Microdissection and microcloning of rye (Secale cereale L.) chromosome 1R
Microdissection and microcloning of rye (Secale cereale L.) chromosome 1R
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DOI：
10.1007/s004120050375
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发表时间：
1999-02
期刊：
影响因子：
1.6
通讯作者：
Yihua Zhou;Zanmin Hu;B. Dang;Huai Wang;Xiangdong Deng;Lanlan Wang;Zheng-hua Chen
中科院分区：
文献类型：
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作者：
Yihua Zhou;Zanmin Hu;B. Dang;Huai Wang;Xiangdong Deng;Lanlan Wang;Zheng-hua Chen
从黑麦(Secale graaleL.)有丝分裂中期铺片上显微解剖收集1R染色体。通过使用玻璃针。用Sau3A接头介导的聚合酶链式反应(PCR)对分离的染色体进行体外扩增。扩增后，Southern杂交证实了黑麦特异DNA的存在。将来自5条1R染色体的第二轮聚合酶链式反应产物克隆到一个质粒载体上，建立了一个染色体特异性文库，产生了大约22万个重组克隆。对微克隆文库的鉴定表明，所评价的172个克隆的大小在300-1800bp之间，平均大小为950bp，其中约42%为中/高拷贝，58%为低/独特拷贝克隆。染色体原位杂交证实，显微切割染色体的扩增产物来源于1R染色体，表明文库中存在许多1R染色体特异序列。
Chromosome 1R was microdissected and collected from mitotic metaphase spreads of rye (Secale cerealeL.) by using glass needles. The isolated chromosomes were amplified in vitro bySau3A linker adaptor-mediated polymerase chain reaction (PCR). After amplification, the presence of rye-specific DNA was verified by Southern hybridization. The second-round PCR products from five 1R chromosomes were cloned into a plasmid vector to create a chromosome-specific library, which produced approximately 220,000 recombinant clones. Characterization of the microclone library showed that the 172 clones evaluated ranged in size from 300–1800 bp with an average size of 950 bp, of which approximately 42% were medium/high copy and 58% were low/unique copy clones. Chromosome in situ hybridization confirmed that the PCR products from microdissected chromosomes originated from chromosome 1R, indicating that many chromosome 1R-specific sequences were present in the library.