2 Base Encoding - General Features

General Features

The general steps common to many of these next-generation sequencing techniques include:

1. Random fragmentation of genomic DNA
2. Immobilization of single DNA fragments on a solid support like a bead or a planar solid surface
3. Amplification of DNA fragments on the solid surface using PCR and making polymerase colonies
4. Sequencing and subsequent in situ interrogation after each cycle using fluorescence scanning or chemiluminescence.

In 1988, Whiteley et al. demonstrated the use of fluorescently labeled oligonucleotide ligation for the detection of DNA variants. In 1995 Macevicz demonstrated repeated ligation of oligonucleotides to detect contiguous DNA variants. In 2003, Dressman et al. demonstrated the use of emulsion PCR to generate millions of clonally amplified beads which one could perform these repeated ligation assays on. In 2005, Shendure et al. performed a sequencing procedure which combined Whiteley and Dressman techniques performing ligation of fluorescent labeled "8 base degenerate" 9-mer probes which distinguished a different base according to the probes label and non degenerate base. This process was repeated (without regenerating an extendable end as in Macevicz) using identical primers but with probes with labels which identified different non-degenerate base to sequence 6bp reads in 5->3 direction and 7bp reads in the 3->5 direction.