Mao Y W, Liang S Y, Song W Q, Li X L, Chen R Y
Biology Department, Nankai University, Tianjin, China.
Cell Res. 1998 Dec;8(4):285-93. doi: 10.1038/cr.1998.28.
A simple method to create a chromosome-specific DNA library of rice, including microdissection, amplification, characterization and cloning, is described. Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR). The PCR products were labeled as probes with DIG-11-dUTP using the random priming method. Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4. A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed. Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences. The size of inserts generated by PCR ranged from 140bp to 500bp. This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.
本文描述了一种创建水稻染色体特异性DNA文库的简单方法，包括显微切割、扩增、表征和克隆。已通过接头衔接子PCR（LA-PCR）从一个中期细胞中分离并扩增了水稻第4号染色体。使用随机引物法将PCR产物用DIG-11-dUTP标记为探针。用水稻基因组DNA和特定的RFLP标记进行Southern印迹分析表明，PCR产物源自水稻第4号染色体。构建了一个包含超过100,000个来自水稻第4号染色体的重组质粒微克隆的大型文库。菌落杂交显示，58%的克隆包含单拷贝或低拷贝序列，42%包含重复序列。PCR产生的插入片段大小范围为140bp至500bp。该方法将有助于克隆水稻特定染色体的DNA标记和重要基因。
