Patent Application
20180127813
This invention relates to techniques, methods, apparatus, reagents and materials which together form a nucleic acid sequencing system that utilizes an indicating polymerase molecule.
The sequencing of nucleic acids, such as deoxyribose nucleic acid (“DNA”) includes determining the order of the nucleotide bases, (e.g., A, C, T and G), along a direction of a nucleic acid strand. The sequence provides detailed molecular level genetic information about the organism. Although many new sequencing technologies have been developed during recent years to sequence DNA more accurately, less expensively and faster than previous techniques, it is still a laborious, expensive and time consuming process to obtain sequencing information. For example, sequencing instruments using clonal amplification in drops or on slide colonies cost $300,000-600,000 and single molecule sequencing instruments cost above $750,000, which does not include the constantly-required stream of very expensive chemicals, reagents and sample preparation protocols. Much of the high cost of these sequencing systems is due to (a) the optical components (microscopes or wave guides) for systems which employ light detection, (b) the custom chip fabrication required for sequencing systems based on electrical detection and (c) the high cost of special labeled chemicals and reagents required in the single molecule-based systems. Widespread use of such valuable sequencing information is greatly hindered by these high costs. Accordingly, there is a great need to develop hardware and reagents that are vastly less expensive and allow the sequencing information to be obtained in a more efficient manner.
Several known sequencing techniques rely on primer extension to sequence the DNA. Primer extension includes a Primer that is in solution or attached to the solid support, a Target that contains the sequence to be determined, dNTP molecules (which will extend the primer and form the synthesized DNA) and a Polymerase molecule. These techniques are often referred to as sequencing-by-synthesis (SBS).
An example of one such primer extension-mediated technique is pyrosequencing. During pyrosequencing, as the primer is undergoing extension, various chemical species are released into the surrounding solution including pyrophosphate (P2O74−) molecules from the cleavage of the triphosphate moiety associated with the dNTP molecules during strand incorporation and protons (H+). By treating the released pyrophosphate ion with a pyrophosphatase enzyme, additional chemical energy can be obtained from this hydrolysis to drive various subsequent chemical reactions. In one case, the pyrophosphate ions are coupled through various chemical species to luciferin, which emits light in proportion to the number of pyrophosphate ions released during primer extension. Therefore, the sequence of the target DNA strand is determined by noting how much light is released upon incorporation of the proper nucleotides.
Another example of DNA sequencing involves electrochemical detection. In this type of sequencing, when the Primer-Target-Polymerase complex (PTP) is undergoing primer extension protons (W) are also released. These protons may be detected using a pH meter to transduce the amount of protons released into an electrical signal. While it is not difficult to detect protons electrochemically, the relatively large distance between the PTP complex and the electrodes may be up to many microns or even millimeters. This large distance between the sample and detector, which affects the diffusion and signal response rates associated with typical pH electrodes, are much slower than techniques where the diffusion distances are shorter. Longer diffusion distances can lead to lower analyte concentrations at the detector and longer, more expensive analysis times.
In these above examples, the signal generated during SBS is not transduced by the polymerase itself but reagents in solution (pyrosequencing example) or a pH-measuring instrument (electrochemical example).
Accordingly, there is a need in the art for a sequencing technique that utilizes a shorter diffusion distance, is easy to use, has inexpensive hardware, uses unlabeled nucleotides and inexpensive reagents and provides a more efficient high throughput screening process.
The instant invention describes methods and compositions to sequence DNA one component of which is an indicating polymerase. When sequencing nucleic acids using sequencing-by-synthesis (SBS), primer extension or other methods, all four dNTP (deoxynucleotide triphosphate) molecules are sequentially added one at a time. When the correct dNTP is added, it is incorporated into the DNA strand being synthesized by action of a polymerase and P2O74− and H+ ions are released into the surrounding solution. Signals from these P2O74− and H+ ions in solution, or the chemical reaction products of these ions, are then measured chemically, instrumentally or optically to identify which dNTP molecule was incorporated from which the nucleic acid sequence may eventually be determined. Rather than detecting these reaction products remote to the polymerase from which they emanate, the present invention discloses an indicating polymerase molecule which itself detects the incorporation of the correct dNTP. The indicating polymerase has an attached moiety R1 which, when the correct dNTP is incorporated in the SBS procedure, transforms into R2. Detection of the change in the physical or chemical properties of the indicating polymerase from R1 to R2 may be correlated with the sequence of the nucleic acid being sequenced.
In one illustrative embodiment, a composition for sequencing a nucleic acid by primer extension or SBS that uses an indicating polymerase comprises a suitable buffer; a nucleic acid to be sequenced; at least one dNTP; a priming sequencing; and an indicating polymerase. In some embodiments, the indicating polymerase changes its physical or chemical properties when the correct dNTP is incorporated. In other embodiments, the indicating polymerase moiety R1 changes its physical or chemical properties and, when the correct dNTP is incorporated, becomes indicating polymerase moiety R2.
To address the current limitations discussed above, disclosed herein are compositions and methods that include a system where the chemical sensor that detects the sequencing reaction the polymerase enzyme itself that is performing the primer extension. The polymerase enzyme, which detects the primer extension by changing its physical or chemical properties upon and concomitant with incorporation of the correct dNTP during SBS and primer extension, is called an indicating polymerase. As described above, all known sequencing systems have the sequencing-detecting sensor or reagents external to and physically separated from the sequencing reactions. By eliminating the optical components, external transducing sensors and highly specialized labeled reagents, a high throughput sequencing instrument may be built, using standard, commercially available components and unlabeled nucleotide reagents, which is at least 100 times less expensive than current sequencing instruments.
Referring to
The smallest and fastest possible sequencing method or protocol would comprise only the three essential S1, PS and P components (steps A and B of
All SBS methods for sequencing nucleic acids detect the incorporation of the correct dNTP into the extending primer by measuring a change in some characteristic property that indicates when the correct dNTP is provided but the property does not change when presented with an incorrect dNTP. In the case of pyrosequencing, the energy released from the pyrophosphate hydrolysis by an added pyrophosphatase enzyme is converted to light emitted via luciferase which may be correlated with correct dNTP incorporation. When using an electrochemical sequencing method such as Ion Torrent, the protons released when the correct dNTP is incorporated are measured with a pH electrode. Haushalter previously taught that the protons released may be detected with a pH-sensitive fluorogenic dye molecule, which, in one embodiment, is attached to a bead along with the nucleic acid being sequenced, which is non-fluorescent at higher pH but fluorescent at lower pH.
In the nucleic acid sequencing method of the present invention, the incorporation of the correct dNTP induces a change in the polymerase molecule itself mediating the primer extension. As illustrated in
The R1 reporter group is attached to, bonded to or otherwise intimately associated with the polymerase. The R1 group may be attached to or associated with the polymerase after the polymerase molecule has been prepared or is attached as the protein is being expressed during synthesis or a combination thereof. The R1 group may be attached to or associated with the polymerase by means of a covalent bond, ionic bond, hydrophobic-hydrophobic or hydrophilic-hydrophilic interactions, van der Waals, magnetic interactions or any other type of bonding or associative interaction or combinations thereof.
The polymerase may be designed, synthesized or modified by many different means in order to detect the dNTP sequencing reaction including chemical and physical means. Some possible R1 materials are listed in Table 1 where the possibilities discussed are for illustrative purposes only and are not meant to limit the scope of the invention in any way. The protons and pyrophosphate anions released during correct dNTP incorporation may react directly with R1 converting R1 into detectable R2. Alternatively, the protons and pyrophosphate ions may react with another molecule or entity (not R1), which may be attached to the polymerase, the priming sequence PS or the nucleic acid being sequenced S1, as illustrated in
In one illustrative embodiment, R1 is a fluorogenic dye covalently bonded to the polymerase that is colorless at higher pH but turns fluorescent when the protons are released and the pH becomes lowered. When the dye becomes fluorescent against a dark background, the amount of light released from the fluorophore indicates correct dNTP incorporation.
Since the instant sequencing method of
In yet another illustrative embodiment, the sequence-indicating (R1→R2) transformation could involve electrochemical detection of R1 modified by reaction with the protons and pyrophosphate ions. This transformation could be measured by measuring the change in conductivity, capacitance, resistance, inductance, voltage, current or combinations thereof when R1 converts into R2. As illustrated for example, but not limitation in
In
In some embodiments, R1 may not necessarily transform from a direct reaction with the protons or pyrophosphate ions but could be transformed into R2 by a mediator or transfer molecule which reacts directly itself with the protons or pyrophosphate ions and then subsequently reacts with R1 to transform R1 into R2.
In still another embodiment, when R1 reacts with the protons or pyrophosphate ions (or the mediator molecule which reacts initially with the protons and pyrophosphate ions, subsequently reacts with R1), then changes in the emission or absorption bands of the vibrational spectrum of R1, such as infrared, Raman or other vibrational measurement techniques, will indicate the correct incorporation of a dNTP.
In a further embodiment, upon reaction with protons or pyrophosphate ions, R1 changes to R2 with a concomitant change in the wavelength or molar absorptivity of a visible or ultraviolet absorption of emission property thereby indicating the incorporation of a correct dNTP in the SBS sequencing protocol.
It should be noted that it would also be possible to use the heat released upon correct dNTP incorporation to drive the R1→R2 transformation instead of the pyrophosphate and protons. Detection of this heat could be combined with the proton and pyrophosphate reactions to detect correct dNTP incorporation.
One should not construe these embodiments, or the embodiments in Table I, as limiting the scope of the invention and many other types of R1 to R2, as well as R1 to R2 transformations and detection schema are possible.
The polymerase proteins used for sequencing are often expressed in hosts such as bacteria, viruses or other cells or organisms. In order to express an indicating polymerase which can be used for SBS or other primer extension methods, a gene to synthesize fluorophores such as Phycoerythrin (PE) or Green Fluorescent Protein (GFP) is inserted into the host gene so that when the polymerase is expressed the PE or GFP is also expressed. These GFP or PE examples represent R1 in
Next, the polymerase-fluorophore moiety is provided with a nucleic acid sequence to be determined and a priming sequence suitable for SBS or primer extension. After measuring the fluorophore under non-acidic conditions, the different dNTP molecules are sequentially added and when the correct dNTP is added, the fluorophore R1 transforms into R2 which has a different absorption, emission and fluorescence spectrum than R1. R2 is therefore distinguished from R1 and this information is used to determine which dNTP were incorporated (i.e., sequencing the nucleic acid).
A polymerase expressed in a bacterium is combined with a fluorogenic organic dye like pHrodo® from Life Technologies which is available as a succinimidyl ester and is non-fluorescent at pH=10 but strongly florescent at pH=≤7. The pHrodo succinimidyl ester reacts with groups on the surface of the polymerase protein and covalently binds the fluorophore to the polymerase.
Next, the polymerase-fluorophore moiety is provided with a nucleic acid sequence to be determined and a priming sequence suitable for SBS or primer extension. After setting the fluorogenic pHrodo to its non-fluorescent state R1, as illustrated in
A reporter molecule R1 as illustrated in
After setting the reporter molecule R1 to its unprotonated state, the different dNTP molecules are sequentially added and, when the correct dNTP is added, protons and pyrophosphate ions are released in step C of
It should be understood that the invention is not limited to the embodiments illustrated and described herein. Rather, the appended claims should be construed broadly to include other variants and embodiments of the invention, which may be made by those skilled in the art without departing from the scope and range of equivalents of the invention. It is indeed intended that the scope of the invention should be determined by proper interpretation and construction of the appended claims and their legal equivalents, as understood by those of skill in the art relying upon the disclosure in this specification and the attached drawings.
This application claims the benefit of U.S. Provisional Application No. 62/385,709 filed Sep. 9, 2016, the entire disclosure of which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62385709 | Sep 2016 | US |
