FLUORESCENT AMINO ACID PROBE OF TEMPLATE-STRAND BASES

描述(申请人提供)：为了探索大规模测序DNA的新方法的可能性，我们建议根据几种X射线晶体结构，在DNA聚合酶的内部装饰一个或多个荧光报告探针，预计这些探针实际上会接触到模板碱基。晶体结构表明，在反应坐标中，一些氨基酸残基处于非常不同的位置和非常不同的角度。同样，每个模板碱基似乎被翻出90度，然后才被摆回碱基配对位置，据报道，它堆积在特定的氨基酸残基上。如果我们能观察到位置固定的、连续的、特殊荧光的DNA聚合酶的单分子，当它们使用正常的dNTP合成DNA时，我们相信会有一些可重现的闪烁，并且这种闪烁模式可能依次对每个模板碱基的同一性进行经验诊断。预计信号电平(猝灭或增强)、波长(荧光峰的移动)和/或极性的改变。作为替代或补充，如果接触碱基的荧光的空间位置和角度对于每个模板碱基是不同的，我们期望位于10-15埃之外的一个或多个FRET受体荧光的荧光发生有用的改变。如果我们的某种形式的想法奏效，它可能会相对便宜，因为不需要加工芯片，不需要特殊的dNTP，也不需要涉及聚合酶链式反应乳剂。(但会被允许)。应该对视频(通过棱镜和/或偏振滤光片拍摄)进行大量的计算分析。在一种情况下，目标DNA可以是有间隙的基因组DNA，然后梳理出来，这样在间隙排成一行的被分析的DNA聚合酶分子将指示它们正在闪烁的序列的相对基因组图位置。

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.