Borodin A, Kopatnzev E, Wagner L, Volik S, Ermolaeva O, Lebedev Y, Monastyrskaya G, Kunz J, Grzeschik K H, Sverdlov E
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow.
Genet Anal. 1995 Mar;12(1):23-31. doi: 10.1016/1050-3862(95)00106-9.
A simple technique for preparation of libraries of the human chromosome specific transcribed sequences is developed. It uses hnRNA from human-rodent hybrid cell lines containing particular human chromosomes or their fragments and includes three stages: (i) reverse transcription of the hnRNA with Alu-specific primers directing the transcription beyond the Alu-repeats to flanking non-repetitive sequences of the chromosome; (ii) nested primer PCR strategy with specifically designed primers; (iii) direct selective cloning of the second-stage nested primer PCR products. An arrayed hncDNA library was prepared from a hybrid cell line containing chromosome 19 and fragments of 22 and X chromosomes. The library contains around 98% of human-specific transcribed sequences. Sequences of 52 human-specific, according to PCR analysis, clones differed from each other and had no close analogs in the EMBL Data Bank. Of 17 clones assigned to certain human chromosomes, 9 belonged to chromosome 19, 5 to chromosome 22 and 3 to chromosome X. Some of the human specific clones contained repetitive elements scattered over different human chromosomes. Clones from hncDNA libraries are useful as STSs/ESTs, as probes for detecting full-size genes in genomic libraries, for RFLP analysis and for identification of chromosome specific cDNAs.
开发了一种制备人类染色体特异性转录序列文库的简单技术。它使用来自含有特定人类染色体或其片段的人-鼠杂交细胞系的核不均一RNA（hnRNA），并包括三个阶段：（i）用Alu特异性引物对hnRNA进行逆转录，该引物引导转录超出Alu重复序列至染色体的侧翼非重复序列；（ii）使用专门设计的引物的巢式引物聚合酶链反应（PCR）策略；（iii）对第二阶段巢式引物PCR产物进行直接选择性克隆。从一个含有19号染色体以及22号和X染色体片段的杂交细胞系制备了一个排列的hncDNA文库。该文库包含约98%的人类特异性转录序列。根据PCR分析，52个人类特异性克隆的序列彼此不同，并且在EMBL数据库中没有紧密的类似物。在分配到某些人类染色体的17个克隆中，9个属于19号染色体，5个属于22号染色体，3个属于X染色体。一些人类特异性克隆含有散布在不同人类染色体上的重复元件。来自hncDNA文库的克隆可用作序列标签位点（STSs）/表达序列标签（ESTs），用作检测基因组文库中全长基因的探针，用于限制性片段长度多态性（RFLP）分析以及用于鉴定染色体特异性cDNA。
