DNA Sequencing with Reversible dNTP and Cleavable Fluorescent ddNTPTerminators

描述(申请人提供)：聚合酶反应过程中固体表面的DNA合成测序(SBS)为破译DNA序列提供了一种新的范例。在拨款申请中，我们将利用分子工程方法开发一种DNA测序系统，该系统是桑格双脱氧链终止反应和SBS的杂交。在这种方法中，四个核苷酸被修饰为可逆终止子(RTS，3‘-O-R1-dNTPs)，通过用小的可逆部分(-R1)覆盖3’-OH以使它们仍然被DNA聚合酶识别为底物，与少量四个标记双脱氧核苷酸永久终止子(PTS，ddNTPS-R2-荧光团)的可切割荧光团结合使用在DNA芯片上进行SBS。DNA序列将由ddNTP上每个荧光团的独特荧光发射来确定。当除去RTS上的3‘-OH封端基团和PTS上的荧光团时，聚合酶反应将重新启动，DNA序列可以连续测定。我们最近已经证明了使用这种方法产生高质量测序数据的可行性。我们将进一步发展这种新的方法，以便它可以很容易地用于基于荧光检测的合成系统的新一代DNA测序。此外，我们将为SBS开发一种行走策略，多次使用固定化DNA模板来增加SBS的阅读长度。我们预计这种方法将产生高达100个碱基的连续序列，可用于开展各种生物医学研究项目。

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.