ABSTRACT
The ability of various nucleoside triphosphate analogues of deoxyguanosine and deoxycytidine with 7-deazadeoxyadenosine (A1 ) and 5-chlorodeoxyuridine (T1 ) to serve as substrates for Taq DNA polymerase was evaluated. The triphosphate set composed of A1 , T1 , and 7-deazadeoxyguanosine with either 5-methyldeoxycytidine or 5-fluorodeoxycytidine was successfully employed in the polymerase chain reaction (PCR) of 1.5â kb fragments as well as random oligonucleotide libraries. Another effective combination of triphosphates for the synthesis of a 1â kb PCR product was A1 , T1 , deoxyinosine, and 5-bromodeoxycytidine. In vivo experiments using an antibiotic-resistant gene containing the latter set demonstrated that the bacterial machinery accepts fully modified sequences as genetic templates. Moreover, the ability of the base-modified segments to selectively protect DNA from cleavage by restriction endonucleases was shown. This approach can be used to regulate the endonuclease cleavage pattern.
Subject(s)
Bromodeoxycytidine/chemistry , Deoxyadenosines/chemistry , Deoxycytidine/analogs & derivatives , Deoxyuridine/analogs & derivatives , Taq Polymerase/chemistry , Base Sequence , Biotechnology , DNA/chemistry , Deoxycytidine/chemistry , Deoxyuridine/chemistry , Gene Library , Nucleic Acids/chemistry , Polymerase Chain Reaction , Synthetic BiologyABSTRACT
The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate inâ vitro and to serve as genetic templates inâ vivo was evaluated. A nucleotide triphosphate set of 5-chloro-2'-deoxyuridine, 7-deaza-2'-deoxyadenosine, 5-fluoro-2'-deoxycytidine, and 7-deaza-2'deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (exo-) DNA polymerases as catalysts. Furthermore, a fully morphed gene encoding a dihydrofolate reductase was generated by PCR using these fully substituted nucleotides and was shown to transform and confer trimethoprim resistance to E. coli. These results demonstrated that fully modified templates were accurately read by the bacterial replication machinery and provide the first example of a long fully modified DNA molecule being functional inâ vivo.
