Smiraglia D J, Frühwald M C, Costello J F, McCormick S P, Dai Z, Peltomäki P, O'Dorisio M S, Cavenee W K, Plass C
Department of Medical Microbiology and Immunology, The Ohio State University, Columbus, Ohio 43210, USA.
Genomics. 1999 Jun 15;58(3):254-62. doi: 10.1006/geno.1999.5840.
Restriction landmark genome scanning (RLGS) is an effective genome-scanning technique capable of identifying DNA amplification and aberrant DNA methylation. Previously published methods for the cloning of human DNA fragments from RLGS gels have been successful only for high-copy-number fragments (repetitive elements or DNA amplifications). We present here the first technique capable of efficiently cloning single-copy human DNA fragments ("spots") identified in RLGS profiles. This technique takes advantage of a plasmid-based, human genomic DNA, NotI/EcoRV boundary library. The library is arrayed in microtiter plates. When clones from a single plate are pooled and mixed with genomic DNA, the resultant RLGS gel is a normal profile with a defined set of spots showing enhanced intensity for that particular plate. This was performed for a set of 32 plates as well as their pooled rows and columns. Thus, we have mapped individual RLGS spots to exact plate, row, and column addresses in the library and have thereby obtained immediate access to these clones. The feasibility of the technique is demonstrated in examples of cloning methylated DNA fragments identified in human breast tumor and testicular tumor RLGS profiles and in the cloning of an amplified DNA fragment identified in a human medulloblastoma RLGS profile.
限制性地标基因组扫描（RLGS）是一种有效的基因组扫描技术，能够识别DNA扩增和异常DNA甲基化。此前发表的从RLGS凝胶中克隆人类DNA片段的方法仅对高拷贝数片段（重复元件或DNA扩增）有效。我们在此展示了第一种能够有效克隆在RLGS图谱中鉴定出的单拷贝人类DNA片段（“斑点”）的技术。该技术利用了基于质粒的人类基因组DNA NotI/EcoRV边界文库。该文库排列在微量滴定板中。当将来自单个板的克隆汇集起来并与基因组DNA混合时，所得的RLGS凝胶是具有一组特定斑点的正常图谱，这些斑点在该特定板上显示出增强的强度。对一组32个板及其汇集的行和列进行了此操作。因此，我们已将各个RLGS斑点定位到文库中确切的板、行和列地址，从而能够立即获取这些克隆。该技术的可行性在克隆人类乳腺肿瘤和睾丸肿瘤RLGS图谱中鉴定出的甲基化DNA片段以及克隆人类髓母细胞瘤RLGS图谱中鉴定出的扩增DNA片段的实例中得到了证明。
