Leal Nicole A, Sukeda Makoto, Benner Steven A
Foundation for Applied Molecular Evolution, Ayers Medical Plaza, Suite 208, 720 SW 2nd Avenue, Gainesville, FL 32601, USA.
Nucleic Acids Res. 2006;34(17):4702-10. doi: 10.1093/nar/gkl625. Epub 2006 Sep 8.
A strategy is presented that uses dynamic equlibria to assemble in situ composite DNA polymerase primers, having lengths of 14 or 16 nt, from DNA fragments that are 6 or 8 nt in length. In this implementation, the fragments are transiently joined under conditions of dynamic equilibrium by an imine linker, which has a dissociation constant of approximately 1 muM. If a polymerase is able to extend the composite, but not the fragments, it is possible to prime the synthesis of a target DNA molecule under conditions where two useful specificities are combined: (i) single nucleotide discrimination that is characteristic of short oligonucleotide duplexes (four to six nucleobase pairs in length), which effectively excludes single mismatches, and (ii) an overall specificity of priming that is characteristic of long (14 to 16mers) oligonucleotides, potentially unique within a genome. We report here the screening of a series of polymerases that combine an ability not to accept short primer fragments with an ability to accept the long composite primer held together by an unnatural imine linkage. Several polymerases were found that achieve this combination, permitting the implementation of the dynamic combinatorial chemical strategy.
本文提出了一种策略，该策略利用动态平衡从长度为6或8个核苷酸的DNA片段原位组装长度为14或16个核苷酸的复合DNA聚合酶引物。在本实施方案中，这些片段在动态平衡条件下通过亚胺连接子瞬时连接，该亚胺连接子的解离常数约为1 μM。如果聚合酶能够延伸复合引物而不是片段，则有可能在结合了两种有用特异性的条件下引发靶DNA分子的合成：（i）短寡核苷酸双链体（长度为四至六个核碱基对）特有的单核苷酸识别，这有效地排除了单个错配；（ii）长（14至16聚体）寡核苷酸特有的引发总体特异性，这在基因组中可能是独特的。我们在此报告了一系列聚合酶的筛选，这些聚合酶兼具不接受短引物片段的能力和接受由非天然亚胺连接连接在一起的长复合引物的能力。发现了几种实现这种组合的聚合酶，从而允许实施动态组合化学策略。
