Synthesis of Long DNA

Producing Natural DNA with Phospholinked Nucleotides

Phospholinked nucleotides enable the polymerase to synthesize DNA in a fast and processive manner. When the DNA polymerase encounters the nucleotide complementary to the next base in the template, it is incorporated into the growing DNA chain. During incorporation, the enzyme holds the nucleotide in the ZMWs detection volume for tens of milliseconds, orders of magnitude longer than the average diffusing nucleotide. The system detects this as a flash of bright light because the background is very low. The polymerase advances to the next base and the process continues to repeat (Figure 10) View animation.
Processive Synthesis with Phospholink Nucleotides (PSPN)

Figure 10. Processive Synthesis with Phospholink Nucleotides.

Step 1: Fluorescent phospholinked labeled nucleotides are introduced into the ZMW. Step 2: Polymerase holds the base being incorporated in the detection volume for tens of milliseconds, producing a bright flash of light. Step 3: The enzyme cleaves the phosphate chain, also releasing the attached dye molecule. Step 4-5: The process repeats.

Synthesis of long DNA

Figure 11. Synthesis of long DNA.

DNA polymerase processively incorporates nucleotides producing long, natural DNA.

With the use of phospholinked nucleotides, a long, natural strand of DNA is produced. This processive synthesis is extremely efficient, consuming only one molecule per base sequenced in reagents (Figure 11). Researchers at PacBio have demonstrated this approach has the capability to produce reads tens of thousands of nucleotides in length.   « previous 1 | 2 | 3

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