A series of 2-alkylamino-2'-deoxyadenosine triphosphates (dATP) was prepared and found to be substrates for the Therminator DNA polymerase, which incorporated only one modified nucleotide into the primer. Using a template encoding for two consecutive adenines, conditions were found for incorporation of either one or two modified nucleotides. In all cases, addition of a mixture of natural dNTPs led to primer extension resulting in site-specific single modification of DNA in the minor groove. The allylamino-substituted DNA was used for the thiol-ene addition, whereas the propargylamino-DNA for the CuAAC click reaction was used to label the DNA with a fluorescent dye in the minor groove. The approach was used to construct FRET probes for detection of oligonucleotides.
Keywords: DNA; fluorescent probes; nucleotides; oligonucleotides; polymerases.
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