CN113234803A

CN113234803A - Novel nucleic acid amplification method

Info

Publication number: CN113234803A
Application number: CN202110671284.7A
Authority: CN
Inventors: 江雷; 宋波; 张峰; 李娜
Original assignee: University of Shanghai for Science and Technology
Current assignee: University of Shanghai for Science and Technology
Priority date: 2021-06-17
Filing date: 2021-06-17
Publication date: 2021-08-10

Abstract

The present invention proposes a novel nucleic acid amplification method, which uses 30THz-90THz infrared light to irradiate the PCR system, reduces the melting temperature of double-stranded DNA, enhances the reaction efficiency of PCR, and drives the 3'-terminal phosphoric acid at the 5' end of the nucleotide and the primer. The 'terminal hydroxyl group generates a new phosphodiester bond, enhancing the yield and specificity of nucleic acid amplification. The invention can be applied to the fields of nucleic acid amplification with high specificity and high fidelity under the condition of low template number, and rapid nucleic acid detection requiring ultra-sensitivity in forensic identification, clinical and environmental detection and the like.

Description

Novel nucleic acid amplification method

Technical Field

The invention relates to the technical field of PCR amplification, in particular to a novel nucleic acid amplification method.

Background

The Polymerase Chain Reaction (PCR) instrument is a device which controls temperature change by a program and has certain signal detection capability and is applied to biomedical detection and nucleic acid amplification. Usually, a small amount of template deoxyribonucleic acid (DNA), a special primer, thermostable DNA polymerase, deoxyribonucleoside triphosphate (dNTP), and magnesium ions (Mg)²⁺) Mixing Tris-hydrochloric acid buffer solution according to a certain proportion, then carrying out denaturation treatment on double-stranded (ds) DNA by raising the temperature to 95 ℃, then cooling to about 60 ℃ for annealing, carrying out hybridization reaction on template single-stranded (ss) DNA and a primer in the process, then raising the temperature to 72 ℃ for extension reaction of the primer, and then repeating the circulation of the three temperaturesThe program can realize the exponential amplification of dsDNA. Due to the ultra-high specificity and signal amplification effect of PCR, the PCR is widely applied to biomedical detection and large-scale clone preparation of nucleic acid samples at present.

In the prior art, in order to shorten the time of PCR, scientists developed a PCR instrument capable of realizing rapid temperature control by heating with microwave, infrared laser and nano gold rod, and cooling with flowing air. It can be said that these methods focus on the regulation of the thermal effects of the solvent, rather than directly on chemical bonds and intermolecular interactions. In order to improve the efficiency of PCR and the fidelity of amplification, researchers in the past have focused on improving the content, properties, etc. of each component in the PCR system and adding a proper amount of an enhancer. In fact, if the denaturation temperature of dsDNA cannot be lowered, PCR cannot be free of high temperature polymerase, and high temperature also limits the lifetime of the polymerase. In addition, since dNTPs in PCR are used as both raw materials and driving energy, excessive addition of dNTPs will compete for chelating Mg²⁺Thereby affecting the reaction efficiency of Taq polymerase, and thus providing a great resistance to precise adjustment. Finally, due to the limitations of energy supply efficiency, the temperature increase and decrease rate of the metal module, and the selection of the thermostable enzyme, PCR is limited in terms of fidelity, amplification rate, length of the target fragment, and time consumption, which are also major problems to be improved or solved by the present invention.

Disclosure of Invention

The invention aims to provide a novel nucleic acid amplification method for greatly improving PCR efficiency.

In order to achieve the purpose, the invention provides a novel nucleic acid amplification method, which comprises the steps of irradiating a PCR system by using 30-90THz light, and carrying out denaturation treatment on a dsDNA template to ensure that a DNA double strand is uncoiled into a single strand; and irradiating the PCR solution assisted system by using 30-90THz infrared light to perform an extension reaction, and driving 5 'end phosphate of the nucleotide and 3' end hydroxyl of the primer to generate a new phosphodiester bond.

Further, the infrared light is obtained by emitting from a laser source or by passing through an optical filter from a common light source.

Further, the method specifically comprises the following steps:

step 1: during PCR amplification, firstly irradiating a PCR system by using 30-90THz light to perform denaturation treatment on a dsDNA template;

step 2: removing 30-90THz infrared light irradiation to anneal the system;

and step 3: irradiating the PCR solution system by using 30-90THz infrared light to perform an extension reaction so as to complete PCR amplification;

and 4, step 4: repeating the steps to realize exponential amplification of the template DNA.

Further, in step 2, ssDNA template and primer are combined by hybridization reaction.

Further, in step 3, the irradiation with 30 to 90THz light drives dNMP and the 3' -terminal hydroxyl group of the primer to form a new phosphodiester bond together with the energy of photon released by cleavage of phosphodiester bond in dNTP molecule.

Compared with the prior art, the invention has the advantages that:

1. the invention provides driving energy for the hydrolysis of dNTP and the generation of phosphodiester bond during primer extension by utilizing light irradiation of a specified Hertz. The primer and dNMP molecules are subjected to esterification reaction to form a phosphodiester bond through 30-90THz light irradiation, and the hydrolysis of the dNTP molecules is accelerated, so that more reaction driving energy is released.

2. The denaturation of the template DNA is facilitated, and the hydrogen bonds between the bases can be broken under the condition of lower than the conventional denaturation temperature when the template DNA is irradiated by 30-90THz light. After 30-90THz light irradiation is withdrawn, the ssDNA can generate a renaturation hybridization reaction with the primer, thereby promoting the PCR.

3. The DNA denaturation temperature is below 90 ℃, the temperature dependence (high temperature resistance) limit of the traditional DNA polymerase is broken, and more polymerases which work in a low-temperature region can be used. The invention greatly reduces the denaturation temperature of dsDNA by irradiating the PCR system with mid-infrared light, thereby relaxing the selection limit of PCR on DNA polymerase and simultaneously realizing PCR with more fidelity, rapidness and simplified operation steps.

4. Reduce dNTP consumption and reduce PCR cost. The invention utilizes the mid-infrared light of the two regions to be respectively positioned in the interval region of the absorption peak of water to the mid-infrared region, and can also be regarded as a non-absorption region, so that the water can be used as the optical filter for further purifying the two regions protected by the invention, and the cost of the PCR instrument can be further reduced.

5. The energy of 30-90THz light far exceeds the energy generated by dNTP hydrolysis and can realize stable and durable supply, and the reaction is not limited by energy any more, so that the amplification efficiency of PCR can be greatly improved, and the PCR time can be shortened.

Drawings

FIG. 1 is a schematic flow chart of a novel PCR amplification method according to an embodiment of the present invention;

FIG. 2 is a schematic diagram illustrating the mechanism of the infrared light assisted PCR instrument using mid-infrared light according to an embodiment of the present invention;

FIG. 2A is a diagram showing the use of mid-infrared light to assist the hydrolysis reaction of dNTPs in a novel PCR instrument;

FIG. 2B is a graph of the use of mid-infrared light to aid in the denaturation of DNA in a novel PCR instrument;

FIG. 2C shows the generation of phosphodiester bonds by using mid-infrared light to provide energy for DNA replication and to assist in the hydrolysis of dNTPs in a novel PCR instrument.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be further described below.

First, in order to facilitate understanding of those skilled in the art, the English abbreviations and formula symbols in the present invention represent the following Chinese meanings:

DNA: deoxyribonucleic acid; dNTP: deoxyribonucleoside triphosphates; dNMP: a deoxyribonucleotide; PPi: pyrophosphoric acid; Δ E: energy; and (3) PCR: polymerase chain reaction.

As shown in figure 1, the invention provides a novel PCR amplification method, during PCR amplification, a PCR system is irradiated by 30-90THz light firstly, a dsDNA template is denatured, a DNA double strand is uncoiled into a single strand, then the irradiation of the 30-90THz light is cancelled, annealing treatment is carried out, the ssDNA template and a primer are combined together through hybridization reaction, then the irradiation of the 30-90THz light is carried out, extension reaction is carried out, wherein a phosphodiester bond in a dNTP molecule is broken to release photon energy, and then the phosphodiester bond and a 3' terminal hydroxyl group of the primer are driven together with the irradiation light to generate a new phosphodiester bond. The specific reaction formula is as follows: (1) dNTP → dNMP + PPi + Δ E; (2) primer-OH (3') + dNMP (5') → primer-O-P-dNMP + PPi + Δ E. By repeating the cyclic procedure, exponential amplification of the template DNA can be achieved. The energy of the permeable infrared light is used for assisting the PCR, so that the efficiency can be improved, the experimental time can be shortened, the cost of reaction materials can be reduced, the method is a brand new PCR innovation technology, and the in vitro DNA replication technology can be simpler and more efficient.

In the present embodiment, the infrared light used is obtained by emission of a laser source or by transmission of a filter through a common light source.

From the technical point of view, the PCR technology can be simpler, more efficient, more sensitive and more fidelity (accurate), so that the false positive can be greatly reduced in biochemical and clinical detection, and the PCR detection kit has the potential to be applied to early screening of major diseases, degenerative diseases and the like.

Using mid-infrared light to assist PCR, the following effects can be produced:

1. provides driving energy for the hydrolysis of dNTP and the generation of phosphodiester bond during primer extension. Traditional PCR technology requires the addition of a large amount of dNTPs, as shown in FIG. 2A, dNTPs undergo hydrolysis reaction to generate dNMP, PPi and Δ E. Wherein the dNMP provides raw materials for primer extension, and the energy generated in the reaction can further drive the primer extension reaction. The invention can provide additional accurate reaction driving energy by using mid-infrared light irradiation. As shown in FIG. 2C, the primer and dNMP undergo esterification reaction to form phosphodiester bond by 30-90THz light irradiation, which also helps to accelerate the hydrolysis of dNTP molecules, thereby releasing more driving energy for reaction.

2. Contributes to the denaturation of the template DNA, i.e.the dsDNA is dissociated into single strands. As shown in FIG. 2B, DNA generally maintains its double helix structure through hydrogen bonds formed between bases. When irradiated with 30-90THz light, hydrogen bonds between bases can be cleaved at temperatures below conventional denaturation temperatures. After 30-90THz light irradiation is withdrawn, the ssDNA can generate a renaturation hybridization reaction with the primer again (as shown in FIG. 2C), thereby driving the PCR.

4. Can reduce dNTP consumption and PCR cost. The commonly used Taq enzyme can keep activity at the temperature of more than 75 ℃, but actually has half-life of about 40min at the temperature of 95 ℃, and the high mismatch rate of the Taq enzyme can not stably amplify the fragment with the length of more than 5 kb; in contrast, Pfu enzyme derived from archaea (Pyrococcus furious) was used, which had a stability 20 times higher than that of Taq enzyme and a fidelity 10 times higher than that of Taq enzyme (the error rate of Taq enzyme was 1.1X 10)^-4-8.9×10^-5Bp), but at a cost. If a non-thermostable polymerase is used, the cost of PCR is expected to be greatly reduced. The invention utilizes the mid-infrared light of the two regions to be respectively positioned in the interval region of the absorption peak of water to the mid-infrared region, and can also be regarded as a non-absorption region, so that the water can be used as the optical filter for further purifying the two regions protected by the invention, and the cost of the PCR instrument can be further reduced.

5. The energy of 30-90THz light far exceeds the energy generated by dNTP hydrolysis and can realize stable and durable supply, and the reaction is not limited by energy any more, so that the amplification efficiency of PCR can be greatly improved, and the PCR time can be shortened. The reaction rate of Taq enzyme was 60 bases/sec, while that of Pfu enzyme was about 25 bases/sec, and the target fragment that can be amplified by Pfu enzyme was not longer than 3kb in PCR using 95 ℃ as a denaturation temperature. The use of the mid-infrared light in the invention can accurately assist in synthesizing the phosphodiester bond, and can greatly improve the reaction rate, thereby realizing the purpose of prolonging the target length of amplification while reducing the PCR time.

From the economic and social aspects, the invention is expected to become the core technology of a new generation PCR instrument, and can occupy a great share in the market to create huge economic and social effects.

The above description is only a preferred embodiment of the present invention, and does not limit the present invention in any way. It will be understood by those skilled in the art that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A novel nucleic acid amplification method is characterized in that a PCR system is irradiated by 30-90THz infrared light, the denaturation or melting temperature of a dsDNA template is reduced, and a DNA double strand is uncoiled into a single strand at a lower temperature; and irradiating the PCR solution assisted system by using 30-90THz infrared light to perform an extension reaction, and driving 5 'end phosphate of the nucleotide and 3' end hydroxyl of the primer to generate a new phosphodiester bond.

2. The novel nucleic acid amplification method according to claim 1, wherein the infrared light is obtained by emission from a laser source or by transmission through a filter from a common light source.

3. The novel nucleic acid amplification method according to claim 1, comprising the steps of:

step 1: during PCR amplification, firstly, irradiating a PCR system by adopting 30-90THz infrared light to perform denaturation treatment on a dsDNA template;

step 2: removing 30-90THz infrared light irradiation to anneal the system;

4. The novel nucleic acid amplification method of claim 3, wherein in step 2, the ssDNA template and the primer are combined by hybridization.

5. The novel nucleic acid amplification method of claim 3, wherein in step 3, the 5 'phosphate of the nucleotide and the 3' hydroxyl of the primer are driven to form a new phosphodiester bond by irradiation with 30-90THz light together with the energy of a photon released by cleavage of the phosphodiester bond in the dNTP molecule.