Romanov M N, Price J A, Dodgson J B
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Mich, USA.
Cytogenet Genome Res. 2003;102(1-4):277-81. doi: 10.1159/000075763.
The alignment of genome linkage maps, defined primarily by segregation of sequence-tagged site (STS) markers, with BAC contig physical maps and full genome sequences requires high throughput mechanisms to identify BAC clones that contain specific STS. A powerful technique for this purpose is multi-dimensional hybridization of "overgo" probes. The probes are chosen from available STS sequence data by selecting unique probe sequences that have a common melting temperature. We have hybridized sets of 216 overgo probes in subset pools of 36 overgos at a time to filter-spotted chicken BAC clone arrays. A four-dimensional pooling strategy, including one degree of redundancy, has been employed. This requires 24 hybridizations to completely assign BACs for all 216 probes. Results to date are consistent with about a 10% failure rate in overgo probe design and a 15-20% false negative detection rate within a group of 216 markers. Three complete rounds of overgo hybridization, each to sets of about 39,000 BACs (either BAMHI or ECORI partial digest inserts) generated a total of 1853 BAC alignments for 517 mapped chicken genome STS markers. These data are publicly available, and they have been used in the assembly of a first generation BAC contig map of the chicken genome.
基因组连锁图谱（主要由序列标签位点（STS）标记的分离定义）与BAC重叠群物理图谱及全基因组序列的比对，需要高通量机制来鉴定包含特定STS的BAC克隆。用于此目的的一项强大技术是“overgo”探针的多维杂交。通过选择具有共同解链温度的独特探针序列，从可用的STS序列数据中选择探针。我们每次将216个overgo探针的集合以36个overgo的子集池形式进行杂交，以筛选点样的鸡BAC克隆阵列。采用了一种四维合并策略，包括一度冗余。这需要24次杂交才能完全为所有216个探针分配BAC。迄今为止的结果表明，overgo探针设计的失败率约为10%，在一组216个标记中假阴性检测率为15 - 20%。三轮完整的overgo杂交，每次针对约39,000个BAC（BAMHI或ECORI部分消化插入片段），为517个已定位的鸡基因组STS标记产生了总共1853个BAC比对。这些数据是公开可用的，并且已用于构建鸡基因组的第一代BAC重叠群图谱。
