Developing in vitro high throughput splicing assays

描述(由申请人提供)： 前mRNA剪接是一个关键的和受调控的加工事件，其中内含子被精确地从新生的RNA转录本中切除。在所有可遗传疾病突变中，多达三分之一会导致剪接缺陷。为了更好地理解剪接的机制，我们提出了大规模实施经典的体外剪接试验和组装试验。我们建议使用这项技术来定位3‘s周围的剪接元件，定位3’s上游的分支点，并将废弃的外显子组测序文库重新定位为体外剪接底物。这一最终目标是在鉴定的、基因分型的样本上进行体外剪接试验，目的是从基因分型工具中提取表型数据。数百万个特征测序文库的存在可能会潜在地将体外发现与临床相关的患者诊断联系起来。

DESCRIPTION (provided by applicant): Pre-mRNA splicing is a critical and regulated processing event where introns are precisely excised from nascent RNA transcripts. As many as one third of all heritable disease mutations result in splicing defects. To better understand the mechanism of splicing we propose a large-scale implementation of classic in vitro splicing assays and assembly assays. We propose to use this technology to map splicing elements around the 3'ss, to map branchpoints upstream of the 3'ss and also to repurpose spent exome sequencing libraries as in-vitro splicing substrates. This final goal stages an in-vitro splicing assay on characterized, genotyped samples with the aim of extracting phenotypic data from a genotyping tool. The existence of millions of characterized sequencing libraries could potentially connect in-vitro discovery to clinically relevant patient diagnosis.