Construction of a BAC contig map of chromosome 16q by two-dimensional overgo hybridization
Construction of a BAC contig map of chromosome 16q by two-dimensional overgo hybridization
复制标题
DOI：
10.1101/gr.10.5.714
复制
发表时间：
2000-05-01
期刊：
影响因子：
7
通讯作者：
Doggett, NA
中科院分区：
文献类型：
--
作者：
Han, CS;Sutherland, RD;Doggett, NA
我们使用基于序列的标记从整合YAC STS-内容/体细胞杂交断点物理图和辐射杂交图的人类16号染色体的长臂构建一个新的序列准备BAC地图。综合物理图谱是以前在我们的实验室中产生的，包含1150个STS，在16号染色体的常染色质臂上平均每78 kb提供一个标记。用于这项工作的另外两个图谱是怀特黑德研究所和斯坦福大学的16号染色体辐射杂交图谱。为了创建该染色体的大的可测序靶标，我们使用了一种系统的方法来筛选具有从重叠寡核苷酸（overgos）生成的探针的高密度BAC过滤器。我们首先确定了三个图谱中所有可用的序列。这些包括来自基因、EST、STS和粘粒末端序列的序列。然后，我们使用BLAST来鉴定用于overgo探针的36-bp独特DNA片段。从第16号染色体长臂上共选出906个染色体。杂交发生在三个阶段：（I）针对12 X覆盖度人BAC文库（RPCI-11）的超级池杂交;（2）针对超级池杂交中鉴定的反向阳性BAC的二维杂交;和（3）针对在二维杂交后具有不明确阳性剩余的那些overgos的合并三级杂交。对于超级库杂交，在针对12 x BAC文库的单次杂交中汇集了多达236个overgos。5次超级池杂交共鉴定出5187个来自16号染色体q的阳性BAC。将这些阳性克隆在膜上重新照射，并与161个overgos的二维亚池杂交，以确定哪些BAC克隆对单个overgos呈阳性。需要额外的46个三级杂交来解决模糊的overgo-BAC关系。因此，在总共212次杂交后，我们构建了染色体16 q的初始探针含量BAC图谱，该图谱由828个overgo标记和3363个BAC组成，提供了该染色体长臂的>85%覆盖率。该图谱已被指纹图谱数据和BAC末端PCR筛选所证实。
We have used sequence-based markers From an integrated YAC STS-content/somatic cell hybrid breakpoint physical map and radiation hybrid maps of human chromosome 16 to construct a new sequence-ready BAC map of the long arm of this chromosome. The integrated physical map was generated previously in our laboratory and contains 1150 STSs, providing a marker on average every 78 kb on the euchromatic arms of chromosome 16. The other two maps used for this effort were the radiation hybrid maps of chromosome 16 from Whitehead Institute and Stanford University. To create large sequenceable targets of this chromosome, we used a systematic approach to screen high-density BAC Filters with probes generated from overlapping oligonucleotides (overgos). We first identified all available sequences in the three maps. These include sequences from genes, ESTs, STSs, and cosmid end sequences. We then used BLASTto identify 36-bp unique fragments of DNA for over go probes. A total of 906 overgos were selected from the long arm of chromosome 16. Hybridizations occurred in thr ee stages: (I) superpool hybridizations against the 12x coverage human BAC library (RPCI-11); (2) two-dimensional hybridizations against rearrayed positive BACs identified in the superpool hybridizations; and (3) pooled tertiary hybridizations for those overgos that had ambiguous positives remaining after the two-dimensional hybridization. For the superpool hybridizations, up to 236 overgos have been pooled in a single hybridization against the 12x BAC library. A total of 5187 positive BACs from chromosome 16q were identified as a result of five superpool hybridizations. These positive clones were rearrayed on membranes and hybridized with 161 two-dimensional subpools of overgos to determine which BAC clones were positive for individual overgos. An additional 46 tertiary hybridizations were required to resolve ambiguous overgo-BAC relationships. Thus, after a total of 212 hybridizations, we have constructed an initial probe-content BAC map of chromosome 16q consisting of 828 overgo markers and 3363 BACs providing >85% coverage of the long arm of this chromosome. The map has been confirmed by the fingerprinting data and BAC end PCR screening.