT quenching group. Further, the sequence structure of the probe is: 5'-FAM-GGCCGCG-AGCTTCGAAAGCCTTAGCGGTTCCAACTAC-CGCGGCC-BHQ-3' (sequence as shown in SEQ ID NO: 1), the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a GT quenching group.

[0016] In a third aspect, the present invention seeks to protect the use of the above-mentioned GT quenching group or probe in multiple melting curve analysis.

[0017] In a fourth aspect, the present invention seeks to protect the use of the above-mentioned GT quenching group or probe in the preparation of a cervical cancer detection reagent.

[0018] In a fifth aspect, the present invention seeks to protect the use of the above-mentioned GT quenching group or probe in genotyping or mutation detection.

[0019] Beneficial effects: The modification method of the present invention significantly improves the application performance of fluorescent groups and quenching groups in multiple melting curve analysis, and provides a powerful tool for research and application in the field of gene diagnosis represented by HPV detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figures 1 to 12 This is the chemical formula of the modified probe sequence during verification in Example 1.

[0021] Figures 13 to 24 This is the original melting curve after the modified probe sequence was used for verification in Example 1. DETAILED DESCRIPTION

[0022] The present invention preferably uses a fluorescent group with high brightness and low light drift. The improvement direction is as follows: 1. Introducing long-chain alkyl or aromatic groups: By introducing long-chain alkyl groups into the structure of the fluorescent group, its hydrophobicity is increased, thereby reducing nonspecific binding; 2. Expansion of the conjugated system: By expanding the conjugated system of the fluorescent group, its absorption and emission intensity are enhanced to improve the detection sensitivity; 3. Rare earth element doping: Rare earth elements are doped into fluorescent groups to improve the emission efficiency and stability of fluorescence.

[0023] By comparison, we designed a quenching group by introducing a long-chain alkyl group, which is named GT quenching group.

[0024] The modified quenching group of the present invention enhances the photostability of the fluorescent group and the quenching efficiency of the quenching group by introducing a specific functional group or structural change. The modification reduces the hydrophobicity value and improves the spatial accessibility, so that the background signal can still be lower at 90 degrees, thereby improving the reliability and accuracy of the experiment.

[0025] The GT quenching group of the present invention, in the absence of target DNA, the molecular beacon is in a stem-closed form, the organic fluorophore is close to the GT quenching group, the fluorescence of the organic fluorophore FAM, TAMRA or multiple channels is quenched by the GT quenching group, and the fluorescence signal of the organic fluorophore is very weak. In the presence of target DNA, the molecular beacon hybridizes with the target DNA, the stem complementary part of the molecular beacon is melted, and the fluorophore and the GT quenching group cause the fluorescence recovery of the fluorophore. In addition, the Stokes shift of FAM and TAMRA is very close, so they can obtain fluorescence signals simultaneously by synchronous fluorescence analysis. Therefore, the molecular beacon can be used to realize the simultaneous detection of two or more target DNAs by synchronous fluorescence analysis.

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0027] Example 1 Design and application of GT quenching group Specific steps: Select fluorescent groups and quenching groups: Select fluorescent groups available on the market such as FAM, HEX, CY5, etc., and commonly used quenching groups. Take FAM fluorescent group and BHQ quenching group as an example.

[0028] Sequence (SEQ ID NO: 1): 5'-FAM-GGCCGCG-AGCTTCGAAAGCCTTAGCGGTTTCCAACTAC-CGCGGCC-BHQ-3' Modification of quenching groups: by introducing long-chain alkyl groups (CH 3 - to C 12 H 25 -) to change its structure and improve the quenching efficiency of the quenching group, thereby reducing background fluorescence. The modified sequence is as follows Figures 1 to 12 shown. Probe design and application: The modified quencher group was applied to the probe design for multiple melting curve analysis to optimize the probe length, sequence and labeling position.

[0029] Experimental verification: A series of experiments were performed, including modifying CH 3 - to C 12 H 25 -groups, and then experiments were conducted to generate melting curves and data analysis, and the performance of the unmodified and modified quenching groups was compared to verify the application effect in multiple melting curve analysis. The modified sequences are shown in the figure. 6 H 13 —C 10 H 21 The effect of reducing the signal-to-noise ratio of background noise is the best. The melting curves of the modified sequences are as follows Figures 13 to 24 Shown like Figures 13 to 17 As shown, when the modifying group is CH 3 - to C 5 H 11 -, the quenching group fails to stably bind to the probe; Figures 18 to 22 As shown, when the modifying group is C 6 H 13 - to C 10 H 21 -, the original melting curve is gentle and stable, indicating that the quenching group and the probe are stably combined. Figure 23 to Figure 24 As shown, when the modifying group is C 11 H 23 - to C 12 H 25 -, the original melting curve fluctuated again, indicating that the quenching group failed to stably bind to the probe.

[0030] Through the above technical improvements, the GT quenching group provided by the present invention has the following advantages: 1. Reduce background signals: The quenching group can absorb the fluorescence emitted by the adjacent fluorescent group, thereby reducing background noise and improving the signal-to-noise ratio of the experiment.

[0031] 2. Improve detection sensitivity: Due to the reduction of background signals, weak fluorescent signals can be more easily detected, making the detection of low-abundance targets possible.

[0032] 3. Enhanced specificity: In some applications, such as molecular beacons or TaqMan probes, the distance between the quencher and the fluorescent group is very close. The fluorescent signal will only be detected when the target sequence is present and causes the probe structure to change, which increases the specificity of the detection.

[0033] 4. Multiplex detection: In experiments such as multiplex PCR, multiple targets can be detected simultaneously using different fluorescent groups and corresponding quenching groups. Each fluorescent group-quenching group pair corresponds to a specific target sequence.

[0034] 5. Prevent fluorescence energy transfer: In fluorescence resonance energy transfer (FRET) experiments, quenching groups can prevent fluorescence energy from being transferred between non-target molecules, ensuring that energy transfer only occurs in target events.

[0035] 6. Stable fluorescent labels: Certain quenching groups can improve the photostability of fluorescent labels, reduce photobleaching, and make the fluorescent signal more durable.

[0036] 7. Adjusting fluorescence lifetime: In some fluorescence detection techniques, such as time-resolved fluorescence (TRF) or fluorescence lifetime imaging (FLIM), quenching groups can be used to adjust the lifetime of fluorescent groups to meet different detection requirements.

[0037] 8. Improve experimental repeatability: By reducing background signals and improving specificity, quenching groups help improve the repeatability and reliability of experimental results.

[0038] 9. Avoid signal crosstalk: In multiple detection experiments, quenching groups help avoid signal interference between different detection channels and ensure that the signal of each channel is clearly distinguishable.

[0039] It should be noted that the above-described embodiments should be understood as illustrative rather than limiting the scope of protection of the present invention, and the scope of protection of the present invention shall be subject to the claims. For those skilled in the art, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention without departing from the essence and scope of the present invention.

Claims

1. A GT quenching group, characterized in that: The GT quenching group is obtained by modifying the conventional quenching group by introducing a long-chain alkyl group to change its structure.

2. The GT quencher group according to claim 1, characterized in that: The carbon chain length of the long chain alkyl is C6H 13 -—C 10 H 21 -.

3. The GT quencher group according to claim 2, characterized in that: The conventional quenching group is BHQ, and the long-chain alkyl is C 10 H 21 -.

4. A probe, characterized in that: The GT quencher group of claim 1 is labeled at the 3' end of the probe.

5. The probe according to claim 4, characterized in that: The sequence of the probe is shown in SEQ ID NO:

1. The 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a GT quenching group.

6. Use of the GT quencher group according to any one of claims 1 to 3 or the probe according to any one of claims 4 to 5 in multiple melting curve analysis.

7. Use of the GT quenching group according to any one of claims 1 to 3 or the probe according to any one of claims 4 to 5 in the preparation of a cervical cancer detection reagent.

8. Use of the GT quencher group according to any one of claims 1 to 3 or the probe according to any one of claims 4 to 5 in genotyping or mutation detection.

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