24-Capillary Reveal Mutation Discovery System

描述（由申请人提供）：使用SpectuMedix Reveal-2410突变发现系统的温度梯度毛细管电泳（TGCE）将加速识别生物表型和发育过程背后的DNA序列。将温度梯度电泳与毛细管电泳相结合，可以根据熔融温度快速鉴定核苷酸序列不匹配的DNA片段。通过运行足够宽的温度梯度，可以在单次运行中以95%以上的效率检测到大小和核酸序列不同的多个片段的不匹配。这是识别功能性核苷酸序列变化的主要优势。突变发现系统不是对整个候选基因进行测序，而是优先考虑突变型和野生型之间不匹配的DNA外显子或区域进行核苷酸测序。然后，Reveal-2410可用作标准核苷酸测序仪来鉴定核苷酸的变化。由于扩大了基于噬菌体的大肠杆菌同源重组的使用，以快速修饰基因组DNA的大片段，以产生用于敲除和转基因小鼠的构建物，因此还需要增加核苷酸测序。所申请的仪器将有利于NIH资助的研究项目，包括雪旺细胞髓鞘形成的遗传控制、朊病毒疾病易感性的功能基因组学、分子马达在听力中的作用以及听觉系统和先天性耳聋的发育生物学。除了证明该技术能够揭示200 - 500bp片段中单个碱基的变化，如ENU诱导的等基因突变外，新的用途，如对QTL的候选基因进行排序，可能会证明是富有成效的。Spectrumedix Reveal 2410不仅将加速McLaughlin研究所的现有研究，而且有助于在神经遗传学领域招募新的生物医学研究人员。

DESCRIPTION (provided by applicant): Temperature gradient capillary electrophoresis (TGCE) using the SpectuMedix Reveal-2410 Mutation Discovery System will accelerate identification of the DNA sequence underlying biological phenotypes and developmental processes. By combining temperature gradient electrophoresis with capillary electrophoresis it is possible to rapidly identify DNA fragments with mismatches in nucleotide sequence based on the melting temperature. By running sufficiently broad temperature gradients, mismatches in multiple fragments differing in size and nucleic acid sequence can be detected at efficiencies above 95% in a single run. This is a major advantage over other approaches to identify functional nucleotide sequence changes. Rather than sequencing an entire candidate gene, the Mutation Discovery System prioritizes the exon or region of DNA with mismatch between mutant and wild-type for nucleotide sequencing. The Reveal-2410 can then be used as a standard nucleotide sequencer to identify the nucleotide changes. Increased nucleotide sequencing is also needed due to expanded use of phage-based E. coli homologous recombination for rapid modification of large segments of genomic DNA for generation of constructs for knockout and transgenic mice. The instrument requested will benefit NIH funded research programs in the genetic control of rnyelination by Schwann cells, in the functional genomics of susceptibility to prion diseases, in the role of molecular motors in hearing, and in the developmental biology of the auditory system and congenital deafness. In addition to the demonstrated ability of this technology to reveal single base changes in fragments between 200 to 500 bp, such as isogenic mutations induced by ENU, novel uses, such ranking candidate genes underlying QTL, are likely to prove fruitful. The Spectrumedix Reveal 2410 will not only accelerate current research at McLaughlin Research Institute, but help in recruiting new biomedical researchers in the field of neurogenetics.