Single molecule real-time sequencing

Mechanism Dideoxy chain termination Pyrosequencing Sequencing by synthesis (SBS) Single molecule real-time SBS Semiconductor-based SBS... 

Figure 17.21 Single nanometric aperture for real time single molecule DNA sequencing. A DNA polymerase molecule attached to the bottom surface is used to successively Incorporate fluorescent nucleotides complementary to the DNA strand, causing fluorescence bursts for each incorporation process (see text for details). Image copyright of Pacific Biosciences Inc., reprinted with permission.

Enzyme microreactors have also received wide application not only in the analysis but also in the synthesis of DNA. A nanoscale platform for real-time single-molecule sequencing by recording the DNA synthesis process by an immobilized DNA polymerase has been developed and has been covered by US patent application number 7,329,492 [62]. The process relies on fluorescence resonance energy transfer (FRET) between the fluorescence receptor moiety on the dNTPs and the fluorescent dye attached to the donor DNA that emits an enhanced signal upon nucleotide incorporation. Immobilization of polymerase enables increased sensitivity detection as the DNA strand extends imattached. 

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