Advances in Single Molecule Nucleic Acid Sequencing

The ability to quickly and accurately obtain sequence information from single molecules of DNA and RNA has far-reaching implications for our understanding of biology. In the work presented here, we have made several advances in the area of single-molecule DNA and RNA sequencing. Specifically, in attempting to increase the read length of DNA polymerase, we have assayed several custom synthesized fluorescent nucleotides containing longer dye–base linkers. We have validated the efficacy of these nucleotides at both bulk and single-molecule levels. Furthermore, we have screened several commercially available DNA polymerases for their ability to incorporate these nucleotides. We also show that reverse transcriptase is able to synthesize a complimentary DNA strand of 28 bases in length from an RNA template, using solely fluorescently labeled nucleotides. Additionally, we show that reverse transcriptase is able to incorporate a fluorescently labeled nucleotide into an RNA template at the single-molecule level. Finally, we demonstrate automated reagent exchange for our single-molecule sequencing system

Effects of a Modified Dye-Labeled Nucleotide Spacer Arm on Incorporation by Thermophilic DNA Polymerases

The ability of eight commercially available thermophilic DNA polymerases to sequentially incorporate fluorescently labeled nucleotides sequentially was analyzed by a gel based primer extension assay. Cy5-dUTP or a variant nucleotide in which the linker had been lengthened by 14 atoms between the dye and the nucleobase were compared. We found that the Cy5-dUTP with a longer linker resulted in longer primer extension lengths. Furthermore, some of the assayed polymerases are capable of extending the primer to the full or near full length of 30 nucleotides using dye-labeled nucleotides exclusively

Effects of a Modified Dye-Labeled Nucleotide Spacer Arm on Incorporation by Thermophilic DNA Polymerases

The ability of eight commercially available thermophilic DNA polymerases to sequentially incorporate fluorescently labeled nucleotides sequentially was analyzed by a gel based primer extension assay. Cy5-dUTP or a variant nucleotide in which the linker had been lengthened by 14 atoms between the dye and the nucleobase were compared. We found that the Cy5-dUTP with a longer linker resulted in longer primer extension lengths. Furthermore, some of the assayed polymerases are capable of extending the primer to the full or near full length of 30 nucleotides using dye-labeled nucleotides exclusively