可逆末端终止测序技术是目前市场占有率较高应用前景较被看好的一种二代高通量测序技术。它属于合成测序法，利用DNA聚合酶链延伸反应，边合成边测序。传统的具有可逆末端终止功能的核苷酸类似物在引物上合成延伸测序过程中，切除荧光基团之后，都会在碱基上残留一部分连接臂（疤痕）。这种带有疤痕的dNTPs在引物上延伸累积，会破坏DNA双螺旋结构的稳定性，影响其在引物上的延伸和测序读长。基于可逆末端合成测序法的读长短的这一缺点，本项目设计一种可完全除疤可逆末端终止功能核苷酸，使其能够在引物上更高效地合成延伸和有效终止，从而实现可逆末端终止测序法的长读长。另外，本项目拟通过对商业化酶以及"重建自适应进化路径"手段所建库的筛选，获取高效延伸、有效终止可完全去疤可逆末端终止功能核苷酸的酶；同时也进一步在方法学上丰富和完善应用"重建自适应进化路径"设计构建蛋白突变库获取特殊功能蛋白的手段和策略。

Sequencing using reversible terminator is a new second-generation high-throughput DNA sequencing technology with higher market share and great promise. It belongs to "Sequencing by synthesis" using polymerase primer extension as its core biochemistry. Traditional reversible terminator nucleotide analogs leave behind a vestige ("scar") after cleavage of the linker carrying the fluorophore. During repeated cycling, these scars accumulate on the growing primer, lowering duplex stability and impeding with further primer extension. This limits the overall read length of the sequencing process. To achieve the long read length of "reversible-terminator sequencing" architecture, we design and plan to synthesize a "Scarless reversible terminator" having a special linker holding a fluorescent tag that, after being read, can be cleaved to leave behind no "scar". Here the commercial DNA polymerases and reverse transcriptases will be first screened for their ability to well primer extension with only one scarless reversible terminator and then stop the reaction. Furthermore, to get the better enzymes to accept the scarless reversible terminator, we plan to construct several REAP polymerase libraries using "Reconstructed evolutionary adaptive paths (REAP)" approach. During this process, the REAP approach as a new methodology to construct functional protein mutant library will be further enriched and improved.