Exon Specific Sequencing of Whole Genomic DNA

描述（由申请人提供）： 本研究的长期目标是提供一种方法，通过该方法可以同时对来自基因组DNA样品的1000个不同外显子进行测序，每个外显子的覆盖率为30至50倍。这将为研究人员提供关于癌前、早期癌细胞和癌细胞的基因组成和多样性的重要新信息。 RainDance Technologies（Guilford，CT）已经开发出微流体技术，以每天超过10 ~ 9个液滴的速率操纵亚纳升体积的试剂。我们建议使用这种技术来处理基因组DNA，用于对来自个体样品的1000个外显子进行测序。我们将使用单个微流控芯片进行多个处理步骤。第一个芯片上处理步骤是以每秒1000 s的速率将基因组DNA逐滴地与1000 s引物对文库的成员结合联合收割机。引物对确保在聚合酶链式反应（PCR）步骤期间在给定液滴中扩增单个外显子;该步骤的产物被捕获在珠上用于测序。最后的芯片上步骤是质量控制步骤，其中在将剩余的珠送至454 Life Sciences（Branford，CT）仪器上进行测序之前，去除具有少于1000万个外显子拷贝的珠。该项目的成功完成将允许使用一个汇集的引物对文库乳液来经济地对来自几乎无限数量的基因组（包括肿瘤）DNA样品的数千个单个外显子进行测序。

DESCRIPTION (provided by applicant): The long-term objective of this research is to provide a means by which to sequence 10OOs of different exons simultaneously from a genomic DNA sample with 30 to 50 times coverage of each exon. This will provide researchers with significant new information about the genetic makeup and diversity of pre- cancerous, early-cancerous, and cancerous cells. RainDance Technologies (Guilford, CT) has developed microfluidic technology to manipulate sub-nanoliter volumes of reagents at rates exceeding 10A9 droplets per day. We propose to use this technology to process genomic DNA for the sequencing of 1000s of exons from individual samples. We will use a single microfluidic chip for multiple processing steps. The first on-chip processing step is to combine genomic DNA on a droplet-by-droplet basis with a member of a library of 1000s of primer pairs at rates of 1000s per second. The primer pairs ensure that a single exon is amplified in a given droplet during a polymerase chain reaction (PCR) step; the product of this step is captured on a bead for sequencing. The final on-chip step is a quality control step where beads with less than 10 million copies of an exon are removed before the remaining beads are sent for sequencing on the 454 Life Sciences (Branford, CT) instrument. Successful completion of this project will allow one pooled-primer-pair-library emulsion to be used to economically sequence thousands of individual exons from an almost unlimited number of genomic, (including tumor), DNA samples.