染色体绘画是一种以整条或染色体区带特异性 DNA 作为探针池进行染色体荧光原位杂交的方法。我们建立了一种简单的染色体区带特异性绘画方法,这种方法主要步骤包括:(1)染色体或染色体区带的显微切割。(2)用多聚酶链式反应(PCR)扩增 DNA...染色体绘画是一种以整条或染色体区带特异性 DNA 作为探针池进行染色体荧光原位杂交的方法。我们建立了一种简单的染色体区带特异性绘画方法,这种方法主要步骤包括:(1)染色体或染色体区带的显微切割。(2)用多聚酶链式反应(PCR)扩增 DNA。(3)生物素标记 PCR 产物。(4)染色体荧光原位杂交。(5)用连有 FITC 的 Avidin 检测杂交信号。用这一方法我们已经建立了整条 X 和整条 Y 染色休以及2号染色体长臂远侧1/4区及8q24.1带的 DNA 探针池并经染色体区带特异性绘画及分子杂交所证实。展开更多
荧光原位杂交技术(fluorescence in situ hybridization,FISH)是20世纪80年代末在已有的放射性原位杂交技术的基础上发展起来的一种非放射性DNA分子原位杂交技术,该技术目前广泛应用于动植物领域内的DNA重复序列或多拷贝的基因家族... 荧光原位杂交技术(fluorescence in situ hybridization,FISH)是20世纪80年代末在已有的放射性原位杂交技术的基础上发展起来的一种非放射性DNA分子原位杂交技术,该技术目前广泛应用于动植物领域内的DNA重复序列或多拷贝的基因家族的染色体定位、杂种亲本染色体的鉴定,染色体的结构分析与染色体物理图谱构建,外源染色质检测,物种进化及亲缘关系等的研究.本文主要介绍了荧光原位杂交技术及其衍生技术,并对其在禽类遗传学中的应用加以综述.……展开更多
The material T240_6 derived from SC 2 young embryo of the combination CA9211/RW15 (6D/6V alien substitution) was telosomic substitution line of 6VS identified by GISH (genomic in situ hybridization) analysis. The 6V...The material T240_6 derived from SC 2 young embryo of the combination CA9211/RW15 (6D/6V alien substitution) was telosomic substitution line of 6VS identified by GISH (genomic in situ hybridization) analysis. The 6VS was microdissected with a needle and transferred into a 0.5 mL Ep tube. In the 'single tube', all the subsequence steps were conducted. After two round of LA (Linker adaptor)_PCR amplification, the size of PCR bands ranged from 100 to 3 000 bp, with predominate bands 600-1 500 bp. The products were confirmed by Southern blotting analysis using Haynaldia villosa (L.) Schur. genomic DNA labeled with 32 P as probe. The PCR products were purified and ligated into clone vector-pGEM_T easy vector. Then, the plasmids were transformed into competence E. coli JM109 with cool CaCl 2. It was estimated that there were more than 17 000 white clones in the library. The size of insert fragments distributed from 100-1 500 bp, with average of 600 bp. Using H. villosa genomic DNA as probe, dot blotting results showed that 37% clones displayed strong and medium positive signals, and 63% clones had faint or no signals. It is demonstrated that there were about 37% repeat sequence clones and 67% single/unique sequence clones in the library. Eight H. villosa_specific clones were screened from the library, and two clones pHVMK22 and pHVMK134 were used for RFLP analysis and sequencing. Both of them were H. villosa specific clones. The pHVMK22 was a unique sequence clone, and the pHVMK134 was a repeat sequence clone. When the pHVMK22 was used as a probe for Southern hybridization, all the powdery mildew resistance materials showed a special band of 2 kb, while all the susceptible ones not. The pHVMK22 may be applied to detect the existence of Pm21.展开更多
文摘染色体绘画是一种以整条或染色体区带特异性 DNA 作为探针池进行染色体荧光原位杂交的方法。我们建立了一种简单的染色体区带特异性绘画方法,这种方法主要步骤包括:(1)染色体或染色体区带的显微切割。(2)用多聚酶链式反应(PCR)扩增 DNA。(3)生物素标记 PCR 产物。(4)染色体荧光原位杂交。(5)用连有 FITC 的 Avidin 检测杂交信号。用这一方法我们已经建立了整条 X 和整条 Y 染色休以及2号染色体长臂远侧1/4区及8q24.1带的 DNA 探针池并经染色体区带特异性绘画及分子杂交所证实。
文摘 荧光原位杂交技术(fluorescence in situ hybridization,FISH)是20世纪80年代末在已有的放射性原位杂交技术的基础上发展起来的一种非放射性DNA分子原位杂交技术,该技术目前广泛应用于动植物领域内的DNA重复序列或多拷贝的基因家族的染色体定位、杂种亲本染色体的鉴定,染色体的结构分析与染色体物理图谱构建,外源染色质检测,物种进化及亲缘关系等的研究.本文主要介绍了荧光原位杂交技术及其衍生技术,并对其在禽类遗传学中的应用加以综述.……
基金国家"8 6 3"计划资助项目 (Z 17 0 4 0 1) 国家转基因植物研究与产业化资助项目 (J0 0 A 0 0 2 )~~
文摘The material T240_6 derived from SC 2 young embryo of the combination CA9211/RW15 (6D/6V alien substitution) was telosomic substitution line of 6VS identified by GISH (genomic in situ hybridization) analysis. The 6VS was microdissected with a needle and transferred into a 0.5 mL Ep tube. In the 'single tube', all the subsequence steps were conducted. After two round of LA (Linker adaptor)_PCR amplification, the size of PCR bands ranged from 100 to 3 000 bp, with predominate bands 600-1 500 bp. The products were confirmed by Southern blotting analysis using Haynaldia villosa (L.) Schur. genomic DNA labeled with 32 P as probe. The PCR products were purified and ligated into clone vector-pGEM_T easy vector. Then, the plasmids were transformed into competence E. coli JM109 with cool CaCl 2. It was estimated that there were more than 17 000 white clones in the library. The size of insert fragments distributed from 100-1 500 bp, with average of 600 bp. Using H. villosa genomic DNA as probe, dot blotting results showed that 37% clones displayed strong and medium positive signals, and 63% clones had faint or no signals. It is demonstrated that there were about 37% repeat sequence clones and 67% single/unique sequence clones in the library. Eight H. villosa_specific clones were screened from the library, and two clones pHVMK22 and pHVMK134 were used for RFLP analysis and sequencing. Both of them were H. villosa specific clones. The pHVMK22 was a unique sequence clone, and the pHVMK134 was a repeat sequence clone. When the pHVMK22 was used as a probe for Southern hybridization, all the powdery mildew resistance materials showed a special band of 2 kb, while all the susceptible ones not. The pHVMK22 may be applied to detect the existence of Pm21.
