Sapolsky R J, Lipshutz R J
Affymetrix, 3380 Central Expressway, Santa Clara, California, 95051, USA.
Genomics. 1996 May 1;33(3):445-56. doi: 10.1006/geno.1996.0219.
We have developed a high-density DNA probe array and accompanying biochemical and informatic methods to order clones from genomic libraries. This approach involves a series of enzymatic steps for capturing a set of short dispersed sequence markers scattered throughout a high-molecular-weight DNA. By this process, all the ambiguous sequences lying adjacent to a given Type IIS restriction site are ligated between two DNA adapters. These markers, once amplified and labeled by PCR, can be hybridized and detected on a high-density oligonucleotide array bearing probes complementary to all possible markers. The array is synthesized using light-directed combinatorial chemistry. For each clone in a genomic library, a characteristic set of sequence markers can be determined. On the basis of the similarity between the marker sets for each pair of clones, their relative overlap can be measured. The library can be sequentially ordered into a contig map using this overlap information. This new methodology does not require gel-based methods or prior sequence information and involves manipulations that should allow for easy adaptation to automated processing and data collection.
我们开发了一种高密度DNA探针阵列以及配套的生化和信息学方法，用于对基因组文库中的克隆进行排序。这种方法涉及一系列酶促步骤，用于捕获散布在高分子量DNA中的一组短分散序列标记。通过这个过程，与给定的IIS型限制性位点相邻的所有模糊序列都连接在两个DNA衔接子之间。这些标记一旦通过PCR扩增和标记，就可以在高密度寡核苷酸阵列上进行杂交和检测，该阵列带有与所有可能标记互补的探针。该阵列是使用光导向组合化学合成的。对于基因组文库中的每个克隆，可以确定一组特征性的序列标记。根据每对克隆的标记集之间的相似性，可以测量它们的相对重叠。利用这些重叠信息，可以将文库顺序排列成一个重叠群图谱。这种新方法不需要基于凝胶的方法或先前的序列信息，并且涉及的操作应该便于轻松适应自动化处理和数据收集。
