Method and kit for nucleic acid sequencing
A nucleic acid and sequencing technology, applied in the field of DNA sequencing and fragment length analysis, can solve problems such as complexity and time requirements
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Embodiment 1-C
[0171] Embodiment 1-CMOS is synthesized
[0172] To develop nanopores or nanochannels, thicker Al2O3 (or SiO2) layers are deposited on the active NW region, typically but not limited to 35 nm in thickness. Some designs were constructed to have 35nm high NWs on the base oxide. The 3nm AlO3 dielectric layer is the overlay deposited, creating the inter-nanowire regions (valleys) of the device, with a 3nm AlO3 layer and 35nm NWs on oxide, with a combined height of 38nm.
[0173] Following this construction method, a second 35nm AlO3 (or SiO2) is deposited, resulting in a valley height of 38nm AlO3 on the oxide and a height of about 70nm including AlO3 and NW. One non-limiting embodiment uses a focused ion beam (FIB) to remove 20nm of material in the valley region of the channel in AlO3 and 50nm on the NW, making the channel flat. This can have the effect of including 20nm fluidic channels in AlO3 and thinning the NW to 20nm (removing 15nm Si and 35nm AlO3 from the surface). T...
Embodiment 2-
[0175] Example 2 - Next Generation Sanger Sequencing (NSS)
[0176] Sanger sequencing primers are designed against the template DNA molecule, involving multiple primers along the length of the region of interest. Each primer has a unique reporter moiety (based on charge reporting - or, if buffer exchange is the detection mode used, on size reporting). The primers and template are added to a sequencing mix along with dNTPs, some of which are chain-terminating dNTPs. Each of the four chain-terminating dNTPs carries a unique reporter moiety. The concentration of chain-terminating dNTPs is such that as in Sanger sequencing, amplification (using standard thermocycling, or isothermal) ( Figure 4 , b) chains of different lengths ( Figure 4 , c). Introducing these different lengths via nanochannels ( Figure 4, d), whereby each amplified fragment is brought into contact with an array of nanowires (only one nanowire is shown in this picture, however, in the final device there ...
Embodiment 3-
[0177] Example 3 - Next Generation Probe-Based Sequencing (NPS)
[0178] All variants of short oligomeric probes (2, 3, 4, 5 or 6-mers) were synthesized. Probes are optionally synthesized without attachment of reporter moieties or other ligands, or each can carry a different reporter. These probes are added to the DNA-containing solution. The solution is heated to melt the DNA and then cooled to allow hybridization of the probes along the length of the ssDNA target molecule. ( Figure 5 , b) Then, one or more target molecules with attached probes are introduced into the nanochannel. Sensitive nanostructured structures (eg, nanowire FETs) detect the probes, and / or reporter moieties attached to the probes. Since the speed at which the DNA passes the sensor and / or sensors is known, the position of the probe can be mapped along the target molecule. Since the sequences of the probes are known, these can be deduced to target molecules. Multiple passes of the target molecule ...
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