RT Conference Proceedings
T1 Evolution of genome sequencing techniques
A1 Henao Mejía, Luisa María
A1 Goebel Vázquez, Cristina
A2 Biología Molecular y Bioquímica, 
K1 Genomas
AB The quality and the speed for genome sequencing has advanced at the same time that technology boundaries arestretched. This advancement has been divided so far in three generations. The first-generation methods enabledsequencing of clonal DNA populations. The second-generation massively increased throughput by parallelizing manyreactions while the third-generation methods allow direct sequencing of single DNA molecules.The first techniques to sequence DNA were not developed until the mid-1970s, when two distinct sequencing methodswere developed almost simultaneously, one by Alan Maxam and Walter Gilbert, and the other one by Frederick Sanger.The first one is a chemical method to cleave DNA at specific points and the second one uses ddNTPs, which synthesizesa copy from the DNA chain template. Nevertheless, both methods generate fragments of varying lengths that are furtherelectrophoresed.Moreover, it is important to say that until the 1990s, the sequencing of DNA was relatively expensive and it was seen asa long process. Besides, using radiolabeled nucleotides also compounded the problem through safety concerns andprevented the automation. Some advancements within the first generation include the replacement of radioactive labelsby fluorescent labeled ddNTPs and cycle sequencing with thermostable DNA polymerase, which allows automation andsignal amplification, making the process cheaper, safer and faster. Another method is Pyrosequencing, which is based onthe “sequencing by synthesis” principle. It differs from Sanger sequencing, in that it relies on the detection ofpyrophosphate release on nucleotide incorporation.By the end of the last millennia, parallelization of this method started the Next Generation Sequencing (NGS) with 454 asthe first of many methods that can process multiple samples, calling it the 2º generation sequencing. Hereelectrophoresis was completely eliminated. One of the methods that is sometimes used is SOLiD, based on sequencingby ligation of fluorescently dye-labeled di-base probes which competes to ligate to the sequencing primer. Specificity ofthe di-base probe is achieved by interrogating every 1st and 2nd base in each ligation reaction. The widely usedSolexa/Illumina method uses modified dNTPs containing so called “reversible terminators” which blocks furtherpolymerization. The terminator also contains a fluorescent label, which can be detected by a camera.Now, the previous step towards the third generation was in charge of Ion Torrent, who developed a technique that isbased in a method of “sequencing-by-synthesis”. Its main feature is the detection of hydrogen ions that are releasedduring base incorporation.Likewise, the third generation takes into account nanotechnology advancements for the processing of unique DNAmolecules to a real time synthesis sequencing system like PacBio; and finally, the NANOPORE, projected since 1995,also uses Nano-sensors forming channels obtained from bacteria that conducts the sample to a sensor that allows thedetection of each nucleotide residue in the DNA strand.The advancements in terms of technology that we have nowadays have been so quick, that it makes wonder: ¿How dowe imagine the next generation?
YR 2016
FD 2016-06-07
LK http://hdl.handle.net/10630/11581
UL http://hdl.handle.net/10630/11581
LA spa
DS RIUMA. Repositorio Institucional de la Universidad de Málaga
RD 20 ene 2026