FLUORESCENT AMINO ACID PROBE OF TEMPLATE-STRAND BASES

描述（由申请人提供）：为了探索一种大规模测序DNA的新方法的可能性，我们建议用一个或多个荧光报告探针装饰DNA聚合酶的内部，根据几个x射线晶体结构，这些探针预计会实际接触模板碱基。晶体结构显示了一些氨基酸残基在反应座标中处于非常不同的位置和角度。类似地，每个模板碱基在回到碱基配对位置之前似乎被翻转了90度，据报道，它们堆积在某种氨基酸残基上。如果我们能观察到单个分子的位置固定，进程，特殊荧光DNA聚合酶，当它们使用正常的dNTPs合成DNA时，我们相信会有一些可重复的闪烁，这种闪烁模式可能是经验诊断每个模板碱基的身份。预计信号电平（猝灭或增强）、波长（荧光峰的移动）和/或极性的变化。另外，如果每个模板碱基的触底荧光的空间位置和角度不同，我们预计10-15埃之外的一个或多个FRET受体荧光的荧光会发生有用的改变。如果我们的想法在某种程度上起作用，它可能相对便宜，因为不需要加工芯片，不需要不寻常的dNTPs，也不需要PCR乳剂。（但会被允许）。大量的视频计算分析（通过棱镜和/或偏振滤光片）应该涉及。在一种情况下，受试者DNA可以是有缺口的基因组DNA，梳理出来，这样在缺口处被分析的DNA聚合酶分子，排成一行，将表明它们闪烁的序列的相对基因组图谱位置。

DESCRIPTION (provided by applicant): To explore the possibility of a new way to massively sequence DNA, we propose to decorate the inside of a DNA polymerase with one or more fluorescent reporter probes that are predicted to actually touch the template bases, according to several X-ray crystal structures. The crystallographic structures show some of the amino acid residues in very different positions and at very different angles during the reaction coordinate. Similarly, each template base appears to be flipped out by 90 degrees before it is swung back into base-pairing position, reportedly stacking on a certain amino acid residue. If we can observe single molecules of positionally fixed, processive, specially-fluorescent DNA polymerases as they synthesize DNA using normal dNTPs, we believe there will be some reproducible flickering, and that this flickering pattern might be empirically diagnostic of the identity of each template base in turn. Signal level (quenching or enhancement), wavelength (a shift of the peak of fluorescence) and/or a change in polarity are anticipated. Alternatively or additionally, if the spatial position and angle of the base- touching fluor is different for each template base, we expect useful alteration of the fluorescence of one or more FRET acceptor fluors positioned 10-15 angstroms away. If some form of our idea works, it could be relatively inexpensive, since no machined chips, no unusual dNTPs, and no PCR emulsions need be involved. (but would be allowed). A great deal of computational analysis of video (taken through a prism and/or a polarizing filter) should be involved. In one scenario, the subject DNA could be gapped genomic DNA, combed out, so that the analyzed DNA polymerase molecules at the gaps, lined up in a row, would indicate relative genomic map position of the sequences that they are flashing.