Invented by Stephen Turner, Benjamin Flusberg, Pacific Biosciences of California Inc
The Pacific Biosciences of California Inc invention works as followsThe invention is a method for nanopore sequence. The substrate, which can be a semiconductor, has a nanopore (or an array of them) connecting an upper and lower solution. The polymerase-nucleic complex comprises a circular template of nucleic acids and a DNA polymerase strand that displaces the latter. It also includes components for DNA synthesis. A polymerase complex containing a circular template of nucleic acids and DNA polymerase, and the components for DNA synthesis is provided. The polymerase creates a complementary nascent nucleic strand and the nascent nucleic strand moves through the nanopore as it is produced. While the nascent strand is translating through the nanopore, the sequence of that strand can be determined by measuring the current. The polymerase continues to produce nascentDNA strands as it moves around the circular templates. This allows a sequence of the nascentstrand that corresponds with a particular sequence on the circular template to be determined multiple times. “By determining the sequence of the circular template multiple times, greater accuracy can be achieved.
Background for Method for sequencing multiple copies of the same sequence in a circle template
Researchers who are attempting to determine the genome sequence of an organism have set a high priority on determining the nucleotide order of DNA and RNA. It is important to be able to identify genetic polymorphisms and mutations by being able determine the nucleic acid sequence in DNA orRNA. The concept of using nanopores (holes smaller than a nanometer), or “nanopores”, has recently been developed. Recently, the concept of using nanopores (or nanometer-sized holes) to characterize polymer and biological macromolecules has been developed.
Nanopore based analysis methods involve the passing of a polymeric molecular, such as single-stranded (?ssDNA?) DNA through a nanoscopic opening while monitoring a signal. The molecule is passed through the opening in a nanopore while a signal, such as an electric signal, is monitored. The nanopore size is usually designed so that the polymer can only pass in sequential order. The nanopore converts the differences in chemical and physical properties between the monomeric units of the polymer molecule, such as the nucleotides which make up ssDNA.
The structural differences between different nucleotides cause the current to be interrupted in different ways. Due to structural differences, different nucleotides can interrupt the current differently. Each type of nucleotide in ssDNA produces a different modulation of the current.
Nanopores have been used to sequence DNA, including protein nanopores within lipid bilayers membranes such as? -hemolysin, and solid state micropores created, for example, through ion beam sculpture of a thin solid state film. The devices that use nanopores for sequencing DNA and RNA molecules are not able to read sequences at a single nucleotide level.
While the nanopores have shown promise in detecting sequence information, it is necessary to develop accurate and reliable devices and methods that can measure sequences, such as RNA or DNA. There is therefore a need to develop a manufacturing-friendly method for fabricating nanopore arrays. There is also a need for devices that can sequence molecules with nanoscale dimensions quickly and with high resolution.
In certain aspects, the invention provides an apparatus for determining polymer sequencing information. The device comprises: a substrate having an array of micropores; wherein every nanopore is fluidically coupled to an upper and lower fluidic regions; wherein each upper liquid region is fluidically linked through an upper resistive aperture to an upper volume. In some embodiments, the upper liquid is fluidically linked to two or three upper fluidic regions. “In some embodiments, each lower fluidic area is fluidically linked through a lower resistant opening to a liquid volume and the liquid volume is connected fluidically to two or more liquidic regions.
In some embodiments, the substrate is a semiconductor that contains circuit elements. In some embodiments, either the upper or lower fluidic regions of each nanopore are electrically connected with a circuit element. In some embodiments, the circuit element is an amplifier, an Analog-to-Digital Converter, or a Clock Circuit.
In some embodiments, the resistive openning comprises one or more channel. In certain embodiments, the width and length of the channels are chosen to ensure a sufficient resistance across the resistive aperture. In some embodiments, the conduit is a channel that runs through a layer of polymer. In some embodiments, the polymeric layer comprises polydimethylsiloxane.
In some embodiments, the device also includes an upper drive electrode in the upper volume of liquid, a lower driving electrode in lower volume of liquid, and either a measurement or upper volume liquid electrode.
In some embodiments, the device also includes an upper drive electrode in the upper volume of liquid, a low drive electrode for the lower volume of liquid, an upper measurement electrode in the upper volume of liquid, and a low measurement electrode for the lower volume.
In some embodiments, the upper fluidic and lower fluidic reserviors are located within a channel extending through the substrate. In some embodiments, the upper fluidic and lower fluidic reserves each open on the same side of substrate.
The invention includes a polymer sequencer that consists of: “a) A nanopore layer with an array nanopores. Each nanopore has a cross-sectional dimension between 1 and 10 nanometers and has a top and bottom opening. The bottom opening of each micropore leads to a discrete storage reservoir.
In some embodiments, the array of nanopores is an array of holes on a solid substrate. Each hole contains a protein-nanopore. In certain embodiments, each protein nanopore in its respective hole is held in place by a lipid-based bilayer. In some embodiments, the top openings of the nanopores are open to an upper reservoir. Circuit elements can be amplifiers, clock circuits or analog to digital converters in some embodiments.
The invention, in some aspects, provides a method for fabricating a polymer sequence device that includes: a. obtaining a substrate of semiconductor; b. processing the substrate to create a microfluidic array, where the array is capable of supporting a nanopore array; c. producing circuit elements onto the substrate which are electronically coupled with the microfluidic array; and d. introducing nanopores to the microfluidic array.
In some embodiments, the circuit elements comprise CMOS circuit components. In some embodiments, the CMOS elements are amplifiers and analog to digital converters.
The invention, in some aspects, provides a method for fabricating a polymer sequence device that includes the following steps, presented in order: a) obtaining a substrate of semiconductor; b), processing the substrate of semiconductor to create an array CMOS, without performing an aluminum dilution step; c), processing the substrate of semiconductor with the CMOS to produce microfluidic elements, wherein these microfluidic elements are capable of supporting micropores; d), subsequently performing a step
The invention includes a method of fabricating a polymer sequence device that involves: a. producing an insulator with microfluidic features that include an array of holes extending through it; b. bonding the insulator to a semiconductor; c. exposing the semiconductor to etchant via the pores of the insulator to create discrete reservoirs within the layer; d. removing portions of this layer to isolate these discrete reserves from each other; e. incorporating electrical contact into the semiconductor
In some embodiments, the method also includes the step of adding micropores to each of the pores.
In some embodiments, the method also includes two or more electrodes in each of the discrete receptacles.
The invention includes a method of fabricating a polymer sequence device that involves: a. producing an insulator with microfluidic features that include an array corresponding wells in the insulator, b. bonding the insulator with a semiconducting, which has an array corresponding wells in the insulator, c. removing portions from the semiconductor to isolate the discrete reservoirs one from another, d. adding electrical contacts to allow current to flow to
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