Zhang M, Yu L, Hu P R, Bi A D, Xia J H, Deng H X, Zhao S Y
Genetic Institute of Fudan University, Shanghai.
Shi Yan Sheng Wu Xue Bao. 1997 Sep;30(3):241-6.
The strategy of isolating the band-specific expression fragments from the probe pool of human chromosome generated by microdissection was reported in present paper. A chromosome 14 q 24.3 band-specific single copy DNA library was constructed based on this probe pool. Using this pool DNA as probe to hybridize the human bone marrow cell cDNA library, 68 primary positive clones were selected from 5 x 10(5) cDNA clones. Of them 32 clones were got in second-round screening and designed as cFD 14-1-32. Finally, 24 bandspecific expression fragments were identified from these 32 positive clones by analysing the results of DNA hybridization. Those band-specific clones can hybridize to both 14 q 24.3 DNA and human genomic DNA, but have no hybridization signal with 17 q 11-12 DNA. Partial sequences of 13 fragments of them were sequenced and were identified as novel cDNA sequences as well as have some homology with known genes in NCBI database. Analysis of expression spectrum of cFD 14-1 suggested that the cDNA fragments thus obtained can be used to isolate the genes not yet be cloned in 14 q 24.3 region.
本文报道了从显微切割产生的人类染色体探针库中分离带特异性表达片段的策略。基于该探针库构建了一个14号染色体q24.3带特异性单拷贝DNA文库。以该库DNA为探针与人骨髓细胞cDNA文库杂交,从5×10(5)个cDNA克隆中筛选出68个初级阳性克隆。其中32个克隆在第二轮筛选中获得,并命名为cFD 14-1-32。最后,通过分析DNA杂交结果,从这32个阳性克隆中鉴定出24个带特异性表达片段。这些带特异性克隆可与14 q24.3 DNA和人类基因组DNA杂交,但与17 q11-12 DNA无杂交信号。对其中13个片段的部分序列进行了测序,鉴定为新的cDNA序列,且与NCBI数据库中的已知基因有一定同源性。对cFD 14-1表达谱的分析表明,由此获得的cDNA片段可用于分离14 q24.3区域中尚未克隆的基因。