The Human Genome Project, initiated in the year 1991, generated numerous activities in the field of DNA sequencing. The project not only generated data on humans and model genomes but also paved way for the development of better technology and automated the process for obtaining sequenced data. The sequencing technique was developed by Sanger and he used dideoxynucleotide terminators for automated DNA sequencing. Today, automated DNA sequencers encompass a wide array of technologies. Initially, the technology was developed and marketed by PE Biosystems (Applied Biosystems).
The sequence fragments are detected in the electromagnetic spectrum with the use of fluorophores. The visible dyes found attached to the fragments of the DNA are primarily from rhodamine and fluorescein classes of dyes. The technology in the near infrared became popular during the 1980s; especially in the data recording and digital audio media.
DNA Sequencing Methods
DNA fragments are sequenced by two fundamental methods namely: the Sanger method and the Maxam-Gilbert method. In the Maxam-Gilbert method, the DNA is chemically degraded to identify its sequence. While, in the Sanger method, which is an enzymatic method uses the chain termination method mediated by dideoxynucleotide. Out of the methods, the Sanger process is the most widely used one by automated DNA sequencers.
Automated DNA Sequencing
Understanding automated DNA sequencing provided exciting opportunities in interpreting results, maximizing applications utility, and aid in correcting faulty data errors. All the automated DNA sequencers consist of three basic parts:
(1) a source to excite and a device to detect emission;
(2) a device separating the terminated DNA fragments (capillary electrophoresis or slab gel electrophoresis) working in conjugation with emission and the excitation device; and
(3) an emission signal processing unit, which give data output.
Among the many automated DNA sequencers, three major brands cover the near-infrared region. Model 4000, the first automated DNA sequencer, was introduced by LI-COR in 1993 and was later upgraded to Model 4200 in 1997. The other near-infrared sequencers include CEQ 2000, the Beckman Coulter Capillary System, and the Open Gene System. All these systems utilize infrared laser diodes (solid-state) and other detection systems. Among these systems, only the Beckman Coulter Capillary System is capillary electrophoresis based, while the other two systems are slab gel electrophoresis based. Since the first introduced DNA automated DNA sequencer was the LI-COR Model 4000, more data have been extracted from the LI-COR DNA sequencing system, when compared to the other two near-infrared systems.
The LI-COR instrument consists of three basic elements. The source of excitation is a solid-state near-infrared laser while the detector source is a solid-state silicon- avalanche diode.
The upgraded model, LI-COR Model 4200 has either a single-laser scanning instrument (single excitation source) or a dual-laser scanning instrument (dual excitation source). The detector, a microscope, is mounted at 56 degrees (Brewster's angle) to the light emitted from the laser diode beam at the observation point. Among the two laser diodes, one is maximized of about 40 nm with maximum at 780 nm, while the other has a maximum at 680nm. Using appropriate filters, the detectors sensitivities are maximized 820 nm and 720 nm respectively.
The detector and the belt are driven by the laser back and forth scanning the electrophoretic gel apparatus mounted in the front. Since the lasers are solid-state diodes and detectors are silicon-avalanche photodiodes, they have long durability.
A small dialysis system was introduced by Visible Genetics in the year 1997 and it was called as OpenGene System. This technology uses 16 diode lasers the entire same wavelength (676 nm) for all the 16 lanes. The two primary dyes used with this system are Cy5.5 (absorption maximum at 760 nm and emission maximum at 694 nm) and Cy5 (absorbance maximum at 650 nm and emission maximum at 670 nm). Since the instrument is small, it is used in diagnostic applications.
Beckman Coulters' Technology
In 1998, the CEQ 2000 was introduced by Beckman Coulters' Bioresearch Division. This instrument has eight capillaries and has laser diodes at a maximum range of 700 and 800 nm. This system utilizes four-dye one lane chemistry like the Applied Systems Model 3700.
The use of near-infrared dyes in detecting fluorescence in automated DNA sequencing systems has many advantages. Near-infrared detection offers researchers with potential alternatives to use visible-wavelength fluorescent dyes.
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