Chapter 7 Single-Molecule Fluorescence Microscopy and its Applications to Single-Molecule Sequencing by Cyclic Synthesis

Abstract

Abstract Single-molecule DNA sequencing (SMDS) had been proposed well before genomic research had advanced to the point where the DNA sequences of a few human individuals became available. Skepticism arose as to whether or not there was a need to replace methods that had been proven to be productive by a new technology. However, DNA information from thousands of individuals is needed to connect genomic information to the function it serves. Direct extensions of current methods are expected to be still much too expensive and slow to collect the amount of DNA and RNA sequence information that is required to enter the next phase in genomic research. Single-molecule techniques show great promise, as the next generation of DNA sequencing methods will allow the required amount of sequence information to be gathered in a timely and inexpensive manner. While several SMDS methods are under development, currently only single-molecule sequencing by cyclic synthesis advanced to the point where sequence information is produced in a massively parallel way directly from single DNA molecules. This sequencing technology relies on incorporation of fluorescently labeled nucleotides by DNA polymerase into complementary strands of DNA that are immobilized to a surface. The individual DNA strands are separated by a few microns and can be monitored as independent entities. The fluorescent signal of each incorporated labeled nucleotide is then sequentially detected using fluorescent microscopy. Because each DNA molecule is sequenced separately there is no need for synchronization between different molecules. Tens of millions of molecules can be sequenced in parallel in single small reaction volume, and thus this method readily produces high-throughput sequencing at a minimal cost. Currently this technique produces short-reading lengths, which make it suitable to re-sequencing applications in which a reference sequence is given. A single reference genome can serve as a template for the thousands of genomes produced by the short DNA fragments. These data can be used to find rare mutations and genetic heterogeneity in multiple target environments with great accuracy, high rates, and low cost. The ability to extract a massive amount of sequence information will equip cancer research with a powerful tool needed to defeat genetic diseases. In this chapter, different aspects of SMDS by cyclic synthesis will be discussed.