DNA Sequencing with Reversible dNTP and Cleavable Fluorescent ddNTPTerminators

描述（由申请人提供）：聚合酶反应期间固体表面上的合成DNA测序（SBS）提供了一种新的模式来破译DNA序列。 在基金申请中，我们将继续开发一种DNA测序系统，该系统是使用分子工程方法的桑格双脱氧链终止反应和SBS之间的混合物。 在该方法中，通过用小的可逆部分（-R1）加帽3 '-OH而修饰为可逆终止子（RT，3'-O-R1-dNTP）的四种核苷酸，使得它们仍然被DNA聚合酶识别为底物，与少量的四种可裂解的荧光团标记的双脱氧核苷酸永久终止子（PT，ddNTPs-R2-荧光团）组合使用，以在DNA芯片上进行SBS。 DNA序列将通过ddNTP上每个荧光团的独特荧光发射来确定。 在去除RT上的3 '-OH封端基团和PT上的荧光团后，聚合酶反应将重新启动，并且可以连续测定DNA序列。 我们最近已经证明了使用这种方法生成高质量测序数据的可行性。 我们将进一步发展这种新方法，使其可以很容易地用于新一代的DNA测序合成系统的基础上荧光检测。 此外，我们将开发SBS的步移策略，以多次使用固定化的DNA模板来增加SBS的读长。 我们预计，通过这种方法将产生高达100 bp的连续序列，可用于进行各种生物医学研究项目。 项目健康相关性 获得个性化医疗的完整基因蓝图的能力取决于以高精度和低成本对整个基因组进行测序的技术的发展。为了实现这一目标，我们提出了一种新的测序方法，使用可逆的脱氧核苷酸终止子（RT）和可切割的荧光双脱氧核苷酸永久终止子（PT）的发展。这种RT/PT测序方法，沿着获得多个串联序列的新型步移策略，预计将以非常高的准确性和相对较低的成本并行产生数百万个100个碱基的读段。

DESCRIPTION (provided by applicant): DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a new paradigm to decipher DNA sequences. In the grant application, we will pursue the development of a DNA sequencing system that is a hybrid between Sanger dideoxy chain terminating reaction and SBS using molecular engineering approaches. In this approach, four nucleotides, modified as reversible terminators (RTs, 3'-O-R1-dNTPs) by capping the 3'-OH with small reversible moiety (-R1) so that they are still recognized by DNA polymerase as substrates, are used in combination with a small amount of four cleavable fluorophore labeled dideoxynucleotide permanent terminators (PTs, ddNTPs-R2-fluorophore) to perform SBS on a DNA chip. DNA sequences will be determined by the unique fluorescence emission of each fluorophore on the ddNTPs. Upon removing the 3'-OH capping group on the RTs and the fluorophore from the PTs, the polymerase reaction will reinitiate and the DNA sequence can be continuously determined. We have recently demonstrated the feasibility of generating good quality sequencing data using this method. We will further develop this new method so that it can be readily used in the new generation of DNA sequencing by synthesis systems based on fluorescence detection. In addition, we will develop a walking strategy for SBS to use the immobilized DNA templates multiple times to increase the readlength of the SBS. We anticipate that up to 100 bp of continuous sequences will be produced by this approach, which can be used to pursue a variety of biomedical research projects. PROJECT HEALTH RELEVANCE The ability to obtain the complete genetic blueprint for personalized medicine depends on the development of the technology to sequence the entire genome with high accuracy and at low cost. To achieve this goal, we are proposing the development of a new sequencing approach that uses reversible deoxynucleotide terminators (RT) and cleavable fluorescent dideoxynucleotide permanent terminators (PT). This RT/PT sequencing method, along with novel walking strategies to obtain multiple tandem sequences, is expected to produce millions of 100 base reads in parallel with very high accuracy and at relatively low cost.