SHAC: ISOLATION OF CHROMOSOME REGION-SPECIFIC CDNA

该建议描述了制备cDNA亚文库特异性的方法， 某些染色体区域。这个过程，我们称之为选择 通过亲和捕获（SHAC）的杂交，可以分两个阶段描述。在 第一阶段，显微切割带状有丝分裂染色体，PCR- 使用通用简并引物扩增。这是一种通用技术 在我们的实验室使用，以获得染色体区域特异性基因组DNA; 所得产物的特异性常规地通过荧光检测来验证。 原位杂交（FISH）。这种染色体区域特异性生物素 标记的基因组DNA被称为靶基因组DNA。在 第二阶段，具有独特接头-接头末端的cDNA文库， 称为源cDNA，在溶液中变性并与 靶基因组DNA已经在存在下变性和再退火， 基因组竞争者的DNA。在适当的杂交条件下， 生物素标记的靶DNA将结合到其同源对应物， 源cDNA。产生的DNA双链体被链霉亲和素捕获- 通过生物素和生物素之间的强亲和力， 链霉亲和素 cDNA（称为SHACcDNA）从 它们的生物素标记的靶基因组对应物通过碱变性 和PCR扩增。 SHAC技术应有助于 细胞遗传学定义的染色体区域特异性cDNA的构建 子库，并应协助基因发现和基因组领域 映射.这项新技术将使用人类染色体进行演示 带L3 Q12 -13作为测试情况。 拟议的商业应用：染色体区域特异性cDNA 子文库将大大加速分子遗传学研究。 特别是III疾病基因发现。我们计划开发工具包， 含有PCR扩增的人类染色体带和组织的试剂- 分别具有PCR引物结合位点的特异性cDNA文库， 全世界的研究人员可以随意选择他们的目标染色体 区域和表达该感兴趣基因的cDNA文库。

This proposal describes a method for preparing cDNA sublibraries specific to certain chromosome regions. The procedure, which we call selection of hybrids by affinity capture (SHAC), can be described in two stages. In the first stage, banded mitotic chromosomes are microdissected and PCR- amplified using a universal degenerate primer. This is a general technique employed in our lab to obtain chromosome region-specific genomic DNA; the specificity of the resulting products is routinely verified by fluorescent in situ hybridization (FISH). This chromosome-region specific, biotin- labeled genomic DNA is referred to as the target genomic DNA. In the second stage, a cDNA library with unique linker-adapter ends, which we call the source cDNA, is denatured and hybridized in solution with the target genomic DNA which has been denatured and reannealed in the presence of genomic competitor DNA. Under appropriate hybridization conditions, the biotin-labeled target DNA will bind to its homologous counterpart from the source cDNA. The resulting DNA duplexes are captured by streptavidin- coated magnetic beads via the strong affinity between biotin and streptavidin. The cDNAs (referred to as SHACcDNAs) are recovered from their biotin-labeled target genomic counterparts by alkaline denaturation and PCR amplification. The SHAC technique should facilitate the construction of cytogenetically-defined, chromosome region-specific cDNA sublibraries and should assist in the fields of gene discovery and genome mapping. This new technology will be demonstrated using human chromosome bands l3ql2-13 as a test case. PROPOSED COMMERCIAL APPLICATION: Chromosome region-specific cDNA sublibraries will greatly accelerate molecular genetic research. particularly iii disease gene finding. We plan to develop kits and reagents containing PCR-amplified human chromosome band(s) and tissue- specific cDNA libraries with PCR primer-binding sites separately, so that researchers worldwide can select at will their targeted chromosome region(s) and the cDNA libraries that express this gene(s) of interest.