Development of Novel Fluorescent Dyes for DNA Sequencing

用于 DNA 测序的新型荧光染料的开发

• 批准号: 6692875
• 负责人: MATTHEW MAHINDARATNE
• 金额: $ 21.4万
• 依托单位: LASERGEN
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-21 至 2004-07-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6692875
• 关键词: DNA; DNA directed DNA polymerase; X ray crystallography; fluorescence; fluorescent dye /probe; mass spectrometry; nuclear magnetic resonance spectroscopy; nucleic acid sequence; single nucleotide polymorphism; stereochemistry; technology /technique development

DESCRIPTION (provided by applicant): The introduction of novel fluorescent dyes has significantly impacted DNA sequencing technologies by improving accuracy and read lengths, increasing throughput, and decreasing costs. Despite these advances, however, sequencing reactions labeled by dye-terminator chemistries show lower, although acceptable phred quality values. This results from sequence-dependent bias incorporation by DNA polymerase resulting in non-uniform peak heights in the chromatographic data. Variation in peak heights is particularly problematic when attempting to quantitate different base mixtures for heterozygote analyses of single nucleotide polymorphisms (SNPs). Bias incorporation can be attributed to steric hindrance from the large fluorescent dye structures attached to DNA bases via 3-amino-l-propynyl (AP-3) linkers. Here, we propose the development of fluorobases, which are modified DNA bases that fluoresce. We anticipate that the elimination of the AP-3 linkers and the compactness of the proposed fluorobase nucleotides will significantly reduce bias incorporation by DNA polymerase and therefore, significantly improve data quality and heterozygote identification. To test this concept, a prototypical series of novel fluorobase dye structures will be synthesized and their physical and spectral properties will be fully characterized. It is anticipated that these compounds will be highly soluble in aqueous solutions due to their polar structures. Although the development of the dyes proposed in this research are a priori intended for DNA sequencing applications, the commercial potential of these novel compounds is extremely high for general fluorescent labeling and detection of nucleic acids. Specifically, the scope of research outlines the synthesis of two different fluorobases, each consisting of either a purine or pyrimidine base. Following each successful synthesis and structure analysis, the fluorobases will be characterized spectroscopically to determine their excitation and emission maxima and respective half-bandwidths, and their molar extinction coefficients, quantum yields, photostability and solubility in a variety of aqueous and organic solvents. Subsequently, fluorobases, which show good fluorescent and physical properties, will be coupled to a 2',3'- dideoxyribose molecule and phosphorylated to create fluorobase nucleoside triphosphates. Oligo-template incorporation and conventional sequencing assays will be performed to examine DNA polymerase performance and bias incorporation effects. Successful outcome of this Phase I research could lead to the development of a set of spectrally resolvable fluorobase nucleotides configured as single dye or energy-transfer dye cassettes. The work described here could potentially impact the field of DNA sequencing technologies by increasing accuracy and read lengths, reducing costs, and improving heterozygote detection.

描述（由申请人提供）：新型荧光染料的引入通过提高准确性和读取长度，增加通量和降低成本，显著影响了DNA测序技术。然而，尽管取得了这些进展，用染料终止物化学标记的测序反应显示出较低的，尽管可以接受的phred质量值。这是由于DNA聚合酶的序列依赖性偏置导致色谱数据中峰高不均匀。当试图为单核苷酸多态性（SNPs）的杂合子分析定量不同碱基混合物时，峰高的变化尤其成问题。偏置掺入可归因于通过3-氨基-丙基（AP-3）连接剂附着在DNA碱基上的大荧光染料结构的空间位阻。在这里，我们提出开发含氟碱基，这是修饰的DNA碱基荧光。我们预计，AP-3连接体的消除和所提出的氟碱基核苷酸的紧凑性将显著减少DNA聚合酶的偏倚掺入，从而显著提高数据质量和杂合子鉴定。为了测试这一概念，将合成一系列新型氟基染料结构的原型，并对其物理和光谱特性进行充分表征。由于它们的极性结构，预计这些化合物将在水溶液中高度可溶。虽然本研究中提出的染料的开发是先验的用于DNA测序应用，但这些新化合物的商业潜力对于核酸的一般荧光标记和检测是非常高的。