Stallings R L, Torney D C, Hildebrand C E, Longmire J L, Deaven L L, Jett J H, Doggett N A, Moyzis R K
Center for Human Genome Studies, Los Alamos National Laboratory, University of California, NM 87545.
Proc Natl Acad Sci U S A. 1990 Aug;87(16):6218-22. doi: 10.1073/pnas.87.16.6218.
We have developed an approach for identifying overlapping cosmid clones by exploiting the high density of repetitive sequences in complex genomes. Individual clones are fingerprinted, using a combination of restriction enzyme digestions followed by hybridization with selected classes of repetitive sequences. This "repeat fingerprinting" technique allows small regions of clone overlap (10-20%) to be unambiguously assigned. We demonstrate the utility of this approach, using the fingerprinting of 3145 cosmid clones (1.25 x coverage), containing one or more (GT)n repeats, from human chromosome 16. A statistical analysis was used to link these clones into 460 contiguous sequences (contigs), averaging 106 kilobases (kb) in length and representing approximately 54% (48.7 Mb) of the euchromatic arms of this chromosome. These values are consistent with theoretical calculations and indicate that 150- to 200-kb contigs can be generated with 1.5 x coverage. This strategy requires the fingerprinting of approximately one-fourth as many cosmids as random strategies requiring 50% minimum overlap for overlap detection. By "nucleating" at specific regions in the human genome, and exploiting the high density of interspersed sequences, this approach allows (i) the rapid generation of large (greater than 100-kb) contigs in the early stages of contig mapping and (ii) the production of a contig map with useful landmarks for rapid integration of the genetic and physical maps.
我们开发了一种通过利用复杂基因组中重复序列的高密度来鉴定重叠黏粒克隆的方法。使用限制性内切酶消化并结合与选定类别的重复序列杂交对单个克隆进行指纹分析。这种“重复指纹分析”技术能够明确确定克隆重叠的小区域（10%-20%）。我们通过对来自人类16号染色体的3145个含有一个或多个（GT）n重复序列的黏粒克隆（覆盖度为1.25倍）进行指纹分析，展示了该方法的实用性。使用统计分析将这些克隆连接成460个连续序列（重叠群），平均长度为106千碱基（kb），约占该染色体常染色质臂的54%（48.7 Mb）。这些值与理论计算结果一致，表明覆盖度为1.5倍时可生成150至200 kb的重叠群。与检测重叠需要至少50%重叠的随机策略相比，该策略所需指纹分析的黏粒数量约为其四分之一。通过在人类基因组的特定区域“成核”，并利用散布序列的高密度，这种方法能够：（i）在重叠群图谱绘制的早期阶段快速生成大的（大于100 kb）重叠群；（ii）生成一个带有有用标记的重叠群图谱，以便快速整合遗传图谱和物理图谱。
