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A nucleotide triphosphate set of 5\u2010chloro\u20102\u2032\u2010deoxyuridine, 7\u2010deaza\u20102\u2032\u2010deoxyadenosine, 5\u2010fluoro\u20102\u2032\u2010deoxycytidine, and 7\u2010deaza\u20102\u2032deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (<jats:italic>exo<\/jats:italic>\u2010) 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 <jats:italic>E. coli<\/jats:italic>. 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