Patent Application
20150017655
This application claims priority for Taiwan patent application no. 102124556 filed at Jul. 9, 2013, the content of which is incorporated by reference in its entirely.
1. Field of the Invention
The present invention relates to a sequencing technology for a single molecule, particularly to a sequencing method measuring conductance to sequence a single unlabelled nucleic acid molecule.
2. Description of the Related Art
The emergence of personalized medicine indicates evolution from traditional medicine to personal genetic information-dependent medicine. The key of personalized medicine is a technology able to fast sequence DNA in high throughput and low cost. In the past decade, a new-generation sequencing technology has been developed, based on the arrayed reactions that sequence amplified DNA targets. Compared with the former-generation (Sanger) sequencing method, the new-generation sequencing technology can obviously decrease the time required to completely sequence a human genome. However, the short read length and high error rate limits the application of the new technology to sequencing unknown genomes.
One of the third-generation sequencing technologies is the single-molecule sequencing, which does not require amplification, ligation or cloning and is expected to provide single-molecule resolution, long read length and negligible error rate, together with a reduction in cost. Such methods typically involve cyclic reactions using fluorescent substrates that are monitored by optical imaging, and have, for example, been used to sequence the M13 viral genome.
An alternative third-generation technology is the nanopore sequencing, which uses a special protein to perforate nanopores in a membrane, and which identifies the sequence of nucleotides (T, C, G, and A) of a DNA molecule by measuring the modulations in the ionic current across a synthetic or biological pore while the DNA molecule is driven through it under an applied potential. This approach has been used to read DNA at single-nucleotide resolution by using Φ29 DNA polymerase (F29) to control the rate of DNA translocation through an MspA nanopore. Oxford Nanopore Technologies has also reportedly used a prototype nanopore device to decode a viral genome in a single pass of a complete DNA strand.
The commercialized third-generation technology is the only method currently comparable to the next-generation sequencing methods. However, the short read length and high error rate thereof have yet to be solved.
The primary objective of the present invention is to provide a label-free sequencing method for a single nucleic acid molecule, which incorporates unlabelled nucleotides into a nucleic acid template, assembles the template to a polymerase, and measures the conductance of the polymerase to sequence the nucleic acid molecule. The method of the present invention is not only adaptive to different polymerases but also able to decode various difficult nucleic acid sequences with very high accuracy.
To achieve the abovementioned objective, the present invention proposes a label-free sequencing method for a single nucleic acid molecule, which comprises steps: providine, a protein transistor including two electrodes and at least two gold nanoparticles respectively connected with the two electrodes, wherein a bias is applied to the two electrodes to make a first antibody self-assemble to the two gold nanoparticles; connecting a polymerase with the first antibody; introducing a nucleic acid template, pairing a primer with the nucleic acid template, and assembling the template to a polymerase; adding one or more types of unlabelled nucleotides to react with the polymerase to synthesize a complementary nucleic acid; using the protein transistor to synchronically detect the conductance signals generated by the reaction of the polymerase and obtain a conductance trajectory; and determining the sequence of the nucleic acid template according to the conductance trajectory.
In the present invention, the conductance signals of the reaction of the polymerase is detected via connecting the polymerase with the protein transistor, wherein the polymerase is connected with a second antibody molecule firstly; next the second antibody is connected with the first antibody molecule; next the first antibody is connected with the two gold nanoparticles and thus electrically connected with the source electrode and drain electrode of the protein transistor. After addition of the nucleotides, the conductance trajectory presented by the polymerase has reaction plateaux with heights of 3-6 pA, which can be recognized very easily. Each plateau is exactly corresponding to a nucleotide being read. The present invention can read about 22 nucleotides per second. The spikes of the reaction plateaux have obvious features sufficient to discriminate four different types of nucleotides. Further, the present invention can use different polymerases to decode difficult sequences, such as homopolymers. Furthermore, the present invention can arrange several self-assembling protein transistors in an identical chip to simultaneously sequence several nucleic acid templates.
Below, the embodiments are described in detail to make easily understood the objectives, technical contents, characteristics and accomplishments of the present invention.
a shows a conductance trajectory obtained in a reaction catalyzed by Φ29 DNA polymerase connected with a protein transistor according to one embodiment of the present invention;
a-1,
b and
a-4d respectively elementary patterns of reaction plateaux of nucleotides G, T, C and A according to one embodiment of the present invention;
a-5d respectively show conductance trajectories obtained via using Φ29 DNA polymerase (F29), T4 DNA polymerase (T4), T7 DNA polymerase (T7) and DNA polymerase I (Pol I) of colon bacillus to sequence a nucleic acid template of Oligo 3 according to one embodiment of the present invention; and
The present invention provides a label-free sequencing method for a single nucleic acid molecule, which uses a polymerase to pair unlabelled nucleotides with a nucleic acid template and uses conductance signals to sequence a single nucleic acid molecule, such as a single molecule of DNA or RNA. Refer to
In Step S10, Prepare a protein transistor, which provides stable conductance readings and is designed to hold a polymerase during synthesis of a new strand.
Refer to
In Step S20, connect a polymerase 60 with a second antibody molecule 50, and then connect the second body molecule 50 with the first antibody molecule 40. Alternatively, directly connect the polymerase 60 with the first antibody molecule 40. The second antibody molecule 50 may also be an immunoglobulin. The polymerase 60, such as a DNA polymerase, is an enzyme to catalyze the synthesis of DNA. The polymerase 60 is Φ29 DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, or DNA polymerase I.
The Φ29 DNA polymerase is a replicative polymerase with long processivity and low error rate. The Φ29 DNA polymerase is chemically cross-linked to the second antibody molecule 50; next the second antibody molecule 50 is connected with the Fc domain of the first antibody molecule 40 on the protein transistor 100; then the first antibody molecule 40 is bonded to the gold nanoparticles 20 and 30 respectively on the source electrode 11 and the drain electrode 12. The self-assembly process can be monitored with measuring conductance and will be described in detail thereinafter.
In Step S30, introduce a nucleic acid template 70, pair a primer 80 with the nucleic acid template 70, and assemble them to the polymerase 60. In the present invention, the nucleic acid template 70 is a single-strand DND (ssDNA), a double-strand DNA (dsDNA) or an RNA.
In Step S40, add one or more types of unlabelled nucleotides 90 to react with the polymerase 60 and generate a complementary nucleic acid. In the present invention, the unlabelled nucleotides 90 are dNTPs, including dTTP, dATP, dCTP, and dGTP. During the reaction, a nucleotide 90 (dNTP) complementary to the nucleic acid template 70 is chosen according the base-pairing principle to form a phosphodiester bonded to the 3′-OH of the primer 80 and release pyrophosphate. Before dissociating from the nucleic acid template 70, the chain elongates as the DNA polymerase 60 proceeds along the nucleic acid template 70. The interaction between the nucleotides 90 (dNTP) and the DNA polymerase 60 exhibits a classical Michaelis-Menten mechanism consisting of steps of substrate-binding (base-pairing) and bond-formation.
In Step S50, while the nucleotides 90 participate in the synthesis reaction described in Step S40, detect the conductance signals between the source electrode 11 and the drain electrode 12 to learn the conductance variation of the polymerase 60 and obtain the conductance trajectory of the polymerase 60.
In Step S60, determine the sequence of the nucleic acid template 70 according to the conductance trajectory.
Below will be described in detail the experiments of conductance detection and label-free sequencing of a single nucleic acid molecule according to the conductance trajectory.
The present invention uses a protein transistor to monitor the conductance variation of a polymerase and recognize different nucleotides. Refer to
Next, a conjugate of the Φ29 DNA polymerase, which is purified by column chromatography, is carried to the protein transistor and attached to the Fc terminal of the first antibody molecule on the protein transistor. While the source-drain voltage (VSD) is 9.0 V and the gate voltage (VG) is 3.0 V, the attachment of the Φ29 DNA polymerase conjugate induces an irreversible current rise by about 60 pA. Meanwhile, a prominent conductance signal appears in the conductance trajectory. The conductance signal will finally settle at a stable value of 102 pA with a noise level of about 5 pA.
In order to obtain a pico-ampere signal, all measurements are performed in a shielding room to minimize electromagnetic and radiofrequency interference. In order to reduce signal decay, superconducting materials are used for the interface between the transistor and probes of signal-output terminals. The dynamic response of the conductance signals is measured by sending a high-frequency laser pulse to the quantum dots of protein transistor and measuring the photon-induced fluctuation in the conductance signal. The laser waveform at a frequency of 1.7×109s−1 can be detected by means of electrical conductance with fidelity. It indicates that the system of the present invention can provide a sub-nanosecond dynamic response. The turnover rate of Φ29 DNA polymerase ranges from 20 to 150 nucleotides (nt) per second, and the sequencing reaction occurs within a millisecond time scale. Thus, the time bin is set to be 1 nanosecond during the measurement.
The sequential incorporation of nucleotides, as well as the identities of the four different nucleotides, can be detected by their characteristic conductance responses. A synthetic template carrying GATC repeats is annealed with a complementary primer and loaded onto the immobilized Φ29 DNA polymerase. If sufficient time is allowed, the fluctuation of noise will eventually stabilize with the noise level decreasing to 1 pA (shown in
The binding of a nucleotide to the active site of the (Φ29 DNA polymerase promotes conductance. The binding between the nucleotide and the polymerase is followed by bond formation, release of pyrophosphate, sliding down of the double-stranded DNA, active site evacuation (creating room for the next nucleotide, and binding of the nucleotide and the polymerase. One complete reaction cycle appears as a plateau in the conductance trajectory. The four different nucleotides are distinguished by their characteristic spike patterns. Refer to
The present invention performs statistics of more than fifty thousands of the patterns of reaction plateaux and obtains the following results.
Base-calling is verified by giving one type of nucleotide at a time. The characteristic electrical signature appears only when the corresponding nucleotide is provided. The reaction plateaux would not appear unless the correct substrate reacts with the activated site of the polymerase. For example, in synthesis of G nucleotide, injection of dGTP causes appearance of a reaction plateau. While the synthesis reaction of G nucleotide is terminated and the polymerase is displaced, dGTP is nor more the correct substrate. In such a case, addition of dGTP would not cause appearance of a reaction plateau. Furthermore, if the polymerization is terminated by dideoxynucleotide, addition of dNTP gives only binding spikes, without any reaction plateaux. The nucleotides dGTP, dATP and dTTP are used in the sequencing experiment, bonded to the frequently-seen primers according to randomly mixed nucleic acid templates, and assembled to the protein transistor by Φ29 DNA polymerase. The results show that sequencing randomly mixed templates also exhibit the accuracy of base-calling. From shape analysis, it is found that the patterns starting from the beginning of the plateaux and extending over 90% of the plateaux are consistent. Variations in width and shape frequently occur at the end of the plateaux, which is after the last spike in the cases of A nucleotide and C nucleotide. This result demonstrates that single-molecule sequencing can be achieved by monitoring the conductance of a polymerase during the synthesis of a growing DNA strand.
The present invention also makes a research to further study the association between the nucleotides and the corresponding plateau shapes via examining the conductance trajectories of the other DNA polymerases.
Refer to
Nucleic acid templates containing a stretch of a single nucleotide are known to be difficult for sequencing, and such templates frequently give read errors in many sequencing technologies. Refer to
The conductance trajectories measured by the present invention are consistent with previous studies examining the kinetics of DNA polymerases and single-molecular enzymes. The Michaelis-Menten mechanism proposes a reversible binding step that occurs before the formation of an enzyme-substrate complex, which is followed by catalysis and product release. This mechanism is corroborated by the observed binding spikes and groups of reaction plateaux (shown in
The experimental details of the present invention are provided below.
(1) Materials and Methods
Φ29 DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, and DNA polymerase I (E. coli) are purchased from NEB or Invitrogen. The standard reaction buffers for the Φ29 DNA polymerase (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT), T4 DNA polymerase (33 mM Tris-acetate pH 7.9, 66 mM sodium acetate, 10 mM magnesium acetate, 1 mM DTT), 17 DNA polymerase (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT), and DNA Polymerase I (10 mM Tris-HCl pH 7.9, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT) are made according to the supplier's specifications. The reaction buffer used in the experiment is made via diluting the standard buffer 1,000,000 folds. Ensemble experiments are performed to verify if the diluted reaction buffer and diluted magnesium affects polymerase activity. The polymerase activity remains unchanged in the presence of diluted reaction buffer. The activity of Φ29 DNA polymerase is measured via comparing fluorescence in a buffer solution diluted one fold. The sequencing results of a single molecule in the buffer solutions respectively diluted 102, 103, 104, 105 or 106 folds prove that the reaction rate of the polymerase is the same before and after dilution. In the present invention, the experiments of the label-free sequencing method for single nucleic acid molecule are conducted in the diluted buffer.
(2) Conjugation of Polymerases
A rabbit anti-mouse IgG (H+L) antibody (ZyMax™ Grade, Invitrogen, CA) is reconstituted in 10 mM phosphate buffered saline (pH 7.4) to a final concentration of 2 mg/ml. 5% glutaraldehyde (Sigma) is added to the antibody solution at a final concentration of 0.2%. Conjugation is performed by mixing 0.5 mg activated antibody with 1.5 mg DNA polymerases and 100 μl phosphate buffer followed by incubation at 25° C. for 2 hours. The reaction is terminated by adding the phosphate buffer to a final volume of 1 ml. The conjugates are purified by passage through a protein A column. The supernatants are further purified by high pressure liquid chromatography (HPLC) (Discovery BIO GFC 100 HPLC Column L×I.D. 5 cm×4.6 mm; Discovery R BIO GFC 100 L×I.D. 30 cm×4.6 mm).
In conclusion, the present invention proposes a label-free sequencing method for a single nucleic acid molecule, which determines the sequence of a single nucleic acid molecule according to the conductance signals occurring while the polymerase is assembled to the protein transistor. While nucleotides participate in synthesis, the conductance signals generated by assemblage of the polymerase to the protein transistor are used to determine the sequence of the nucleic acid template. The trajectory of the conductance signals includes a plurality of reaction plateaux each containing at least one characteristic spike. The reaction plateaux containing the characteristic spikes are respectively corresponding to four nucleotides and the bases thereof. The present invention is adaptive to different polymerases and able to decode various difficult nucleic acids, including a nucleic acid containing 20 consecutive T nucleotides. Experiments prove that the present invention can read more than 50000 nucleotides without even one error. It indicates that the present invention has remarkable precision. Further, the present invention can use a chip containing a plurality of self-assembling protein transistors to sequence a plurality of nucleic acid templates simultaneously.
The embodiments described above are only to exemplify the present invention but not to limit the scope of the present invention. Any equivalent modification or variation according to the spirit of the present invention is to be also included within the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102124556 | Jul 2013 | TW | national |
