Desoxyribonucleic acid strand sequencing method based on terahertz scanning near-field optical microscope

Abstract

The invention discloses a desoxyribonucleic acid strand sequencing method based on a terahertz scanning near-field optical microscope, which comprises the following steps: preparing a straightened desoxyribonucleic acid strand sample, and obtaining a terahertz near-field spatial position image and a near-field absorption spectrum of the desoxyribonucleic acid strand by using the terahertz scanning near-field optical microscope; according to a comparison database between different arrangement combinations of nucleotides and terahertz near-field signal spectral characteristics, determining a corresponding relation between terahertz near-field absorption spectra changing along with scanning positions and nucleotide combination sequences, accurately positioning the types of the nucleotides, and realizing sequencing. In addition, the methylation region in the desoxyribonucleic acid chain can be visualized. According to the method provided by the invention, accurate and efficient sequencing of the single deoxynucleotide chain and identification of the methylation state of the single deoxynucleotide chain can be realized by a physical lossless method. The method disclosed by the invention is wider in application range and lower in cost, and can adapt to different forms and types of nucleic acid samples.

Description

Technical Field

[0001] The invention relates to a terahertz biological imaging technology method, in particular to a deoxyribonucleic acid chain label-free sequencing method based on a terahertz scanning near-field optical microscope. Background Art

[0002] Deoxyribonucleic acid (DNA) is the main component of chromosomes and also the main genetic material. The deoxyribonucleic acid chain is a double helix structure formed by connecting four deoxynucleotides as units, including adenine deoxynucleotide (A), guanine deoxynucleotide (G), cytosine deoxynucleotide (C) and thymine deoxynucleotide (T). The DNA of each organism follows the complementary base pairing principle of A=T, C=G. By analyzing the nucleotide sequence of DNA fragments, we can know the intrinsic correlation between genes and life characteristics, which has important research value and driving role in many fields such as genomics, disease treatment, drug development, agricultural breeding, etc. At present, DNA sequencing technology has become an indispensable tool for biological research.

[0003] So far, the development of DNA sequencing has gone through three generations: (1) The first generation Sanger method, which has the advantages of a sequencing read length of up to 1000 bp and an accuracy of up to 99.999%, but its sequencing cost is too high and its throughput is extremely low, which seriously affects its truly large-scale application. (2) The second generation of short-read sequencing technology is currently the main sequencing method used worldwide. Compared with the first generation of sequencing technology, it has greatly improved the sequencing speed and reduced the sequencing cost. However, there is a certain degree of error rate in sequencing. (3) The third generation of sequencing technology was developed to solve the shortcomings of the second generation. Since the sequencing process does not require fragment amplification and the ultra-long read length can effectively avoid system errors caused by fragment amplification bias, the sequencing speed is fast, but the overall technology is not mature enough, the sequencing error rate is relatively high, and the cost is also high. While people are constantly improving and upgrading these sequencing technologies, they are also looking for new and better DNA sequencing methods.

[0004] Terahertz waves have key advantages in biomedical testing, such as no ionizing radiation, fingerprint spectrum recognition, no sample loss, high efficiency and speed. It is currently a popular and effective method for safety detection and biomolecular analysis in the world. In recent years, people have tried to use terahertz waves to study and test DNA-related information, including comparative analysis of absorption spectra of DNA fragments, single strands and double strands. However, due to the long wavelength of terahertz waves, traditional far-field detection is limited by the diffraction limit, and the spatial resolution can only reach the sub-millimeter level, which cannot meet the requirements of DNA sequence detection.

[0005] Therefore, an efficient and accurate DNA sequencing method is urgently needed in the market. Summary of the invention

[0006] Purpose of the invention: In view of the problems of slow sequencing speed and low accuracy in traditional DNA sequencing methods, the present invention provides a deoxyribonucleic acid chain sequencing method based on terahertz scanning near-field optical microscope.

[0007] This method uses a terahertz near-field super-resolution system to improve the spatial resolution of terahertz waves to the nanometer level, and combines the fingerprint spectrum recognition characteristics of terahertz waves for four nucleotides A, C, G, and T and various denatured nucleotide molecules to achieve a new technological breakthrough in terahertz wave detection of DNA sequences.

[0008] Technical solution: The method for sequencing a deoxyribonucleic acid chain based on a terahertz scanning near-field optical microscope of the present invention comprises the following steps:

[0009] Step S1: preparing a straightened DNA chain sample;

[0010] Step S2: using a terahertz scanning near-field optical microscope to obtain a terahertz near-field spatial position image of the deoxyribonucleic acid chain and its near-field absorption spectrum;

[0011] Step S3: combining the comparison database between different nucleotide arrangements and combinations and the terahertz near-field signal spectrum characteristics, determining the corresponding relationship between the terahertz near-field absorption spectrum that changes with the scanning position and the nucleotide combination sequence, accurately locating the type of nucleotides, and realizing the sequencing of the deoxyribonucleic acid chain;

[0012] Step S4: Visualizing the methylated regions in the DNA strand.

[0013] Furthermore, the method of step S1 is as follows: diluting the deoxyribonucleic acid chain solution with a buffer solution, dripping the diluted solution onto the substrate (3), depositing it, then rinsing it, and then using high-purity nitrogen gas to blow dry the liquid on the surface of the substrate (3) in a fixed direction, thereby achieving straightening of the deoxyribonucleic acid chain (4).

[0014] Furthermore, the buffer comprises MgCl2 buffer; and the sample used to prepare the sample comprises deoxyribonucleic acid (single / double) strands and ribonucleic acid (single / double) strands of various lengths.

[0015] Furthermore, the substrate (3) is made of a material with high terahertz reflectivity and high surface flatness, preferably a gold sheet or graphene.

[0016] Furthermore, the method of step S2 is as follows: using a radius ultrafine probe (2) to perform a spatial two-dimensional spectral scan on the area where the straightened DNA chain (4) is located, to obtain a terahertz near-field spatial position image of the DNA chain, and to obtain a terahertz near-field absorption spectrum of the DNA chain (4) point by point along the straightening direction from the starting end to the terminal end at a fixed scanning interval.

[0017] Furthermore, an extremely sharp probe (2) is used, the size of the probe (2) includes 1-10 nm, the material used includes platinum and gold, and the size of the cantilever (1) includes 200 µm.

[0018] Furthermore, the method of step S3: considering that the distance between nucleotide molecules is slightly less than 1 nm, two or three adjacent nucleotides may be detected simultaneously when scanning the straightened DNA chain, so according to the correspondence between different nucleotide arrangements and combinations and the spectral characteristics of the terahertz near-field signal, a comparison database is first established. In the subsequent actual measurement, the terahertz near-field absorption spectrum is scanned at different spatial positions of the DNA chain to be sequenced to obtain the position-related terahertz near-field signal spectrum curve, and then combined with the comparison database, a model is established to quickly parse the nucleotide sequence of the DNA chain.

[0019] Furthermore, the terahertz near-field signal spectrum curve can provide information including absorption intensity, amplitude, phase, and area under characteristic peaks.

[0020] Furthermore, based on the terahertz near-field signal spectral characteristics of methylated nucleotides, a terahertz near-field image under these spectral characteristics is constructed from the terahertz near-field absorption spectrum to visualize the spatial distribution of methylated regions in the deoxyribonucleic acid chain. The terahertz near-field image includes a terahertz near-field image obtained using a single-frequency terahertz source and a single-frequency terahertz near-field image constructed based on the terahertz near-field absorption spectrum.

[0021] Furthermore, the comparison database is: the nucleotide combination A, T and C, including ATC, ACT, CTA, CAT, TAC and TCA, has an absorption intensity of Y1 at X1 THz; the nucleotide combination T, C and G, including TCG, TGC, GTC, GCT, CTG, CGT, has an absorption intensity of Y4 at X1 THz; the nucleotide combination C, C and G, including CCG, CGC, GCC, has an absorption intensity of Y5 at X1 THz.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The present invention is based on a DNA chain sequencing method using a terahertz scanning near-field optical microscope. By straightening DNA and combining the terahertz near-field spatial position image and the near-field absorption spectrum, the corresponding relationship between the terahertz near-field absorption spectrum and the nucleotide combination sequence is determined, thereby achieving accurate DNA sequencing and visualizing its methylation region. This has important application value in fields such as biomedical research and clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Schematic diagram of a DNA chain sequencing method based on terahertz scanning near-field optical microscopy;

[0024] Figure 2 Schematic diagram of using terahertz near-field absorption spectroscopy to identify DNA with different nucleotide sequences;

[0025] Figure 3 The terahertz near-field signal spectrum curve related to the position is obtained by scanning the terahertz near-field absorption spectrum of the different spatial positions of the DNA chain armor;

[0026] Figure 4 The position-dependent terahertz near-field signal spectrum curve is obtained by scanning the terahertz near-field absorption spectrum of the deoxyribonucleic acid chain B at different spatial positions. DETAILED DESCRIPTION

[0027] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0028] The deoxyribonucleic acid chain sequencing method based on terahertz scanning near-field optical microscope of this embodiment includes the following specific steps:

[0029] (1) Use MgCl2 buffer to dilute the two DNA chain solutions A and B, and drop the diluted solutions on two gold sheets respectively, and let them settle for five minutes. Then drop ultrapure water to lightly rinse the gold sheets. Then use high-purity nitrogen to gently blow dry the liquid on the surface of the gold sheet in a fixed direction to straighten the DNA chains A and B.

[0030] (2) If Figure 1 As shown, a platinum probe with a radius of 1 nm and a cantilever size of 200 µm is used to perform two-dimensional spectral scanning on the regions where the DNA chains A and B are located after being straightened in step (1), respectively, to obtain the terahertz near-field spatial position images of A and B, and to obtain the terahertz near-field absorption spectrum of the DNA chain point by point along the straightening direction, from the starting end to the terminal end, at a fixed scanning interval;

[0031] (3) Considering that the distance between nucleotide molecules is slightly less than 1 nm, three adjacent nucleotides may be detected simultaneously when scanning the straightened DNA chain. Therefore, a comparison database is first established based on the correspondence between different nucleotide arrangements and the terahertz near-field signal spectral characteristics. According to the comparison database, when the nucleotides are grouped in groups of three, the terahertz near-field absorption spectra of nucleotides with different arrangements have obvious differences in the absorption intensity at X1 THz. Figure 2As shown, taking two deoxyribonucleic acid chains A and B as examples, the present invention describes in detail how to use terahertz near-field absorption spectroscopy to identify DNA with different nucleotide sequences. The sequence of deoxyribonucleic acid chain A is ATCGATCGCGAT, and the sequence of deoxyribonucleic acid chain B is ATCGATGCGAT. The two deoxyribonucleic acid chains differ in the types of nucleotides at the seventh and eighth positions. Considering that three adjacent nucleotides will be detected simultaneously using a 1nm probe, the nucleotide combinations detected by deoxyribonucleic acid chain A include ATC, TCG, CGA, GAT, ATC, TCG, CGC, GCG, CGA and GAT, and the nucleotide combinations detected by deoxyribonucleic acid chain B include ATC, TCG, CGA, GAT, ATG, TGC, GCC, CCG, CGA and GAT. The two deoxyribonucleic acid chains A and B are scanned by terahertz near-field absorption spectroscopy respectively to obtain a terahertz near-field signal spectrum curve related to the position. Taking the absorption intensity of the terahertz near-field signal spectrum extracted at X1 THz as an example, the position-dependent terahertz near-field signal spectrum curves obtained from A and B are as follows: Figure 3 and Figure 4 As shown. Combine the above comparison database and Figure 3 , Figure 4 The results are used to build a model to analyze the types of nucleotides. For example, the terahertz absorption intensities of A at spatial positions Z5, Z6 and Z7 are Y1, Y4 and Y5 respectively. Combined with the comparison database: the absorption intensity of the nucleotide combination A, T and C (including ATC, ACT, CTA, CAT, TAC and TCA) at X1 THz is Y1, the absorption intensity of the nucleotide combination T, C and G (including TCG, TGC, GTC, GCT, CTG, CGT) at X1 THz is Y4, and the absorption intensity of the nucleotide combination C, C and G (including CCG, CGC, GCC) at X1 THz is Y5. It is speculated that the combination of the sixth and seventh nucleotides may be TC or CT, and the combination of the seventh and eighth nucleotides may be CG or GC. Comprehensively judging, the seventh nucleotide of A should be C. Similarly, it can be speculated that the combination of the sixth and seventh nucleotides of B may be TG or GT, and the combination of the seventh and eighth nucleotides may be CG or GC, so its seventh nucleotide should be G.

[0032] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any technician in the relevant technical field, without departing from the scope of the technical solution of the present invention, makes any form of equivalent replacement or modification to the technical solution and technical content disclosed in the present invention, which does not depart from the content of the technical solution of the present invention and still falls within the protection scope of the present invention.

Claims

1. A method for sequencing a deoxyribonucleic acid chain based on a terahertz scanning near-field optical microscope, characterized in that: The steps include: Step S1: preparing a straightened DNA chain sample; Step S2: using a terahertz scanning near-field optical microscope to obtain a terahertz near-field spatial position image of the deoxyribonucleic acid chain and its near-field absorption spectrum; Step S3: combining the comparison database between different nucleotide arrangements and combinations and the terahertz near-field signal spectrum characteristics, determining the corresponding relationship between the terahertz near-field absorption spectrum that changes with the scanning position and the nucleotide combination sequence, accurately locating the type of nucleotides, and realizing the sequencing of the deoxyribonucleic acid chain; Step S4: Visualizing the methylated regions in the DNA strand.

2. The method for sequencing a deoxyribonucleic acid chain based on a terahertz scanning near-field optical microscope according to claim 1, characterized in that: The method of step S1 is as follows: diluting the deoxyribonucleic acid chain solution with a buffer solution, dripping the diluted solution onto the substrate (3), depositing it, then rinsing it, and then using high-purity nitrogen gas to blow dry the liquid on the surface of the substrate (3) in a fixed direction, thereby achieving straightening of the deoxyribonucleic acid chain (4).

3. The method for sequencing a deoxyribonucleic acid chain based on a terahertz scanning near-field optical microscope according to claim 2, characterized in that: The buffer comprises MgCl2 buffer; and the samples used to prepare the samples include deoxyribonucleic acid (single / double) chains and ribonucleic acid (single / double) chains of various lengths.

4. The method for sequencing a deoxyribonucleic acid chain based on a terahertz scanning near-field optical microscope according to claim 2, characterized in that: The substrate (3) is made of a material with high terahertz reflectivity and high surface flatness, preferably a gold sheet or graphene.

5. The method for sequencing a deoxyribonucleic acid chain based on a terahertz scanning near-field optical microscope according to claim 1, characterized in that: The method of step S2 comprises: using a radius ultrafine probe (2) to perform a spatial two-dimensional spectral scan on the area where the straightened DNA chain (4) is located, obtaining a terahertz near-field spatial position image of the DNA chain, and obtaining a terahertz near-field absorption spectrum of the DNA chain (4) point by point along the straightening direction from the starting end to the terminal end at a fixed scanning interval.

6. The method for sequencing a deoxyribonucleic acid chain based on a terahertz scanning near-field optical microscope according to claim 5, characterized in that: An extremely sharp probe (2) is used. The size of the probe (2) ranges from 1 to 10 nm. The materials used include platinum and gold. The size of the cantilever (1) ranges from 200 µm.

7. The method for sequencing a deoxyribonucleic acid chain based on a terahertz scanning near-field optical microscope according to claim 1, characterized in that: The method of step S3: Considering that the distance between nucleotide molecules is slightly less than 1 nm, two or three adjacent nucleotides may be detected simultaneously when scanning the straightened DNA chain. Therefore, a comparison database is first established based on the correspondence between different nucleotide arrangements and combinations and the spectral characteristics of the terahertz near-field signal. In subsequent actual measurements, the terahertz near-field absorption spectrum is scanned at different spatial positions of the DNA chain to be sequenced to obtain the position-related terahertz near-field signal spectrum curve, and then combined with the comparison database, a model is established to quickly resolve the nucleotide sequence of the DNA chain.

8. The method for sequencing a deoxyribonucleic acid chain based on a terahertz scanning near-field optical microscope according to claim 7, characterized in that: The information provided by the terahertz near-field signal spectrum curve includes absorption intensity, amplitude, phase, and area under the characteristic peak.

9. The method for sequencing a deoxyribonucleic acid chain based on a terahertz scanning near-field optical microscope according to claim 1, characterized in that: Based on the terahertz near-field signal spectral characteristics of methylated nucleotides, terahertz near-field images under these spectral characteristics are constructed from the terahertz near-field absorption spectrum to realize visualization of the spatial distribution of methylated regions in the deoxyribonucleic acid chain. The terahertz near-field image includes a terahertz near-field image obtained using a single-frequency terahertz source and a single-frequency terahertz near-field image constructed according to the terahertz near-field absorption spectrum.

10. The method for sequencing a DNA chain based on a terahertz scanning near-field optical microscope according to claim 1, characterized in that: The comparison database is: the nucleotide combination A, T and C, including ATC, ACT, CTA, CAT, TAC and TCA, has an absorption intensity of Y1 at X1 THz; the nucleotide combination T, C and G, including TCG, TGC, GTC, GCT, CTG, CGT, has an absorption intensity of Y4 at X1 THz; the nucleotide combination C, C and G, including CCG, CGC, GCC, has an absorption intensity of Y5 at X1 THz.

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