Producing Natural DNA with Phospholinked Nucleotides
Figure 7. Base-labeled nucleotide of other methods.
Base-labeled nucleotides have fluorophores chemically attached directly to the base.
Most sequencing-by-synthesis approaches utilize nucleotides with fluorophores attached directly to the base (Figure 7). With this labeling approach, as the enzyme incorporates each nucleotide, the fluorophore becomes a permanent part of the DNA strand. Synthesis with base-labeled nucleotides creates difficulties for real-time synthesis and detection because if multiple bases are incorporated, the physical bulk of multiple dye molecules would create steric hindrance, limiting enzyme activity(Figure 8). To work around this issue, most sequencing approaches using base-labeled nucleotides synthesize DNA one single nucleotide at a time, starting and stopping the reaction after each incorporation. The constant disruption of the reaction is time consuming, requires high reagent volumes, and severely limits the processivity of the polymerase.
Figure 8. Issues caused by base-labeled nucleotides.
Incorporation of base-labeled nucleotides into a growing DNA chain creates: 1) steric hindrance that inhibits enzymatic activity and, 2) contributes to an increase in background.
Figure 9. Phospholinked nucleotides.
Phospholinked nucleotides have fluorophores attached to the phosphate chain, which polymerase naturally cleaves after the nucleotide is incorporated.
In contrast, our SMRT™ sequencing is a real-time approach that uses alternatively labeled phospholinked nucleotides. With this strategy, the fluorescent dye is attached to the phosphate chain of the nucleotide rather than to the base (Figure 9). As a natural step in the synthesis process, the enzyme cleaves the phosphate chain when it incorporates the nucleotide into the DNA strand. Thus, upon incorporation of a phospholinked nucleotide, the DNA polymerase naturally cleaves the dye molecule from the nucleotide when it cleaves the phosphate chain. The phosphate chain-dye complex quickly diffuses the short distance out of the detection volume, ensuring the background signal remains at the same low level.
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