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Differential screening of deoxyribozyme probe for specifically sensing triple negative breast cancer

A deoxyribozyme probe, triple-negative breast cancer technology, applied in biochemical equipment and methods, instruments, analysis materials, etc.

Pending Publication Date: 2021-06-29
FUDAN UNIV SHANGHAI CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Researchers have used this method to obtain a DNAzyme probe that can specifically recognize the lysate of TNBC cell line MDA-MB-231 and cleave at a specific RNA base site, but the probe recognizes intracellular molecules , the cells need to be lysed in the actual detection, which has limitations in the clinical diagnosis of TNBC

Method used

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  • Differential screening of deoxyribozyme probe for specifically sensing triple negative breast cancer
  • Differential screening of deoxyribozyme probe for specifically sensing triple negative breast cancer
  • Differential screening of deoxyribozyme probe for specifically sensing triple negative breast cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The construction of embodiment 1 DNA library

[0036] The synthesized substrate chain, enzyme chain and connecting chain were mixed at 1:1:1, incubated at 70°C for 5 minutes, and then annealed at room temperature for 10 minutes; T4 ligase was added and reacted at room temperature for 2 hours. After the reaction, 3 times the volume of -20°C 100% ethanol and 3 μL of 2.5 mg / mL glycogen were added, and placed in a -80°C refrigerator for 10 minutes. The frozen mixture was centrifuged at low temperature and high speed (4°C, 15000rpm, 30min), and the supernatant was removed. After the ethanol volatilized, 20 μL of ultrapure water was added to redissolve, and then subjected to 8% denatured polyacrylamide gel electrophoresis (Denatured Polyacrylamide Gel Electrophoresis, dPAGE ) was purified, and the successfully connected band (full length 119nt) was recovered with a fluorescence imager and a rubber tapping instrument. Add crush-soak buffer (200mM NaCl, 10mM Tris-HCl pH 7.5, 1...

Embodiment 2

[0046] Example 2 Cell culture and extracellular metabolites (extracellular mixtures, EMs) extraction

[0047] Select typical TNBC subtype MCA-MB-231 (ATCC No.HTB-26) as the target cell line, normal breast cell line MCF-10A (ATCC No.CRL-10317) and HMEC (ATCC No.PCS-600-010) as control cells. The above-mentioned cells were cultured and passaged according to the culture protocol recommended by ATCC. When the cells were cultured to about 80% confluence, they were washed with PBS three times and then transferred to serum-free medium for 24 hours, centrifuged to remove cell debris and filtered with a 0.22 μm membrane, and protease inhibitors were added to the resulting supernatant, and placed at -80 Store at ℃, and obtain the EMs required for screening.

Embodiment 3

[0048] Example 3 Differential Screening

[0049] Such as figure 2 As shown, the differential screening is divided into steps such as negative screening, positive screening, PCR amplification, and ligation into a library. Clones were sequenced after 11 rounds of selection. Specific screening steps include:

[0050] 1. Negative screening

[0051] Dissolve the DNA library constructed in Example 1 with 75 μL buffer 1, heat in a metal bath at 70°C for 5 minutes, then cool down to 37°C, add 25 μL of EMs of normal mammary epithelial cells MCF-10A and HMEC, 37°C Incubate for 5min; then add 100μL containing Mg 2+ Buffer 2, incubate at room temperature for 1 h for negative selection. Finally, an equal volume of 2×loading buffer (containing urea) was added to terminate the reaction, and the uncut DNA band, ie, the full-length sequence, was separated and purified by 8% dPAGE, and eluted overnight.

[0052]

[0053] 2. Positive screening

[0054] The full-length sequence obtaine...

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Abstract

The invention discloses a deoxyribozyme probe for specifically sensing triple negative breast cancer and a screening method. According to the invention, the deoxyribozyme probe for specifically sensing the extracellular metabolite of the TNBC cell line MDA-MB-231 is obtained through a'hedging counteracting 'difference screening strategy under the condition that a target molecule is unknown. The probe has the characteristics of high recognition speed and good specificity, and provides a new idea for subsequent development of rapid diagnosis of triple-negative breast cancer.

Description

technical field [0001] The invention belongs to the technical field of deoxyribozyme probes, and in particular relates to a differential screening method to obtain deoxyribozyme probes for specific induction of triple-negative breast cancer. Background technique [0002] Triple-negative breast cancer (TNBC for short) is a type of breast cancer that is negative for estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, accounting for 15% of the total breast cancer -20%. TNBC has the characteristics of younger patients, strong tumor invasiveness, poor prognosis after radiotherapy and chemotherapy, and distant metastasis in the early stage of the disease. It is currently the most malignant breast cancer. Due to the lack of clear markers (that is, therapeutic targets), the existing endocrine therapy and targeted therapy are not effective for TNBC, and conventional radiotherapy and chemotherapy can only benefit some patients. [0003] Deoxyribozy...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10G01N33/569G01N33/574
CPCC12N15/113C12N15/1072G01N33/56966G01N33/57484G01N33/57415C12N2310/127G01N2333/9005
Inventor 顾宏周胡沁沁佟宗轩江一舟邵志敏刘西禹葛丽萍傅彤
Owner FUDAN UNIV SHANGHAI CANCER CENT
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