Head of Service: Anna Pérez Lezaun
Laboratory 117
Tel.: 93 542 29 33
DNA Sequencing
Molecular biology has undergone spectacular changes over the last twenty years thanks to two great discoveries: the development of a process to establish the nucleotide sequence of DNA (Sanger, Nobel Prize 1980), and the invention of the PCR or polymerase chain reaction technique (Mullis, Nobel Prize 1993). Both discoveries have paved the way for development of more recent techniques in modern molecular biology benefiting very diverse fields of study. One of the most important applications is the sequencing of DNA fragments.
The latest automated sequencing technologies employ the Sanger method with a thermostable polymerase using terminators or fluorescent primers that allow cycle sequencing using a simple PCR process. The product of the sequencing is separated by denaturing capillary or gel electrophoresis on extremely sophisticated equipment that allow high-speed automated sequencing of the DNA with a high level of accuracy.
Although gel sequencers are still commonly used, the latest sequencing equipment uses capillary electrophoresis systems, which offer a range of advantages over traditional gel electrophoresis. Capillaries dissipate heat much more efficiently, enabling the use of higher voltages and hence much shorter run times. With this technology, samples are also electrokinetically injected into the capillaries in under 30 seconds and, as the detection system is more sensitive, less DNA per sample is required than with traditional gels.
The most notable applications of these technologies are sequencing of DNA fragments from PCR or cloning (original or comparative sequencing) and analysis of PCR fragments labelled with fluorochromes: "Genescan"(detection of variation in STRs, VNTRs...); "Snapshot"(detection of SNPs...).