1st Generation

Dideoxy Chain Termination Sequencing

Fluorescent DNA sequencing with Capillary Electrophoresis

Dye terminator sequencing is the modern variant of the dideoxy chain termination sequencing method first pioneered by Dr. Frederick Sanger in 1977 (Nobel Prize in Chemistry). The modern method utilizes fluorescent-labeled dideoxy nucleotide chain terminators for use in a single sequencing reaction. During strand synthesis, when polymerase encounters a labeled nucleotide, the strand synthesis is terminated. Use of normal dNTPs in molar excess allows for generation of billions of strands (for each base addition), each labeled with a 3’ fluorescent dideoxy terminator. DNA sequencers separate these strands using capillary electrophoresis, and a laser scanner detects and records the dye fluorescence, outputting the data as a trace chromatogram. This technology produces high-quality reads up to 850 bp, but is unsuitable for high-throughput projects.

2nd Generation

High-throughput Cycling Technologies


Illumina is the most widely adopted Next-Generation Sequencing (NGS) technology. Small DNA fragments are annealed to a flow cell and clonally amplified (clustering) for use as template strands. Following this procedure, additions of fluorescently labeled reversible terminators, cluster imaging, and cleaving of the fluorescent label after dNTP addition occurs for each base synthesized. This technology (sequencing by synthesis) is very accurate, has extremely high output (12-225 million reads per lane), but sequences are of relatively short length (50-300 bp).

pH Sequencing (Ion Torrent)

This technology detects base incorporation by electrical sensing. Microwells containing template DNA are flooded with a single dNTP, which if complementary, release a proton (H+) during incorporation, which is then detected by an ion sensor. Homopolymeric regions, whereby sequential addition of the same base pair, results in an increased signal proportional to the number of bases added. This process repeats sequentially with the addition of different bases until the sequencing run completes.

3rd Generation & Beyond

Single Molecule Sequencing in Real Time

Pacific Biosciences (Single Molecule Real-Time; SMRT)

PacBio SMRT sequencing is the first 3rd generation technology to become widely utilized. In contrast to 2nd generation technologies, PacBio sequences DNA template strands without clonal amplification (single molecule), and detection occurs the moment the base is incorporated (real-time). DNA polymerase is attached to the bottom of a small nanowell (called a Zero-Mode Wave guide, or ZMW) which confines and restricts light signatures, whereby only a single fluorescent signature is observed. As the polymerase incorporates labeled dNTPs the detector identifies the fluorescent signature of each base added. Cleaved fluorophores rapidly diffuse out of the ZMW. While the output and accuracy of PacBio sequencing is less than that of Illumina, this technology is capable of generating very long reads (>20 kb), which is very useful for de novo sequencing applications.

Oxford Nanopore

Oxford Nanopore uses nanopore technology to further refine single molecule sequencing by electrical resistance. Alpha-hemolysin pores are dispersed throughout a proprietary membrane with high electrical resistance. A DNA-enzyme complex feeds the synthesized strand through the nanopore, with each base pair giving a unique electrical signature as it passes through the membrane. This technology is currently being refined, with smaller scale models available for beta-testing and application development.