Ion Semiconductor Sequencing is a method of DNA quencing bad on the detection of hydrogen ions that are relead during the polymerization of DNA. This is a method of "quencing by synthesis", during which a complementary strand is built bad on the quence of a template stand.
A microwell containing a template DNA strand to be quenced is flooded with a single species of deoxyribonucleotide (dNTP). If the introduced dNTP is complementary to the leading template nucleotide it is incorporated into the growing complementary strand. This caus the relea of a hydrogen ion that triggers a hypernsitive ion nsor, which indicates that a reaction has occurred. If homopolymer repeats are prent in the template quence multiple dNTP molecules will be incorporated in a single cycle. This leads to a corresponding number of relead hydrogens and a proportionally higher electronic signal.
This technology differs from other quencing technologies in that no modified nucleotides or optics are ud. Ion miconductor quencing may also be referred to as ion torrent quencing, pH-mediated quencing, silicone quencing, or miconductor quencing. I
t was developed by Ion Torrent Systems Inc. and was relead in February 2010.[1] Ion Torrent have marketed their machine as a rapid, compact and economical quencer that can be utilized in a large number of laboratories as a bench top machine.[2]
Technology
The incorporation of deoxyribonucleotide into a growing DNA strand caus the relea of hydrogen and pyrophosphate.
The relea of hydrogen ions indicate if zero, one or more nucleotides were incorporated.
Relead hydrogens ions are detected by an ion nsor. Multiple incorporations lead to a corresponding number of relead hydrogens and intensity of signal.
Sequencing Chemistry
In nature, the incorporation of a deoxyribonucleotide (dNTP) into a growing DNA strand involves the formation of a covalent bond and the relea of pyrophosphate and a positively charged hydrogen ion.[1] A dNTP will only be incorporated if it is complementary to the leading unpaired template nucleotide. Ion miconductor quencing exploits the facts by determining if a hydrogen ion is relead upon providing a single species of dNTP to the reaction.
Microwells on a miconductor chip that each contain one single-stranded template DNA molecule to be quenced and one DNA polymera are quentially flooded with unmodified A, C, G or T dNTP.[1][3][4] If an introduced dNTP is complementary to the next unpaired nucleotide on the template strand it is incorporated into the growing complementary strand by the DNA polymera.[5] If the introduced dNTP is not complementary there is no incorporation and no biochemical reaction. The hydrogen ion that is relead in the reaction changes the pH of the solution, which is detected by a hypernsitive ion nsor.[1][3] The unattached dNTP molecules are washed out before the next cycle when a different dNTP species is introduced.[3]
Signal Detection
Beneath the layer of microwells is an ion nsitive layer, below which is a hypernsitive ISFET ion nsor.[2] All layers are contained within a CMOS miconductor chip, similar to that ud in the electronics industry.[2][6]
Each relead hydrogen ion triggers the ISFET ion nsor. The ries of electrical puls transmitted from the chip to a computer is translated into a DNA quence, with no intermediate signal conversion required.[3][7] Each chip contains an array of microwells with corresponding ISFET detectors.[3] Becau nucleotide incorporation events are measured directly by electronics, the u of labeled nucleotides and optical measurements are avoided.[2][6]
Sequencing Characteristics
The per ba accuracy achieved in hou by Ion Torrent on the Ion Torrent ion miconductor quencer as of February 2011 was 99.6% bad on 50 ba reads, with
100 Mb per run.[8] The read-length as of February 2011 was 100 ba pairs.[8] The accuracy for homopolymer repeats of 5 repeats in length was 98%.[8]. It should be noted that the figures have not yet been independently verified outside of the company.
