Pacific Biosciences Developed A Revolutionary DNA Sequencing Technique

At the turn of the century, Pacific Biosciences developed a revolutionary DNA sequencing technique in an attempt to help facilitate genetic studies and questions concerning human healthcare.[1] Single molecule real time sequencing or SMRT is a parallelized single molecule DNA sequencing analysis. SMRT sequencing consists of integral sequencing rates of several bases per second and read lengths into the kilobase range.[2]
Preceding cell division, enzymes referred to as DNA polymerases effectively replicate entire genomes within minutes. First, these enzymes interpret the DNA itself and then, they successively create complement strands consisting of matching building blocks termed nucleotides. SMRT sequencing exploits the polymerase’s power as a sequencing device by monitoring its actions while replicating DNA.[3] Two exclusive technological advancements facilitated this breakthrough: phospholinked nucleotides and Zero Mode Waveguide.
Phospholinked nucleotides are used to visualize polymerase activity. Each of the four different nucleotides (A, C, G, T) is tagged with a distinctive fluorescent color. Compared to other sequencing methods, phospholinked nucleotides display their fluorescent tag on the terminal phosphate rather than the nitrogenous base. This modernization allows the enzyme to cleave the fluorescent label during

The ZMW is a tubular, metal chamber roughly 70 nm wide, which sustains high signal to noise. Illumination results from the ZMW’s glass support, which generates an exceedingly small detection volume of only 20 zeptoliters.[4] When each ZMW is illuminated from beneath, the light’s wavelength is too big for passage through the waveguide. The excitation beam creates attenuated light that enters the lower 20-30nm of each ZMW fashioning one of the world’s most powerful