Preparation And Optimization Of HER-2 Gene FISH Probe

Objective:We designed an accurate,low-cost new FISH probe and the HER-2 gene was detected by FISH using this probe.We performed a feasibility analysis of the effectiveness of FISH probes and optimized the hybridization system.Methods:HepG2 cells were cultured for extracting total DNA.The target sequences of dsDNA and ssDNA of the HER-2 gene were amplified by PCR and asymmetric PCR,and the specificity of the fluorescent marker sequence in the novel FISH probe was analyzed by FISH.The hybridization effect of the novel FISH probe was verified by electrophoresis and Dot-blot.Oligonucleotide-based FISH probes were used to locate the distribution of RNA in cells.A long-chain novel FISH probe was designed and prepared by PCR to locate the HER-2 gene intron.The FISH system was optimized for antigen retrieval,enzyme digestion time and formamide concentration.Results:1.Design a new type of FISH probe divided into two parts.One is a fluorescent sequence region that is bound to a fluorescent marker sequence,and the other is a specific sequence that binds to a target gene.Simultaneous addition of fluorescent sequences was achieved during FISH hybridization.Different target genes only need to change the specific sequence part of the target gene binding to achieve the same fluorescent marker sequence to detect different target genes,thereby reducing the probe cost.2.The HER-2 ssDNA fragment was successfully prepared by asymmetric PCR and the specificity of the fluorescent sequence in the novel FISH probe was verified.3.Successful use of novel FISH probes mimics oligonucleotide probes and long-stranded DNA probes.It was proved that the target gene,fluorescent sequence and probe in the novel FISH probe can be combined at the same time,which is proved by using the electrophoresis method and Dot-blot method.4.FISH detection by RNA localization has successfully demonstrated that novel FISH probes can be used for oligonucleotide probes.The HER-2 gene FISH probe was successfully prepared and detected by FISH.It was verified that the novel FISH probe can be used for long-chain probes.5.We found that when the pretreatment conditions of the specimens were 7-15 minutes of pepsin digestion time,the concentration of formamide in the hybridization solution was 50%.At this time,the fluorescence signal hybridization rate was higher,the background was lower,and the signal-to-noise ratio was higher.Conclusions:We designed a novel FISH probe structure that can be applied to FISH in oligonucleotide probes and long-stranded DNA probes.A novel FISH probe was used to locate the RNA.A novel FISH probe of HER-2 gene was designed and designed to detect HER-2 gene.The hybridization system was optimized by the key factors in the hybridization step.In this experiment,the structure of the probe was improved,and the same fluorescent sequence was successfully detected to different target genes.At the same time,the fluorescence,probe and target gene were hybridized by incubation,which not only saves the time of fluorescent labeling,but also reduces the probe cost.