| Objectives:Combinded chemotherapy is one of the main treatments of breast cancer. There is significant improvement for prognosis, but the efficacy and side effects of chemotherapy are quite diverse in different individuals, which have been shown closely related to single nucleotide polymorphism(SNP). However, the exact association between the efficacy and SNP of multiple genes has not been reported at home and abroad as yet. Hairpin probe, immobilized on a support surface, is an important tool suitable for detection of SNP and other single base mutation. But the advantage of fluorescence resonance energy transfer(FRET) for hairpin probe technique is difficult to retain in practice, leaving great constraints for the application of hairpin probes. To solve this problem, the improvement methods worldwide mostly count on modified probe stems arm, but the technical difficulty of this approach also subject to certain restrictions. In this study, we integrated the advantages and disadvantages of various improved designs, and proposed a new fracture-docking hairpin probe technology, with a fluorescein / quencher and fracture-docking / closely-contact fashion, to realize the original core technology-FRET phenomenon in different ways. Moreover, this new technology combines the powerful enrichment function of the magnetic bead to amplify fluorescent signals, establishing a signal amplification system based on fracture-docking hairpin probe binding magnetic beads, which optimize the detection more efficiently.Methods:1.Inquiry in NCBI Gen Bank the CYP2C8 gene sequences that related in liver metabolism of the combinded chemotherapy drugs for breast cancer, paclitaxel and doxorubicin, and use the original sequence of the gene and 139A/G SNP locus as the wild sequence and mutation target sequence in following test. By bioinformatics software, design the fracture-docking hairpin probes detecting CYP2C8 gene SNP mutation. Explore the probe concentration and hybridization temperature scale, and ultimately determine the optimal probe concentration ratio and the optimum temperature detection system, to distinguish between the wild target sequence and mutant target sequence.2.Fix the fracture-docking hairpin DNA probes to the nano-beads obtained above, and establish a combined signal amplification system. The fracture-docking hairpin DNA probe and nano-magnetic beads were collected in centrifuge tubes. Detect the signal amplifying effect by enriched fluorescent signal, while optimize conditions for this system with labeled probes which has been previously selected for modification. Observe and compare fluorescence between the wild target sequence and the mutant sequence under fluorescence microscopy. Explore the best concentration,temperature and time for collection magnetic beads in detection system.3.Collect blood samples of 50 breast cancer patients, and extract their genomic DNA. PCR amplify the human gene CYP2C8 with designed 139A/G SNP locus mutation primers, and recollect the CYP2C8 gene fragments from gel. Verify the 139A/G SNP wild/mutation by signal amplification system combined with fracture-docking hairpin probe and nano-magnetic beads. The accuracy and reliability of the new method applied in clinical testing were also confirmed.Results:1.The 139 th amino acids of CYP2C8 gene sequence is AGG, and mutation occurred for AAG. After probe design and condition optimization, the optimal hybridization temperature for detecting CYP2C8 of 139A/G SNP is 40 ℃, the optimal ratio of fluorescent probe P1/ quenching probe P2 is 1: 4, the best hybridization time is 2h when the probe was fixed on nano-magnetic beads. The optimized CYP2C8 SNP detection system has significantly improved the effect of detecting the mutation 139A/G.2.Successfully constructed nano-magnetic beads for fracture-docking hairpin DNA probe, and the detection effect of signal amplification system by magnetic beads enrichment is significant, and it is more efficient and more sensitive to distinguish the fluorescence signal intensity between wild and mutant sequences of CYP2C8 139A/G SNP loci, suggesting that signal amplification system combined with fracture-docking hairpin probe and magnetic beads was initially established.3.The DNA concentration of breast cancer patients was high, while the amplification effects of CYP2C8 gene 139A/G SNP locus mutation sequence(with a fragment size expected to be 153 bp unanimously) was good as well. The signal amplification system combined with fracture-docking hairpin probe and nano-magnetic beads for detecting CYP2C8 gene 139A/G SNP locus mutation was fine.Conclusions:1.This study successfully established a new response system with hairpin DNA probe, and can efficiently detect the CYP2C8 139A/G SNP site mutation involved in drug metabolism of breast cancer chemotherapy.2.On the basis of the fracture-docking hairpin probe, this study initially established a simple and efficient signal amplification system combined with nano-magnetic beads for detecting CYP2C8 gene 139A/G SNP locus mutation. And it provides a potential means of detection for individual applications and new targets in clinical drug studies. |
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