English
Next Generation Sequencing (NGS) is a powerful technique that allow us to obtain large amount of data from each experiment. The most advanced used system is post-light NGS based on semiconductor technology. Reads are not based on color reactions, thus there is no need to use fluorescent markers. Massive parallel sequencing reaction performed with the use of post-light NGS Ion Torrent (Life Technologies) occurs on small (2.5 cm x 2.5 cm) chips.
There are several types of chips (3xx), which are selected depending on the scale of the planned experiment. The surface of each chip is covered by reaction wells with a diameter of 3.0 µm. Distance between wells vary depending on the type of the chip: chip 314 and 316 – 5.1 µm, chip 318 – 4.1 µm. Chips differ also in the surface on which the reaction wells have been placed: chip 314 – 10.6 mm x 11.0 mm, chip 316 and 318 – 16.9 mm x 17.1 mm. This corresponds to 1.2 million, 6.3 million and 11.3 million of reaction wells for chip 314, 316 and 318 respectively.
Independent sequencing reaction occurs in each well and it course induce the change of pH in reaction environment. This change is read by CMOS which is located on the bottom part of the chip. During the sequencing, chip’s surface is flooded with the solution of certain nucleotide creating new reaction environment. If the following nucleotide on the sequenced DNA chain is complementary to the nucleotide from solution, it is incorporated into the newly forming DNA strand. Nucleotide incorporation is associated with the release into the environment of protons. In each well, there is a single bead with hundreds of thousands of clonally replicated DNA chains attached. Incorporation of complementary nucleotide occurs at the same time on each strand, therefore it causes significant change in pH in the environment. Afterwards, chip’s surface is washed with a neutral pH buffer to restore baseline value. In this way, environment is prepared to be floated with another nucleotide. This finishes the process for a given nucleotide. Four such flows for consecutive nucleotides A, C, T, and G constitute the full cycle. Depending on the planned experiment, it is possible to select the number of flows.
Due to CMOS which analyses the difference in potentials, it is possible to translate chemical code of nucleic acids DIRECTLY to the binary signal. Fluorescent markers are not used during reading and translating of the genetic code obtained in sequencing reaction. As a consequence, there is no need to create a colourful image that would have to be interpreted by optic systems. Therefore, numerous intermediate stages in the processing of source information are eliminated. Each step during data processing is a potential source of errors in the analysed experiment. This may also delay the final result. Post-light NGS with the use of CMOS does not require frequent maintenance and calibration since it does not contain any lasers, lenses, mirrors, etc. Thanks to its simplicity, it is not prone to failures. Post-light NGS system is simple, very effective and resistant to any interference and breaks in use. Thanks to the application of this technology which provides reliable approach to genetic diagnostics, it is now possible to perform highly-reliable tests with a possibly very broad range of applications such as INVICTA’s PGS-NGS 360°™.
