SHAC: ISOLATION OF CHROMOSOME REGION-SPECIFIC CDNA

本方案描述了一种制备特异的cDNA亚库的方法。 特定的染色体区域。这个过程，我们称之为选择 通过亲和力捕获(SHAC)的杂交，可以描述为两个阶段。在 第一阶段，有丝分裂染色体显微切割和聚合酶链式反应。 用一种通用的简并引物进行扩增。这是一种通用的技术 用于获得染色体区域特异的基因组DNA； 所得产物的特异性通常用荧光法进行验证。 原位杂交(FISH)。这种特定于染色体区域的生物素- 标记的基因组DNA称为靶基因组DNA。在 第二阶段，具有独特连接子-接头的cDNA库结束，我们 称为源cdna，在溶液中变性并与 在存在的情况下变性和再退火的靶基因组DNA 基因组竞争对手的DNA。在适当的杂交条件下， 生物素标记的靶DNA将与来自 来源：c DNA。由此产生的DNA双链被链霉亲和素捕获- 通过生物素和生物素之间的强大亲和力来包裹磁珠 链霉亲和素。CDNA(称为SHACcDNA)从 碱变性生物素标记靶基因组的研究 和聚合酶链式反应扩增。SHAC技术应该有助于 细胞遗传学定义的染色体区域特异性基因的构建 并应在基因发现和基因组领域提供帮助 映射。这项新技术将用人类染色体进行演示。 13ql2-13带作为测试案例。 建议的商业应用：染色体区域特异性基因 子库将极大地加速分子遗传学研究。 特别是III型疾病基因的发现。我们计划开发套件和 含有聚合酶链式反应扩增的人染色体条带的试剂(S)和组织- 分别带有PCR引物结合位点的特定的cDNA文库，从而 世界各地的研究人员可以随意选择他们的目标染色体 区域(S)和表达该基因的cDNA文库(S)。

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.