A novel procedure was used for cloning large adenovirus genome fragment by the homologous recombination in E.coli strain BJ5183. The 11.2Kb downstream fragment of the CAV-2 strain YCA18 genome was cloned by homologous...A novel procedure was used for cloning large adenovirus genome fragment by the homologous recombination in E.coli strain BJ5183. The 11.2Kb downstream fragment of the CAV-2 strain YCA18 genome was cloned by homologous recombination, the 1029bp left end and the 970bp right end of this fragment were separately amplified by PCR. They were then cloned into plasmid pPoly2 with direction from left fragment to right fragment, obtaining a“rescue”plasmid pT615. The pT615 was liberalized by Hind Ⅲ and PstI digestion and was cotransformed with the purified CAV-2 genome which was cut by BstBI into competent E.coli strain BJ5183. Recombinant plasmids harboring the 11.2Kb downstream fragment of CAV-2 genome were obtained after bacterial intermolecular homologous recombination. The recombinant efficiency of all E.coli strains tested was 78.3%. One of the recombinant plasmids, pT618, was further identified by enzyme digestion analysis and PCR amplification. The results showed the plasmids contained the 11.2kb fragment downstream the genome of CAV-2.展开更多
目的建立阵列式标签标记的高通量测序方法并结合多克隆预筛选实现对点突变单克隆细胞株的高效鉴定。方法通过CRISPR/Cas9介导的同源重组修复(HDR)在K562细胞的rs826415位点引入G到T点突变。侯选细胞按每孔10~20个的密度接种培养多克隆,...目的建立阵列式标签标记的高通量测序方法并结合多克隆预筛选实现对点突变单克隆细胞株的高效鉴定。方法通过CRISPR/Cas9介导的同源重组修复(HDR)在K562细胞的rs826415位点引入G到T点突变。侯选细胞按每孔10~20个的密度接种培养多克隆,以带有标签(barcode)的引物区分各多克隆细胞,PCR扩增含rs826415位点的区段,产物合并进行二代测序。生物信息学分析各多克隆细胞的同源重组率及插入/缺失率。将阳性率最高孔内的细胞进一步单克隆培养并鉴定基因型。结果通过CRISPR/Cas9同源重组法和有限稀释获得96个rs826415位点打靶的多克隆细胞。经阵列式标签高通量测序法分析各孔细胞同源重组且不伴随插入/缺失(HDR without indel)的比率,96个多克隆的平均阳性率为0.21%,最高达4.35%,将该多克隆细胞进一步单克隆培养,从30个单克隆中成功获得rs826415位点由G/G突变为T/G的杂合型细胞株。结论通过阵列式标签高通量测序策略结合多克隆细胞预筛选,实现了定点突变单克隆细胞株的高效鉴定,相比常规方法显著降低了工作量和成本。展开更多
文摘A novel procedure was used for cloning large adenovirus genome fragment by the homologous recombination in E.coli strain BJ5183. The 11.2Kb downstream fragment of the CAV-2 strain YCA18 genome was cloned by homologous recombination, the 1029bp left end and the 970bp right end of this fragment were separately amplified by PCR. They were then cloned into plasmid pPoly2 with direction from left fragment to right fragment, obtaining a“rescue”plasmid pT615. The pT615 was liberalized by Hind Ⅲ and PstI digestion and was cotransformed with the purified CAV-2 genome which was cut by BstBI into competent E.coli strain BJ5183. Recombinant plasmids harboring the 11.2Kb downstream fragment of CAV-2 genome were obtained after bacterial intermolecular homologous recombination. The recombinant efficiency of all E.coli strains tested was 78.3%. One of the recombinant plasmids, pT618, was further identified by enzyme digestion analysis and PCR amplification. The results showed the plasmids contained the 11.2kb fragment downstream the genome of CAV-2.
文摘目的建立阵列式标签标记的高通量测序方法并结合多克隆预筛选实现对点突变单克隆细胞株的高效鉴定。方法通过CRISPR/Cas9介导的同源重组修复(HDR)在K562细胞的rs826415位点引入G到T点突变。侯选细胞按每孔10~20个的密度接种培养多克隆,以带有标签(barcode)的引物区分各多克隆细胞,PCR扩增含rs826415位点的区段,产物合并进行二代测序。生物信息学分析各多克隆细胞的同源重组率及插入/缺失率。将阳性率最高孔内的细胞进一步单克隆培养并鉴定基因型。结果通过CRISPR/Cas9同源重组法和有限稀释获得96个rs826415位点打靶的多克隆细胞。经阵列式标签高通量测序法分析各孔细胞同源重组且不伴随插入/缺失(HDR without indel)的比率,96个多克隆的平均阳性率为0.21%,最高达4.35%,将该多克隆细胞进一步单克隆培养,从30个单克隆中成功获得rs826415位点由G/G突变为T/G的杂合型细胞株。结论通过阵列式标签高通量测序策略结合多克隆细胞预筛选,实现了定点突变单克隆细胞株的高效鉴定,相比常规方法显著降低了工作量和成本。
