DNA sequencing has evolved significantly since the late 1970s, when shotgun cloned libraries were deciphered a couple hundred bases at a time using radioactive labels, hand poured gels, films, and a darkroom.
As the demand for sequence data shifted from genes to genomes, or from kilobases to gigabases, gene cloning was excluded as the sophistication and throughput of DNA sequencing increased.
In the sequencing lab, Escherichia coli lost its job.
The latest in Next Generation Sequencing (NGS) now allows for direct sequencing of individual DNA molecules or clonally amplified DNA.
The primary systems used in NGS, manufactured by Pacific Biosciences and Illumina®, can generate gigabases to terabases of data per run (compared to megabases for the previous generation) by directly measuring bases incorporated during DNA polymerization.
Gone are dideoxy terminators and pyrosequencing, replaced by fluorescent nucleotides that are detected during synthesis (i.e., SBS or sequencing by synthesis).
Furthermore, the single-molecule real-time (SMRT™) technology of Pacific Biosciences measures