Howell M D, Resner J, Austin R K, Kagnoff M F
Gene. 1987;55(1):41-5. doi: 10.1016/0378-1119(87)90246-0.
The presence of repeated elements in restriction fragments used as hybridization probes for chromosomal walking poses a major obstacle to the success of this gene-cloning strategy. This report describes a simple and rapid means of identifying restriction fragments devoid of repeated sequences and therefore useful as hybridization probes for chromosomal walking. Restriction fragments derived from a genomic DNA clone are Southern blotted and hybridized to nick-translated total genomic [32P]DNA. Fragments of the genomic clone that contain high abundance sequences (i.e., repeated elements) hybridize strongly to their nick-translated counterparts, which, due to their high copy number, comprise a significant proportion of the total genomic DNA probe. Conversely, fragments containing single-copy or low-abundance sequences do not hybridize, as their nick-translated counterparts are poorly represented in the total genomic DNA probe. These latter fragments, by virtue of their low-abundance sequences, are well suited as probes for chromosomal walking. Ensuring the absence of repeated elements in restriction fragments prior to their purification and utilization as chromosomal walking probes results in marked savings of time, effort and materials.
在用作染色体步移杂交探针的限制性片段中存在重复元件，这对该基因克隆策略的成功构成了主要障碍。本报告描述了一种简单快速的方法，用于鉴定不含重复序列的限制性片段，因此可作为染色体步移的杂交探针。从基因组DNA克隆中获得的限制性片段进行Southern印迹，并与缺口平移的总基因组[32P]DNA杂交。基因组克隆中含有高丰度序列（即重复元件）的片段与其缺口平移的对应物强烈杂交，由于其高拷贝数，这些对应物在总基因组DNA探针中占很大比例。相反，含有单拷贝或低丰度序列的片段不杂交，因为它们的缺口平移对应物在总基因组DNA探针中的代表性很差。这些后一种片段由于其低丰度序列，非常适合作为染色体步移的探针。在纯化并用作染色体步移探针之前，确保限制性片段中不存在重复元件，可显著节省时间、精力和材料。
