FLUORESCENT AMINO ACID PROBE OF TEMPLATE-STRAND BASES

描述（由申请人提供）：为了探索大规模测序DNA的新方法的可能性，我们提出用一个或多个荧光报告探针装饰DNA聚合酶的内部，根据几种X射线晶体结构，预测这些荧光报告探针实际上接触模板碱基。晶体结构显示在反应坐标期间，一些氨基酸残基处于非常不同的位置和非常不同的角度。类似地，每个模板碱基似乎都翻转了90度，然后才摆回碱基配对位置，据报道堆叠在某个氨基酸残基上。如果我们能观察到位置固定的、进行性的、特异性荧光的DNA聚合酶的单分子，因为它们使用正常的dNTP合成DNA，我们相信会有一些可再现的闪烁，并且这种闪烁模式可能是依次对每个模板碱基的身份进行经验诊断的。预期信号水平（猝灭或增强）、波长（荧光峰的偏移）和/或极性变化。可替代地或另外地，如果碱基接触荧光的空间位置和角度对于每个模板碱基是不同的，则我们预期定位在10-15埃之外的一个或多个FRET受体荧光的荧光的有用改变。如果我们的想法能奏效，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乳液。(but将被允许）。应该涉及大量的视频计算分析（通过棱镜和/或偏振滤波器拍摄）。在一种情况下，受试者DNA可以是梳理出来的有缺口的基因组DNA，使得在缺口处排列成一行的分析的DNA聚合酶分子将指示它们闪烁的序列的相对基因组图谱位置。 公共卫生相关性：当DNA通过DNA聚合酶的“手掌”时，它会通过晶体学定义的残基。一些荧光探针分子的亮度和颜色会根据附近的分子而变化，我们将尝试在DNA聚合酶的几个位置插入一些可变探针。当每个模板DNA碱基（A、G、C或T或甲基C）踩在荧光探针上时，我们希望探针以识别每个碱基的诊断模式闪烁或闪烁，从而提供一种廉价的基因组测序方法

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. PUBLIC HEALTH RELEVANCE: As DNA is caused to step through the "palm" of DNA polymerase in action, it passes by crystallographically defined residues. Some fluorescent probe molecules vary in brightness and color, depending on nearby molecules, and we will try inserting some of these variable probes at several positions on the DNA polymerase enzyme. As each template DNA base (A,G,C or T or methyl C) steps on a fluorescent probe, we expect the probe to flicker or twinkle in a diagnostic pattern that identifies each base, and thus provide an inexpensive way to sequence genomes