Ledbetter S A, Nelson D L, Warren S T, Ledbetter D H
Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030.
Genomics. 1990 Mar;6(3):475-81. doi: 10.1016/0888-7543(90)90477-c.
A method was recently developed for the specific amplification of human DNA sequences from interspecific somatic cell hybrids by the polymerase chain reaction (PCR) using primers directed to Alu, a short interspersed repeat element (SINE). We now show human-specific amplification using a primer to the 3' end of the human long interspersed repeat element L1Hs (LINE). A monochromosomal hybrid containing an intact human X chromosome yielded approximately 25 discrete products, ranging in size from 800 to 4500 bp. Combination of a single Alu primer and the L1Hs primer yielded a large number of smaller products (300-1000 bp) distinct from those observed with either primer alone. Inspection of ethidium bromide-stained gels showed one Alu-Alu and three Alu-L1Hs products which were present in an intact X chromosome but absent in a hybrid containing an X chromosome deleted for the single metaphase band q28. These four fragments were isolated from the gel and used as probes on Southern blots which confirmed their localization to Xq28. These results demonstrate that primers can be constructed to a variety of interspersed repetitive sequences (IRS) and used individually or in combination for the rapid isolation of DNA fragments from defined chromosomal regions by IRS-PCR.
最近开发了一种方法，通过聚合酶链反应（PCR），使用针对短散在重复元件（SINE）Alu的引物，从种间体细胞杂种中特异性扩增人类DNA序列。我们现在展示了使用针对人类长散在重复元件L1Hs（LINE）3'端的引物进行人类特异性扩增。一个含有完整人类X染色体的单染色体杂种产生了大约25个离散产物，大小在800至4500 bp之间。单个Alu引物和L1Hs引物的组合产生了大量较小的产物（300 - 1000 bp），这些产物与单独使用任何一种引物观察到的产物不同。对溴化乙锭染色凝胶的检查显示，有一个Alu - Alu产物和三个Alu - L1Hs产物存在于完整的X染色体中，但在一个含有因单条中期带q28缺失的X染色体的杂种中不存在。从凝胶中分离出这四个片段，并用作Southern杂交的探针，证实它们定位于Xq28。这些结果表明，可以针对多种散在重复序列（IRS）构建引物，并单独或组合使用，通过IRS - PCR从特定染色体区域快速分离DNA片段。
