Abstract
We demonstrate the efficient synthesis of DNA with complete replacement of the four deoxyribonucleoside triphosphate (dNTP) substrates with nucleotides carrying fluorescent labels. A different, spectrally separable fluorescent dye suitable for single molecule fluorescence detection was conjugated to each of the four dNTPs via linkage to the terminal phosphate. Using these modified nucleotides, DNA synthesis by φ29 DNA polymerase was observed to be processive for products thousands of bases in length, with labeled nucleotide affinities and DNA polymerization rates approaching unmodified dNTP levels. Results presented here show the compatibility of these nucleotides for single-molecule, real-time DNA sequencing applications.
Highlights
Nucleotides carrying fluorescent labels are widely used in biological research, molecular biotechnology, and cytogenetics
Complete replacement of every deoxyribonucleotide triphosphate by a dye-labeled dNTP is essential to these methods to avoid the creation of sequence gaps
Several of these sequencing-by-synthesis schemes utilize nucleotides with fluorescent dyes linked to the nucleobases, but their enzymatic incorporation becomes increasingly limited with larger fractions of labeled dNTP
Summary
Nucleotides carrying fluorescent labels are widely used in biological research, molecular biotechnology, and cytogenetics. Several of these sequencing-by-synthesis schemes utilize nucleotides with fluorescent dyes linked to the nucleobases, but their enzymatic incorporation becomes increasingly limited with larger fractions of labeled dNTP Using a large excess of polymerase, complete replacement of all dNTPs by terminal phosphate linked dNTPs was shown, reaching as much as 20 bases on a linear template.[12] It was not determined whether DNA synthesis was processive under the reaction conditions used.
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