Zheng C J, Ma N S, Dorman T E, Wang M T, Braunschweiger K, Soares L, Schuster M K, Rothschild C B, Bowden D W, Torrey D
Department of Human and Molecular Genetics, Collaborative Research, Inc., Waltham, Massachusetts 02154.
Genomics. 1994 Jul 1;22(1):55-67. doi: 10.1006/geno.1994.1345.
A total of 124 new chromosome 10-specific sequence-tagged sites (STSs) were derived from two sources: (1) DNA sequences obtained from anonymous clones in new libraries enriched for human chromosome 10 inserts, and (2) published sequences of genes and other loci already known to map to chromosome 10. Libraries were constructed from a somatic cell hybrid carrying human chromosomes 10 and Y. A cosmid library was made from total DNA of the hybrid and probed with labeled total human DNA to identify clones with human DNA inserts. Two hundred seventeen cosmids were mapped to regions of human chromosome 10 by fluorescence in situ hybridization. Twenty-five cosmids represent probes that have been placed on the genetic map previously. One hundred ninety-two cosmids represent new probes that have not been mapped previously. Cosmids carrying inserts with CA repeats were identified by hybridization with a labeled poly(dC-dA)-poly(dG-dT) probe and subcloned to yield microsatellite STS markers. Two small insert plasmid libraries were made, the first by subcloning inserts from a chromosome 10-enriched lambda phage library (LL10NS01) and the second by cloning Alu element-mediated PCR products amplified from hybrid DNA. STSs were generated from the DNA sequences of clone inserts. Chromosome 10-specific STSs were distinguished from Y chromosome STSs by one or both of the following criteria: (1) successful PCR amplification from a template consisting of DNA from another chromosome 10-containing cell line, NA10926B, or (2) FISH localization to chromosome 10 of the source cosmid or of YACs isolated by PCR screening with the STS. These libraries were the source of 90 new chromosome 10-specific STSs, 42 of which contain CA repeats.
总共124个新的10号染色体特异性序列标签位点（STS）来自两个来源：（1）从富含人10号染色体插入片段的新文库中的匿名克隆获得的DNA序列，以及（2）已定位到10号染色体的基因和其他位点的已发表序列。文库由携带人10号和Y染色体的体细胞杂种构建而成。从杂种的总DNA构建一个黏粒文库，并用标记的总人DNA进行探测，以鉴定含有人DNA插入片段的克隆。通过荧光原位杂交将217个黏粒定位到人类10号染色体区域。25个黏粒代表先前已定位到遗传图谱上的探针。192个黏粒代表先前未定位的新探针。通过与标记的聚（dC-dA）-聚（dG-dT）探针杂交鉴定携带含CA重复序列插入片段的黏粒，并进行亚克隆以产生微卫星STS标记。构建了两个小插入片段质粒文库，第一个通过从富含10号染色体的λ噬菌体文库（LL10NS01）中进行插入片段亚克隆，第二个通过克隆从杂种DNA扩增的Alu元件介导的PCR产物。从克隆插入片段的DNA序列产生STS。10号染色体特异性STS通过以下一个或两个标准与Y染色体STS区分开来：（1）从由另一个含10号染色体的细胞系NA10926B的DNA组成的模板成功进行PCR扩增，或（2）通过用该STS进行PCR筛选分离的源黏粒或YAC在10号染色体上的荧光原位杂交定位。这些文库是90个新的10号染色体特异性STS的来源，其中42个含有CA重复序列。
