Dynamic assembly of primers on nucleic acid templates.
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.
Leal
Nicole A
NA
Foundation for Applied Molecular Evolution, Ayers Medical Plaza, Suite 208, 720 SW 2nd Avenue, Gainesville, FL 32601, USA. nleal@ffame.org
Sukeda
Makoto
M
Benner
Steven A
SA
D020137:Base Pair Mismatch; D020650:Combinatorial Chemistry Techniques; D017931:DNA Primers / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase; D013698:Templates, Genetic
D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase