用生物素-11-脱氧尿苷三磷酸(Biotin-11-dUTP),通过缺口平移法标记提纯的鸭瘟病毒 DNA,制备生物素化鸭瘟病毒 DNA 全基因组探针;以斑点杂交检测固定在硝酸纤维膜上的样品鸭瘟病毒 DNA 同源序列,杂交后用亲和素-碱性磷酸酶孵育,底物显色...用生物素-11-脱氧尿苷三磷酸(Biotin-11-dUTP),通过缺口平移法标记提纯的鸭瘟病毒 DNA,制备生物素化鸭瘟病毒 DNA 全基因组探针;以斑点杂交检测固定在硝酸纤维膜上的样品鸭瘟病毒 DNA 同源序列,杂交后用亲和素-碱性磷酸酶孵育,底物显色,阳性反应呈蓝紫色斑点。试验结果表明,生物素标记核酸探针可检出10pg 提纯的鸭瘟病毒DNA,并检出稀释10~5倍和肝组织鸭瘟病毒 DNA;对鸡马立克氏病毒 DNA,鸡痘病毒DNA 和噬菌体 DNA 无杂交反应;对鸭瘟病毒弱毒 DNA 产生微弱杂交。该技术具有快速、敏感、特异和无放射污染等优点。展开更多
It is often needed to detect the DNA single strand break in biological studies. The four most frequently used methods are alkaline elution, alkaline sucrose gradient sedimentation, hydroxyapatite chromatography and nu...It is often needed to detect the DNA single strand break in biological studies. The four most frequently used methods are alkaline elution, alkaline sucrose gradient sedimentation, hydroxyapatite chromatography and nucleoid sedimentation. All these are not perfect because of a number of disadvantages, such as展开更多
[目的]研究RBM5(RNA-binding motif protein 5)荧光探针的制备方法,确立RBM5荧光探针用于肺癌组织检测的原位杂交技术体系。[方法]以RP11-493K19菌株为材料,提取含有RBM5的质粒进行PCR验证,采用缺口平移法制备RBM5荧光探针,并与人肺癌...[目的]研究RBM5(RNA-binding motif protein 5)荧光探针的制备方法,确立RBM5荧光探针用于肺癌组织检测的原位杂交技术体系。[方法]以RP11-493K19菌株为材料,提取含有RBM5的质粒进行PCR验证,采用缺口平移法制备RBM5荧光探针,并与人肺癌组织石蜡切片进行杂交实验建立肺癌荧光原位杂交(fluorescence in situ hybridization,FISH)的检测体系。[结果]RBM5 15℃标记12h可获得合适的探针,探针与样本杂交后样本细胞内出现清晰明亮的绿色荧光信号,通过与呈橘红色荧光信号的CEP-3探针比较,可以判断肺癌细胞是否存在RBM5的缺失。[结论]RBM5探针制备的最佳条件是15℃标记12h,FISH实验的参数为10μg/ml蛋白酶K处理样本100 min、探针与样本37℃杂交16h、2×SSC/0.3%NP-40洗涤杂交样本5min。该实验体系适用于肺癌组织RBM5的FISH检测。展开更多
IN our previous studies, we have used in situ nick translation techniques for analysis of DNaseⅠ sensitivity and restriction enzymes in situ cleavage on pachytene bivalents of rice-field eels(Monopterus albus Zuiew)....IN our previous studies, we have used in situ nick translation techniques for analysis of DNaseⅠ sensitivity and restriction enzymes in situ cleavage on pachytene bivalents of rice-field eels(Monopterus albus Zuiew). A characteristic construction of bivalents--"the lampbrush-likechromosome", which has not been reported yet, was detected in some pachytene bivalents.展开更多
文摘用生物素-11-脱氧尿苷三磷酸(Biotin-11-dUTP),通过缺口平移法标记提纯的鸭瘟病毒 DNA,制备生物素化鸭瘟病毒 DNA 全基因组探针;以斑点杂交检测固定在硝酸纤维膜上的样品鸭瘟病毒 DNA 同源序列,杂交后用亲和素-碱性磷酸酶孵育,底物显色,阳性反应呈蓝紫色斑点。试验结果表明,生物素标记核酸探针可检出10pg 提纯的鸭瘟病毒DNA,并检出稀释10~5倍和肝组织鸭瘟病毒 DNA;对鸡马立克氏病毒 DNA,鸡痘病毒DNA 和噬菌体 DNA 无杂交反应;对鸭瘟病毒弱毒 DNA 产生微弱杂交。该技术具有快速、敏感、特异和无放射污染等优点。
文摘It is often needed to detect the DNA single strand break in biological studies. The four most frequently used methods are alkaline elution, alkaline sucrose gradient sedimentation, hydroxyapatite chromatography and nucleoid sedimentation. All these are not perfect because of a number of disadvantages, such as
文摘[目的]研究RBM5(RNA-binding motif protein 5)荧光探针的制备方法,确立RBM5荧光探针用于肺癌组织检测的原位杂交技术体系。[方法]以RP11-493K19菌株为材料,提取含有RBM5的质粒进行PCR验证,采用缺口平移法制备RBM5荧光探针,并与人肺癌组织石蜡切片进行杂交实验建立肺癌荧光原位杂交(fluorescence in situ hybridization,FISH)的检测体系。[结果]RBM5 15℃标记12h可获得合适的探针,探针与样本杂交后样本细胞内出现清晰明亮的绿色荧光信号,通过与呈橘红色荧光信号的CEP-3探针比较,可以判断肺癌细胞是否存在RBM5的缺失。[结论]RBM5探针制备的最佳条件是15℃标记12h,FISH实验的参数为10μg/ml蛋白酶K处理样本100 min、探针与样本37℃杂交16h、2×SSC/0.3%NP-40洗涤杂交样本5min。该实验体系适用于肺癌组织RBM5的FISH检测。
文摘IN our previous studies, we have used in situ nick translation techniques for analysis of DNaseⅠ sensitivity and restriction enzymes in situ cleavage on pachytene bivalents of rice-field eels(Monopterus albus Zuiew). A characteristic construction of bivalents--"the lampbrush-likechromosome", which has not been reported yet, was detected in some pachytene bivalents.
